atrial-natriuretic-factor and Cardiomegaly

The heart is involved in the regulation of blood pressure and body fluid homeostasis. As a blood volume sensor and effector for the regulation of the volume and pressure homeostasis, the atria are the central regulator to secrete humoral messenger cardiac natriuretic hormones into the circulation. The primary action of the atria in response to the volume change in the body is to control the secretion of atrial natriuretic peptide (ANP), a member of the family of cardiac natriuretic hormones. Although all cardiac chambers are able to secrete ANP, the major source of the cardiac hormone is the atria until reactivation of the synthesis of the ventricles. In heart disease including hypertension and cardiac hypertrophy, ventricular ANP synthesis and plasma levels of ANP are increased. However, the roles of the atria for the ANP secretion are not well defined in hypertension or heart failure. Under the high concentration of plasma levels of ANP by compensatory and/or pathophysiological reactivation of the ventricular synthesis and release of ANP, with activation of the renin-angiotensin system and changes in the atrial distensibility, the roles of the atria should be reevaluated in the heart disease. The purpose of the present review is to address modulation of the atrial role in the regulation of ANP secretion and its significance in the pathological changes in hypertension and cardiac disease and to strengthen the importance of the role of the interstitial fluid dynamics of the atrial wall in the regulation of ANP secretion.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Bodily Secretions; Cardiomegaly; Heart Atria; Heart Ventricles; Humans

Atrial natriuretic factor and brain natriuretic peptide are two important biomarkers in clinical cardiology. These two natriuretic peptide hormones are encoded by the paralogous genes Nppa and Nppb, which are evolutionary conserved. Both genes are predominantly expressed by the heart muscle during the embryonic and fetal stages, and in particular Nppa expression is strongly reduced in the ventricles after birth. Upon cardiac stress, Nppa and Nppb are strongly upregulated in the ventricular myocardium. Much is known about the molecular and physiological ques inducing Nppa and Nppb expression; however, the transcriptional regulatory mechanisms of the Nppa-Nppb cluster in vivo has proven to be quite complex and is not well understood. In this review, we will provide recent insights into the dynamic and complex regulation of Nppa and Nppb during heart development and hypertrophy, and the association of this gene cluster with the cardiomyocyte-intrinsic program of heart regeneration.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Embryo, Mammalian; Epigenesis, Genetic; Fetus; Heart Ventricles; Humans; Mice; Multigene Family; Myocardium; Myocytes, Cardiac; Receptors, Atrial Natriuretic Factor; Regeneration; Signal Transduction; Zebrafish

Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates salt-water balance and blood pressure by promoting renal sodium and water excretion and stimulating vasodilation. ANP also has an anti-hypertrophic function in the heart, which is independent of its systemic blood pressure-lowering effect. In mice, ANP deficiency causes salt-sensitive hypertension and cardiac hypertrophy. Recent studies have shown that ANP plays an important role in regulating vascular remodeling and energy metabolism. Variants in the human NPPA gene, encoding the ANP precursor, are associated with hypertension, stroke, coronary artery disease, heart failure (HF) and obesity. ANP and related peptides are used as biomarkers for heart disease. Recombinant proteins and small molecules that enhance the ANP pathway have been developed to treat patients with HF. In this review, we discuss the role of ANP in cardiovascular biology and disease.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cardiovascular Diseases; Energy Metabolism; Humans; Mice; Vascular Remodeling

Structural remodelling occurring before, due to the underlying heart disease, and during atrial fibrillation (AF) sets the stage for permanent AF. Current therapy in AF aims to maintain sinus rhythm in symptomatic patients, but outcome is unfortunately poor. Stretch of the atria is a main contributor to atrial remodelling. In this review, we describe different aspects of structural remodelling as seen in animal models and in patients with AF, including atrial enlargement, cellular hypertrophy, dedifferentiation, fibrosis, apoptosis, and loss of contractile elements. In the second part, we describe downstream signals of mechanical stretch and their contribution to AF and structural remodelling. Ultimately, knowledge of mechanisms underlying structural remodelling may help to identify new pharmacological targets for AF prevention.

Topics: Animals; Apoptosis; Atrial Fibrillation; Atrial Natriuretic Factor; Cardiomegaly; Cell Differentiation; Fibrosis; Heart Atria; Humans; Oxidative Stress; Renin-Angiotensin System

Atrial natriuretic peptide (ANP) and brain (B-type) natriuretic peptide (BNP) are circulating hormones of cardiac origin that play an important role in the regulation of intravascular blood volume and vascular tone. The plasma concentrations of ANP and BNP are elevated in heart failure, and they are considered to compensate for heart failure because of their diuretic, natriuretic, and vasodilating actions and inhibitory effects on renin and aldosterone secretion. Evidence is also accumulating from recent work that ANP and BNP exert their cardioprotective functions not only as circulating hormones but also as local autocrine and/or paracrine factors. In studies using cultured neonatal myocytes and fibroblasts, exogenous administration of both ANP and ANP antagonists demonstrated that ANP has antihypertrophic and antifibrotic functions. Corroborating these in vitro results, mice lacking natriuretic receptor-A (NPR-A), the receptor for ANP and BNP, develop cardiac hypertrophy and fibrosis independent of their blood pressure. Recent studies also suggest that the intracardiac natriuretic peptides/cGMP system plays a counter-regulatory role against the intracardiac renin-angiotensin-aldosterone system and TGF-beta mediated pathway. In a clinical setting, human recombinant ANP and BNP may be used for a therapy of heart failure; however, further evaluation is required in the future.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart Failure; Humans; Mice; Mice, Knockout; Models, Animal; Myocardium; Natriuretic Peptide, Brain; Natriuretic Peptides; Receptors, Atrial Natriuretic Factor; Renin-Angiotensin System; Signal Transduction

The serine proteases of the trypsin superfamily are versatile enzymes involved in a variety of biological processes. In the cardiovascular system, the importance of these enzymes in blood coagulation, platelet activation, fibrinolysis, and thrombosis has been well established. Recent studies have shown that trypin-like serine proteases are also important in maintaining cardiac function and contribute to heart-related disease processes. In this review, we describe the biological function of corin, tissue kallikrein, chymase and urokinase and discuss their roles in cardiovascular diseases such as hypertension, cardiac hypertrophy, heart failure, and aneurysm.

Topics: Animals; Aortic Aneurysm, Abdominal; Atrial Natriuretic Factor; Cardiomegaly; Chymases; Heart; Humans; Hypertension; Hypertension, Pulmonary; Myocardial Ischemia; Serine Endopeptidases; Tissue Kallikreins; Urokinase-Type Plasminogen Activator

Topics: Animals; Atrial Natriuretic Factor; Blood Volume; Cardiomegaly; Catecholamines; Humans; Hypertension; Hyperthyroidism; Hypothyroidism; Myocardial Contraction; Receptors, Thyroid Hormone; Renin-Angiotensin System; Thyroxine; Triiodothyronine; Vascular Resistance; Vasopressins

It is well-recognized that atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are raised in conditions with ventricular volume and pressure overload. In addition to this established role in left ventricular congestive cardiac failure, there is good evidence that BNP has a diagnostic role in right ventricular (RV) dysfunction and pulmonary arterial hypertension (PAH). For example, BNP levels can be used to differentiate between dyspneic patients with pure respiratory defects and those with RV dysfunction. Studies in patients with PAH have demonstrated significant correlations between BNP levels and mean pulmonary arterial pressure as well as pulmonary vascular resistance. Additionally, BNP has a prognostic role in patients with RV pressure overload and pulmonary hypertension, and it offers a noninvasive test that can be used to guide therapy in patients with PAH. However, although measured plasma proBNP levels are raised in conditions with RV overload, its biological significance is still not well-understood. In this article, we review the general physiologic and potential therapeutic role of natriuretic peptides in respiratory disease, RV dysfunction, and PAH. Furthermore, we assess the various clues toward natriuretic peptide action coming from laboratory studies. ANP and BNP knockout mice develop cardiac fibrosis and hypertrophy. Potentiation of the natriuretic pathway has been shown to reduce cardiac hypertrophy and PAH. This is likely to take place as a result of increased intracellular cyclic guanosine monophosphate levels and subsequent pulmonary vasorelaxant activity. In view of this evidence, there may be a rationale for the therapeutic use of recombinant BNP or neutral endopeptidase inhibitors under conditions of RV dysfunction and PAH.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Fibrosis; Heart Ventricles; Humans; Hypertension, Pulmonary; Natriuretic Peptide, Brain; Ventricular Dysfunction, Right; Ventricular Pressure

Within the last five years, assay systems for measurement of plasma levels of brain natriuretic peptide (BNP) have been approved as a diagnostic aid for heart failure (HF). Similarly, nesiritide, a recombinant form of human BNP, has been approved for the treatment of acutely decompensated HF. Both BNP as a diagnostic test and a therapeutic modality are rapidly becoming integrated into clinical practice. The purpose of this review is to provide a brief overview of the physiology of the natriuretic peptides relevant to their informed clinical use. The current literature regarding the utility of measuring BNP for the diagnosis and management of HF is reviewed and practical recommendations regarding the interpretation of BNP levels are offered. The clinical literature regarding the use of recombinant BNP for the treatment of HF is reviewed, underscoring current gaps in our knowledge regarding the indications for and benefits of this novel agent.

Topics: Atrial Natriuretic Factor; Cardiomegaly; Diuresis; Heart Failure; Humans; Kidney; Myocardial Infarction; Natriuresis; Natriuretic Agents; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; ROC Curve; Survival Analysis; Treatment Outcome; Vasodilation; Ventricular Remodeling

1. If one was to design a hormone to protect the heart, it would have a number of features shown by the cardiac natriuretic peptides atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). These hormones are made in cardiomyocytes and are released into the circulation in response to atrial and ventricular stretch, respectively. Atrial natriuretic peptide and BNP can reduce the preload and after-load in normal and failing hearts. They reduce blood volume over the short term by sequestering plasma and over the longer term by promoting renal salt and water excretion and by antagonizing the renin-angiotensin-aldosterone system at many levels. Each of these actions affords indirect benefit to a volume- or pressure-threatened heart. 2. Recent studies have identified additional modes of action of the natriuretic peptides that may also confer cardioprotective benefits, especially in heart disease. The emerging findings are: (i) that ANP and BNP antagonize the cardiac hypertrophic action of angiotensin II and continue working under conditions where endothelial nitric oxide (NO) function is compromised, such as in the presence of high glucose in diabetes; (ii) they potentiate the bradycardia caused by inhibitory ('autoprotective') cardio-cardiac reflexes; and, furthermore, (iii) BNP can suppress cardiac sympathetic nerve activity in humans, including those with heart failure. Thus, it appears that natriuretic peptides can shift sympathovagal balance in a beneficial direction (away from the sympathetic). The vagal reflex and antihypertrophic actions of the peptides are mediated by particulate guanylyl cyclase (pGC) natriuretic peptide receptors. 3. The multiple synergistic actions of the natriuretic peptides make them and their pGC receptors attractive targets for therapy in heart disease. Encouragingly, exogenous natriuretic peptides remain effective even when endogenous peptide levels are raised, as is the case in heart failure. They also remain effective in disease states where other protective mechanisms, such as the NO system, have become ineffective, offering yet further encouragement for the therapeutic use of the natriuretic peptides.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiovascular Diseases; Heart; Hormones; Humans; Natriuretic Peptide, Brain; Sympathetic Nervous System; Vagus Nerve

Available data suggest that hypertensive cardiopathy is principally determined by the phenoconversion that allows the myocyte to adapt to the new working conditions by re-expressing a fetal program. Nevertheless, in clinical conditions, the scheme is different. The above phenotype is modified by trophic factors, which originate from ischemia, senescence, diabetes, genetics, or neurohormonal reactions. This review only focuses on some of the most recent advances concerning the permanent changes in the myocyte. Changes in extracellular matrix have been excluded. Recently, emphasis has been on the kinetic basis of the myocardial dysfunction at the myosin level, the potential therapeutic utilization of transferring the adrenergic receptor gene, the participation of NO synthases in the adaptational process, the existence of an abnormal excitation-contraction coupling due to a redistribution of Ca2+ sparks, the role of the microtubule as a determinant of sarcomere motion, and the multifactorial origin of cell death by apoptosis.

Topics: Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Autocrine Communication; Cardiomegaly; Cell Death; Cytoskeleton; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Humans; Hypertension; Models, Cardiovascular; Muscle Proteins; Myocardial Contraction; Myocardium; Nitric Oxide; Phenotype; Rats; Receptors, Adrenergic, beta

Heart failure is a pathophysiological state resulting from disturbed cardiac function. It is based on complex molecular processes, many of which are not fully understood. During heart failure adaptive mechanisms, that reinstall altered cardiac function, are activated. The main mechanisms are: a) Alteration of the structure and composition of myocytes by myocardial hypertrophy, reexpression of fetal and neo-natal proteins and the expression of certain proto-oncogenes; b) Activation of the neuroendocrinal system, specifically the sympathetic nervous system, renin-angiotensin-aldosterone system and vasopressin release; c) Activation of autocrine and paracrine systems. However, when these systems are activated beyond a certain limit they contribute to heart failure aggravation. This can also be promoted by alteration of the calcium metabolism inherent in heart failure. The synthesis of the counterregulator atrial natriuretic factor is also increased.

Topics: Atrial Natriuretic Factor; Calcium; Cardiomegaly; Heart Failure; Humans; Neurosecretory Systems; Protein Biosynthesis; Renin-Angiotensin System; Sympathetic Nervous System; Vasopressins

To study the signaling mechanisms which mediate ventricular hypertrophy, we utilized the induction of the ANF gene as a marker of the hypertrophic response. The induction of the atrial natriuretic factor gene (ANF) is one of the most conserved features of ventricular hypertrophy, occurring in multiple species (mouse, rat, hamster, canine, and human) in response to diverse stimuli (hormonal, mechanical, pressure/volume overload, genetic, IHSS, hypertension, etc.). The ANF gene is expressed in both the atrial and ventricular compartments during embryonic development, but shortly after birth ANF expression is down-regulated to negligible levels in the adult myocardium. Since the reactivation of ANF gene expression in the hypertrophied ventricle is a hallmark of the activation of an embryonic gene program, it has also become of interest to determine if similar mechanisms activate ANF expression during hypertrophy and the initial stages of cardiogenesis. A combination of cotransfection, microinjection, and transgenic approaches has been coupled to well characterized cultured cell systems and in vivo murine models employing normal and transgenic mice. The microinjection of oncogenic RAS proteins into living myocardial cells does not lead to the activation of cell proliferation, but activates ANF gene expression, as assessed by immunofluorescence. Co-transfection of mutant and wild-type RAS expression vectors with a ANF-luciferase fusion gene supports a direct effect of activated RAS on ANF gene transcription. Co-transfection of a dominant negative RAS vector effectively inhibits the induction of the ANF gene during alpha adrenergic mediated hypertrophy of ventricular muscle cells, thereby establishing that a RAS-mediated pathway is required for ANF induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Embryo, Mammalian; Gene Expression Regulation; Heart Ventricles; Humans; Signal Transduction

Experimental myocardial infarction is a model of cardiac overload due to amputation of part of the cardiac muscle. The development of cardiac failure depends on the size of the infarct and the time factor. This model of overload is associated with changes of the phenotype of the remaining healthy muscle and with peripheral vascular modifications partially dependent of the activation of pressor and/or deactivation of dilator systems. These changes are proportional to the size of the infarction at a given time after induction of the model. The degree of right ventricular hypertrophy and the decrease in blood pressure reflect the severity of infarction and the deterioration of the remaining myocardial function, affecting the haemodynamics both before and after the left ventricle. The increases in the 1/3 forms of isomyosins, the amount of subendocardial collagen, the biosynthesis, stocking and secretion of ANF are related to the infarct size and degree of overload. Similarly, the concentration of cyclic GMP is proportional to the infarct size. These parameters reflect ventricular overload, the increase of stress and energy deprivation of the remaining healthy muscle. The activation of peripheral pressor systems is also dependent on the infarct size reflects the effect of cardiac pump dysfunction on the kidney, liver, brain and endothelium. Large infarcts are associated with increased circulating renin and renal concentrations, with a decrease in angiotensinogen levels related to its consumption by the renin and to reduced hepatic synthesis and also with increased secretion and biosynthesis of vasopressin by the hypothalamus. In this model, Perindopril is beneficial by decreasing the cardiac load. It reduces the blood pressure, causes regression of bi-auricular and right ventricular hypertrophy. Changes in myosin isoenzyme configuration regress and subendocardial fibrosis and ANF concentrations are normalised. The effects of ACE inhibitors in this context, though very beneficial, are limited by the impossibility of normalising cardiac load and stress when the initial amputation of cardiac contractile mass exceeds 40%.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Indoles; Models, Biological; Myocardial Infarction; Myosins; Perindopril; Rats; Rats, Inbred Strains; Renin-Angiotensin System

The geometry of both the infarcted and non-infarcted zone of the left ventricle changes after myocardial infarction. Two mechanisms are involved: expansion of the infarcted zone and secondary dilatation of the non-infarcted zone. The necrosed area undergoes an inflammatory reaction followed by fibrosis which end up as a sca within a period of a few days to a few weeks. During this period if fibrous scarring the infarcted, thinned myocardium undergoes progressive expansion which starts in the first hours of the myocardial infarction. The loss of left ventricular systolic function related to the infarct and volumic overload created by expansion of the infarct influence the secondary development of dilatation of the non-infarcted zones. This dilatation results in restoration of left ventricular stroke volume but at the price of increased wall stress, which itself induces compensatory wall hypertrophy. These phenomena are more pronounced when the initial infarction is extensive and if they are sustained, they result in definitive myocardial failure. Several factors influence remodeling: the size of the infarct, arterial patency, wall stress and the quality of the scarring process itself. Therapeutic interventions of each of these factors can influence the remodeling. Limitation of infarct size by thrombolytic therapy, arterial revascularisation, even when performed late, seem capable of limiting expansion of the necrosed zone. Pharmacodynamic intervention of left ventricular afterload also affects ventricular remodeling. Nitrate derivatives, vasodilator therapy in general and converting enzyme inhibitors have been shown to be effective.

Topics: Angiotensin-Converting Enzyme Inhibitors; Atrial Natriuretic Factor; Cardiomegaly; Heart Ventricles; Humans; Myocardial Infarction; Prognosis; Renin-Angiotensin System; Stroke Volume; Time Factors; Ventricular Function, Left

Although ANF research started 30 years ago, the atrial natriuretic factor (ANF) was only discovered recently (1981). The presence of such a factor has been suspected for many years because of histological and physiological arguments. In 1956, Kish found "dense granules" in the atrial walls of guinea pigs. Gauer and Henry could explain some of their experimental results on diuresis and natriuresis only by suggesting the presence of a third hormonal factor, but neither by the renin-angiotensin system, nor the anti-diuretic hormone. Hall et al. were the first to recognize a link between the granules and water and sodium metabolism. But it was De Bold who published the crucial experiment in 1981: injecting right atrial extracts to anaesthetized rats rapidly induced intense and transitory diuresis and natriuresis. ANF was born, and, at the same time, the concept of the heart as an endocrine gland. Indeed, ANF corresponds to the strict definition of a hormone. It has the following properties: natriuresis and diuresis via an increase in glomerular filtration fraction without any major changes in renal plasma flow; direct vasodilation of the large arteries with only few effects on small arterioles and veins. The stimuli for ANF secretion are mechanical and pharmacological, especially drugs currently used by anaesthetists. Atrial distension is the main mechanical stimulus. An increase in atrial transmural pressure is always followed by a release in ANF, but this effect is not constant for increases in intra-luminal pressure. It is the former pressure gradient alone that reflects the volume of the right atrium, the mechanical stimulus for ANF secretion. Tachycardia, or, more precisely, an increase in the atrial contraction rate, also leads to an important release of ANF. Cardiac nerves are not necessary for this, as demonstrated by studies in heart transplant patients. Only few pharmacological agents have been shown to really stimulate ANF secretion. In rats, morphine has a direct secretory effect, whereas ketamine hydrochloride, diethylether and chloral hydrate do so by increasing the release of catecholamines. The effects of alpha, beta adrenergic agonists and calcium agonists remain controversial. ANF, which has diuretic and vasodilator effects, plays a part, together with the renin-angiotensin system and the anti-diuretic hormone, in blood volume control in mammals. However, it has a special role to play, because it is a rapid release hormone: rapid vascular fi

Topics: Anesthetics; Animals; Atrial Natriuretic Factor; Calcium Channel Agonists; Cardiomegaly; Diuresis; Fentanyl; Glomerular Filtration Rate; Humans; Rats; Renin-Angiotensin System; Sympathetic Nervous System; Sympathomimetics; Tachycardia; Vasodilation; Vasopressins

Topics: Actinin; Actins; Animals; Atrial Natriuretic Factor; Calcium-Transporting ATPases; Cardiomegaly; Heart Failure; Humans; Myocardium; Myosins; Phenotype; Rabbits; Rats; Sarcoplasmic Reticulum

Topics: Animals; Arteriosclerosis; Atrial Natriuretic Factor; Biological Products; Blood Platelets; Cardiology; Cardiomegaly; Fibrinolytic Agents; Forecasting; Free Radicals; Humans; Myocardial Infarction; Nitric Oxide; Receptors, Cell Surface; Receptors, Lipoprotein

Much has been learned over the past 25 years concerning the role of the heart in hypertension. In a multiplicity of areas a great deal has been clarified but a number of issues remain unresolved. This personal overview outlines some of these challenging areas for investigation, including questions relating to the cardiogenic reflexes, mechanisms underlying total body autoregulation that may involve not only the adaptation of arterioles but also venoconstriction in hypertension, postcapillary constriction also involving the efferent glomerular arterioles, the mechanisms underlying the development and regression of hypertrophy as well as the function of the hypertrophied and "regressed hypertrophy" heart, and the precise hemodynamic actions of atrial natriuretic factor.

Topics: Atrial Natriuretic Factor; Cardiomegaly; Heart; Hemodynamics; Humans; Hypertension; Vascular Resistance

Topics: Adrenal Cortex; Animals; Atrial Natriuretic Factor; Blood Proteins; Cardiomegaly; Cardiotonic Agents; Chickens; Digoxin; Dogs; Guinea Pigs; Heart Atria; Humans; Hydroxysteroids; Hypertension; Hypothalamus; Kidney; Muscle Proteins; Natriuretic Agents; Proteins; Rabbits; Rats; Sheep; Sodium; Sodium-Potassium-Exchanging ATPase; Vasodilation

The effects of long-term sodium restriction on plasma atrial natriuretic factor (ANF) concentrations, and the role of baseline plasma ANF concentration as an indicator of changes in haemodynamics and left ventricular hypertrophy during this treatment were studied in 40 middle-aged previously untreated mildly to moderately hypertensive men and women in a 6-month controlled randomized study. The main emphasis of the treatment programme was to reduce daily sodium intake to less than 70 mmol. Mean sodium excretion decreased from 148 +/- 74 mmol 24 h-1 to 79 +/- 71 mmol 24 h-1 in the treatment group, but remained unchanged in the control group (173 +/- 68 mmol 24 h-1 vs. 186 +/- 62 mmol 24 h-1; P less than 0.01 for the difference in changes between the groups). Mean plasma ANF concentrations in the treatment group were 52.4 +/- 20.7 (median 50) pg ml-1 at baseline and 38.7 +/- 26.3 (median 42) pg ml-1 at 6 months, and the corresponding values in the control group were 55.5 +/- 20.5 (median 50) pg ml-1 and 46.1 +/- 32.4 (median 50) pg ml-1, respectively (P = NS for the difference in changes). The ANF concentration decreased from 70 +/- 14 pg ml-1 to 32 +/- 26 pg ml-1 in treated subjects with a high baseline plasma ANF concentration (greater than 50 pg ml-1), but increased from 37 +/- 11 pg ml-1 to 45 +/- 27 pg ml-1 in subjects with a low baseline plasma ANF concentration (less than or equal to 50 pg ml-1) (difference in changes P less than 0.001). Compared with treated subjects with low baseline plasma ANF levels and with controls, treated subjects with high baseline plasma ANF levels showed a decrease (P less than 0.05) in interventricular septal and left posterior wall thicknesses, in relative wall thickness, and in peripheral resistance. These results suggest that in mildly to moderately hypertensive subjects long-term sodium restriction decreases high plasma ANF concentrations concomitantly with regression of concentric left ventricular hypertrophy, probably as a result of changes in haemodynamics.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Diet, Sodium-Restricted; Female; Hemodynamics; Humans; Hypertension; Male; Middle Aged; Time Factors; Vascular Resistance

Sixteen patients with severe hypertension were treated for 1 year with extended release nifedipine, during which time serial changes in left ventricular mass index and associated alterations in left ventricular systolic function, left ventricular filling, plasma renin activity, atrial natriuretic peptide and catecholamines were evaluated. Mean seated blood pressure (+/- SE) was significantly reduced from 200 +/- 8/122 +/- 3 to 144 +/- 5/89 +/- 2 mm Hg (p less than 0.0001) at 1 year. After 6 months, left ventricular mass index was significantly reduced by 19% from 121 +/- 8 to 96 +/- 7 g/m2 and this reduction was sustained at 1 year. Septal and posterior wall thickness were reduced from 13.4 +/- 0.1 to 11.2 +/- 0.04 mm and from 12.8 +/- 0.1 to 10.0 +/- 0.03 mm (p less than 0.001), respectively. The prevalence of left ventricular hypertrophy decreased from 63% to 25%. Left ventricular fractional shortening increased from 34 +/- 2% to 41 +/- 3% (p less than 0.05) and the relation between fractional shortening and end-systolic stress did not change. Over the year of sustained blood pressure reduction, the peak velocity of early filling increased from 57 +/- 3 to 63 +/- 4 cm/s (p = 0.07), peak velocity of late filling did not change and the ratio of late to early peak left ventricular filling velocity significantly decreased (p less than 0.05). Plasma atrial natriuretic peptide levels, markedly elevated at entry, decreased from 70 +/- 15 to 41 +/- 8 pg/ml at 1 year (p less than 0.05). Plasma renin activity and catecholamine levels were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Atrial Natriuretic Factor; Cardiomegaly; Chlorthalidone; Delayed-Action Preparations; Echocardiography; Epinephrine; Female; Humans; Hypertension; Male; Middle Aged; Nifedipine; Norepinephrine; Renin; Time Factors; Ventricular Function, Left

The involvement of cardiopulmonary and arterial sinoaortic receptors in the control of antidiuretic hormone (ADH) and atrial natriuretic peptide (ANP) release is still controversial in humans. Moreover, it is not clear if this control may be impaired in hypertensive patients with left ventricular hypertrophy (LVH). We studied 17 male subjects, age 18-58 (6 normotensives and 9 mild hypertensives, 5 without and 4 with LVH). Each subject underwent selective loading and unloading of cardiopulmonary receptors, in a randomized sequence, by application of a positive (LBPP) or negative (LENP) pressure to the lower body (steps: +10, +20, +40, -10, -40 mmHg, each for about 30 min), through a plexyglass-constructed tubular apparatus with a rubber adhesion round the patients' waist. Blood samples were taken at the end of every step for measurement of ADH, ANP, PRA, immunoreactive renin, aldosterone, noradrenaline and adrenaline. Cuff arterial pressure was measured every 5 min, while heart rate was evaluated by continuous ECG recording. Hypertensive subjects underwent right atrial pressure measurement by an iv catheter and forearm blood flow evaluation at rest and during the different steps (venous occlusion plethysmography). During LBNP, ADH plasma levels increased progressively, but the increase became statistically significant only at the step of -40 mmHg. ANP increased significantly during LBPP. Taking into account only hypertensive patients, a consistent reduction in the changes of ADH and ANP plasma levels, respectively during LBNP and LBPP in patients with LVH in respect to those without LVH was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Electrocardiography; Heart; Heart Ventricles; Humans; Lung; Male; Middle Aged; Pressoreceptors; Pressure; Reflex; Vasopressins

Excessive activation of the sympathetic nervous system is involved in cardiovascular damage including cardiac hypertrophy. Natriuretic peptides are assumed to exert protective actions for the heart, alleviating hypertrophy and/or fibrosis of the myocardium. In contrast to this assumption, we show in the present study that both atrial and C-type natriuretic peptides (ANP and CNP) potentiate cardiac hypertrophic response to noradrenaline (NA) in rats. Nine-week-old male Wistar rats were continuously infused with subcutaneous 30 micro-g/h NA without or with persistent intravenous administration of either 1.0 micro-g/h ANP or CNP for 14 days. Blood pressure (BP) was recorded under an unrestrained condition by a radiotelemetry system. Cardiac hypertrophic response to NA was evaluated by heart weight/body weight (HW/BW) ratio and microscopic measurement of myocyte size of the left ventricle. Mean BP levels at the light and dark cycles rose by about 20 mmHg following NA infusion for 14 days, with slight increases in HW/BW ratio and ventricular myocyte size. Infusions of ANP and CNP had no significant effects on mean BP in NA-infused rats, while two natriuretic peptides potentiated cardiac hypertrophic response to NA. Cardiac hypertrophy induced by co-administration of NA and ANP was attenuated by treatment with prazosin or atenolol. In summary, both ANP and CNP potentiated cardiac hypertrophic effect of continuously infused NA in rats, suggesting a possible pro-hypertrophic action of natriuretic peptides on the heart.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Male; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Norepinephrine; Rats; Rats, Wistar

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclooctanes; Echocardiography; Heart Failure; Heart Ventricles; Isoproterenol; Lignans; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Signal Transduction; Ventricular Function, Left

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Mice; Mice, Knockout; MicroRNAs; RNA, Long Noncoding; Smad4 Protein; Stroke Volume; Ventricular Function, Left

Ginkgolides are terpenoids peculiar to Ginkgo biloba, which have protective properties against cardiac diseases. This study aims to explore whether ginkgolide A (GA) could improve cardiac dysfunction of MI mice, and whether it could alleviate cardiac remodeling via binding to matrix metalloproteinase-9 (MMP9) to attenuate inflammation. Cardiac remodeling in mice induced by left coronary artery ligation were used in the in vivo model, and angiotensin (Ang) II-induced cardiac fibroblasts (NRCFs) and cardiomyocytes (NRCMs) isolated from neonatal rats were used in in vitro fibrosis and hypertrophy models, respectively. Cardiac dysfunction and fibrosis in MI mice were alleviated by GA treatment. Upregulations of collagen I (Col I), collagen III (Col III) and fibronectin in NRCFs, and enhanced levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (β-MHC) in NRCMs were inhibited by GA treatment. A total of 100 potential targets were found in 5 databases (TCMSP, BATMAN-TCM, PharmMapper, ETCM and SWISS Target). According to Protein Data Bank database GA could form hydrogen bonds between LYS65, GLU157, ASN17, ARG109, ARG106 of MMP9 protein, a target of GA. The regulatory role of GA in downregulating Col I, Col III, fibronectin in NRCFs, and enhancing levels of ANP, BNP and β-MHC in NRCMs were reversed by MMP9 overexpression, so as the downregulation of IL-1β, IL-6 and TNF-α in Ang II-induced NRCFs and NRCMs. GA could alleviate cardiac dysfunction and remodeling via binding to MMP9 to attenuate inflammation. Therefore, GA is a potential drug for cardiac remodeling therapy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Fibronectins; Fibrosis; Ginkgolides; Heart Diseases; Inflammation; Lactones; Matrix Metalloproteinase 9; Mice; Myocardial Infarction; Myocytes, Cardiac; Rats; Ventricular Remodeling

Senescence-associated pathological cardiac hypertrophy (SA-PCH) is associated with upregulation of foetal genes, fibrosis, senescence-associated secretory phenotype (SASP), cardiac dysfunction and increased morbidity and mortality. Therefore, we conducted experiments to investigate whether GATA4 accumulation induces SA-PCH, and whether Bmi-1-RING1B promotes GATA4 ubiquitination and its selective autophagic degradation to prevent SA-PCH.. Bmi-1-deficient (Bmi-1. Bmi-1-RING1B maintained cardiac function and prevented SA-PCH by promoting selective autophagy for degrading GATA4.. AAV9-CMV-Bmi-1-RING1B could be used for translational gene therapy to ubiquitinate GATA4 and prevent GATA4-dependent SA-PCH. Also, the combined domains between Bmi-1-RING1B and GATA4 in aging cardiomyocytes could be therapeutic targets for identifying stapled peptides in clinical applications to promote the combination of Bmi-1-RING1B with GATA4 and the ubiquitination of GATA4 to prevent SA-PCH and heart failure. We found that degradation of cardiac GATA4 by Bmi-1 was mainly dependent on autophagy rather than proteasome, and autophagy agonists metformin and rapamycin could ameliorate the SA-PCH, suggesting that activation of autophagy with metformin or rapamycin could also be a promising method to prevent SA-PCH.

Topics: Animals; Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Cytomegalovirus Infections; GATA4 Transcription Factor; Metformin; Mice; Myocytes, Cardiac; Polycomb Repressive Complex 1; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Sirolimus; Ubiquitin-Protein Ligases

Midkine levels are related to various diseases, including cardiovascular disease, renal disease and autoimmune disease. The research aimed to investigate the mitigation influence of downregulation of intermediate factors on myocardial hypertrophy induced by angiotensin Ⅱ (Ang), and whether downregulation of midkine could attenuate oxidative stress and autophagy. Induced myocardial hypertrophy of the mice model and treated HL-1 cells with Ang Ⅱ in vitro. The expressions of midkine were increased in the model and HL-1 cells with Ang II treatment. Midkine silence alleviated cardiac hypertrophy induced by Ang II, and inhibited the increases of atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP) and beta-myosin heavy chain (β-MHC) in the heart of mice. The raises of ANP, BNP and β-MHC in Ang II-induced HL-1 cells were also suppressed after midkine downregulation. Downregulating of midkine inhibited the increases of oxidative stress markers 8-OHdG, superoxide anions and MDA in the heart of mice or in the Ang II-treated HL-1 cells. The raises of LC3B, Atg3, Atg5 and Beclin1 in mice heart and in the Ang II.-induced HL-1 cells were attenuated after midkine silence. These outcomes showed that midkine was upregulated in myocardial hypertrophy mice. Targeting of midkine could alleviate cardiac hypertrophy via attenuation of oxidative stress and autophagy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Mice; Midkine; Myocytes, Cardiac; Natriuretic Peptide, Brain; Oxidative Stress

Although the association of elevated homocysteine level with cardiac hypertrophy has been reported, the molecular mechanisms by which homocysteine induces cardiac hypertrophy remain inadequately understood. In this study we aim to uncover the roles of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress and their relationship to advance the mechanistic understanding of homocysteine-induced cardiac cell hypertrophy. H9c2 cells and primary neonatal rat cardiomyocytes are exposed to homocysteine with or without ER stress inhibitor TUDCA or PDE1-specific inhibitor Lu AF58027, or transfected with siRNAs targeting PDE1 isoforms prior to homocysteine-exposure. Cell surface area is measured and ultrastructure is examined by transmission electron microscopy. Hypertrophic markers, PDE1 isoforms, and ER stress molecules are detected by q-PCR and western blot analysis. Intracellular cGMP and cAMP are measured by ELISA. The results show that homocysteine causes the enlargement of H9c2 cells, increases the expressions of hypertrophic markers β-MHC and ANP, upregulates PDE1A and PDE1C, promotes the expressions of ER stress molecules, and causes ER dilatation and degranulation. TUDCA and Lu AF58027 downregulate β-MHC and ANP, and alleviate cell enlargement. TUDCA decreases PDE1A and PDE1C levels. Silencing of PDE1C inhibits homocysteine-induced hypertrophy, whereas PDE1A knockdown has minor effect. Both cAMP and cGMP are decreased after homocysteine-exposure, while only cAMP is restored by Lu AF58027 and TUDCA. TUDCA and Lu AF58027 also inhibit cell enlargement, downregulate ANP, β-MHC and PDE1C, and enhance cAMP level in homocysteine-exposed primary cardiomyocytes. ER stress mediates homocysteine-induced hypertrophy of cardiac cells via upregulating PDE1C expression Cyclic nucleotide, especially cAMP, is the downstream mediator of the ER stress-PDE1C signaling axis in homocysteine-induced cell hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Endoplasmic Reticulum Stress; Enzyme Activation; Homocysteine; Myocytes, Cardiac; Phosphoric Diester Hydrolases; Rats; Taurochenodeoxycholic Acid

Cardiac hypertrophy is an adaptive and compensatory mechanism preserving cardiac output during detrimental stimuli. Circular RNAs (circRNAs) have been illustrated to exert important implications in the pathogenesis of multiple cardiovascular diseases (CVD) including demonstrated cardiac hypertrophy. Toll-like receptor 4 (TLR4) has been previously reported to be a crucial regulator in inflammatory response and cardiac hypertrophy. However, the role of circular isoforms derived from TLR4 in cardiac hypertrophy remains unclear.. Expression of circ-TLR4 and TLR4 in cardiomyocytes was detected by RT-qPCR. The indicators of cardiac hypertrophy responses, including cell surface area, atrial natriuretic factor (ANF), B-type natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were measured by immunofluorescence staining and western blot. RIP assay was used to validate the interaction between circ-TLR4 and TLR4.. Circ-TLR4 and TLR4 was up-regulated in cellular models of cardiac hypertrophy. Circ-TLR4 knockdown attenuated angiotensin II (Ang II)-induced hypertrophy responses in cardiomyocytes. Moreover, circ-TLR4 positively regulated TLR4 expression through recruiting FUS to stabilize TLR4 mRNA. Furthermore, TLR4 overexpression rescued the cardiac responses mediated by circ-TLR4 silencing.. Circ-TLR4 promotes cardiac hypertrophy through recruiting FUS to stabilize TLR4 mRNA.

Topics: Angiotensin II; Atrial Natriuretic Factor; Cardiomegaly; Humans; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; RNA-Binding Protein FUS; RNA, Circular; RNA, Messenger; Toll-Like Receptor 4

Cardiac hypertrophy is a leading risk for heart failure and sudden death. Long non-coding RNAs (lncRNAs) have been implicated in a variety of human diseases, including cardiac hypertrophy. We aimed to investigate the potential role and functional mechanism of lncRNA metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1) in cardiac hypertrophy. C57BL/6 mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy in vivo. The expression of MALAT1, miR-93-5p, and sirtuin 4 (SIRT4) mRNA was detected using a quantitative real-time polymerase chain reaction. The protein levels of cardiac hypertrophy-related markers, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), and SIRT4 were measured via western blotting. The putative interaction between miR-93-5p and MALAT1 or SIRT4 was verified using a dual-luciferase reporter assay, RNA immunoprecipitation assay, or pull-down assay. Consequently, the expression of MALAT1 and SIRT4 was increased in TAC-treated mouse heart and angiotensin II (Ang-II)-induced cardiomyocytes, whereas the expression of miR-93-5p was decreased. Ang-II promoted the expression of ANP, BNP, and β-MHC and the surface area of cardiomyocytes, whereas MALAT1 downregulation impaired their expression and cell area. MiR-93-5p was a target of MALAT1, and its inhibition reversed the effects of MALAT1 downregulation. More importantly, MALAT1 modulated SIRT4 expression by degrading miR-93-5p. The expression of ANP, BNP, and β-MHC suppressed by miR-93-5p restoration was recovered by SIRT4 promotion. Overall, MALAT1 knockdown ameliorated cardiac hypertrophy partly by regulating the miR-93-5p/SIRT4 network, indicating that MALAT1 was a substantial indicator of cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Down-Regulation; Mice; Mice, Inbred C57BL; MicroRNAs; Mitochondrial Proteins; Myocytes, Cardiac; Neoplasms; RNA, Long Noncoding; Sirtuins

To investigate the protective effect of fucoxanthin (FX) against diabetic cardiomyopathy and explore the underlying mechanism.. Rat models of diabetes mellitus (DM) induced by intraperitoneal injection of streptozotocin (60 mg/kg) were randomized into DM model group, fucoxanthin treatment (DM+FX) group and metformin treatment (DM+ Met) group, and normal rats with normal feeding served as the control group. In the two treatment groups, fucoxanthin and metformin were administered after modeling by gavage at the daily dose of 200 mg/kg and 230 mg/kg, respectively for 12 weeks, and the rats in the DM model group were given saline only. HE staining was used to examine the area of cardiac myocyte hypertrophy in each group. The expression levels of fibrotic proteins TGF-β1 and FN proteins in rat hearts were detected with Western blotting. In the cell experiment, the effect of 1 μmol/L FX on H9C2 cell hypertrophy induced by exposure to high glucose (HG, 45 mmol/L) was evaluated using FITC-labeled phalloidin. The mRNA expression levels of the hypertrophic factors ANP, BNP and β-MHC in H9C2 cells were detected using qRT-PCR. The protein expressions of Nrf2, Keap1, HO-1 and SOD1 proteins in rat heart tissues and H9C2 cells were determined using Western blotting. The DCFH-DA probe was used to detect the intracellular production of reactive oxygen species (ROS).. In the diabetic rats, fucoxanthin treatment obviously alleviated cardiomyocyte hypertrophy and myocardial fibrosis, increased the protein expressions of Nrf2 and HO-1, and decreased the protein expressions of Keap1 in the heart tissue (. Fucoxanthin possesses strong inhibitory activities against diabetic cardiomyocyte hypertrophy and myocardial fibrosis and is capable of up-regulating Nrf2 signaling to promote the expression of its downstream antioxidant proteins SOD1 and HO-1 to reduce the level of ROS.

Topics: Animals; Antioxidants; Atrial Natriuretic Factor; Cardiomegaly; Diabetes Mellitus, Experimental; Fibrosis; Kelch-Like ECH-Associated Protein 1; Metformin; NF-E2-Related Factor 2; Oxidative Stress; Rats; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase-1; Xanthophylls

Cardiac hypertrophy (CH) is a pathological phenotype of cardiomyopathy. Epigenetic modification is a mechanism associated with CH. Our study here investigated the histone demethylase KDM3C in relation to epigenetic regulation in CH. We found that KDM3C mRNA silencing alleviated CH, as evidenced by reduced ANP, BNP, and β-MHC mRNAs, increased α-MHC mRNA, decreased cell surface area, and reduced cellular protein/DNA ratios. Specifically, KDM3C upregulated miR-200c-3p expression through demethylation of H3K9me2, leading to enhanced binding of miR-200c-3p to GAS5 and suppression of GAS5 expression; these effects then led to reduced binding of GAS5 to miR-495-3p, increased miR-495-3p expression, and repression of PHF8 transcription. Through these mechanisms, our data indicate that KDM3C-dependent epigenetic modification promotes CH.

Topics: Atrial Natriuretic Factor; Cardiomegaly; DNA; Epigenesis, Genetic; Histone Demethylases; Humans; MicroRNAs; RNA, Long Noncoding; RNA, Messenger; Transcription Factors

Recent studies have demonstrated that hyperglycemia is a major risk factor for the development and exacerbation of cardiovascular disease (CVD). However, the molecular mechanisms involved in diabetic cardiomyopathy (DCM) have not been fully elucidated. In this study, we focused on the underlying mechanism of DCM. Leptin receptor-deficient db/db mice were used to model a type 2 diabetes mellitus (T2DM) model in our study. WT mice and db/db mice received 4-phenylbutyric acid (4-PBA) (25 mg/kg/day) and saline by intraperitoneal injection every other day for 4 weeks. WT and db/db mice were given tail vein injections of 100 μL of rAAV9-Sh-MAPK10 and rAAV9-Sh-GFP at the age of 6-8 weeks. Echocardiography was performed to measure cardiac function, histological examinations were used to evaluate ventricular hypertrophy and fibrosis. Quantitative RT-qPCR was used to assess the mRNA expression of Jun N-terminal kinase 3 (JNK3, MAPK10), atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and collagen I and III. Immunoblotting was performed to measure the levels of cardiac hypertrophy-related proteins, fibrosis-related proteins, endoplasmic reticulum stress (ERS)-related proteins and apoptosis-related proteins. TUNEL staining was performed to examine cardiomyocyte apoptosis. In contrast to 12-week-old db/db mice, 16-week-old db/db mice showed the most severe myocardial dysfunction. The DCM induced by hyperglycemia was largely alleviated by 4-PBA (25 mg/kg/day, intraperitoneal injection). Similarly, tail vein injection of rAAV9-Sh-MAPK10 reversed the phenotype of the heart in db/db mice including cardiac hypertrophy and apoptosis in db/db mice. The mechanistic findings suggested that hyperglycemia initiated the ERS response through the negative regulation of sirtuin 1 (SIRT1), leading to the occurrence of myocardial dysfunction, and specific knockdown of MAPK10 in the heart directly reversed myocardial dysfunction induced by hyperglycemia. We demonstrated that hyperglycemia promotes DCM in db/db mice through the ERS-MAPK10 signaling pathway in diabetic mice.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathies; Collagen; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Endoplasmic Reticulum Stress; Fibrosis; Hyperglycemia; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinase 10; Natriuretic Peptide, Brain; Receptors, Leptin; RNA, Messenger; Signal Transduction; Sirtuin 1

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors that mediate the effects of cardiac diseases. GPR30, also named G-protein-coupled estrogen receptor, shows beneficial effect on female patients with heart failure. This research aimed to probe the role and mechanism of GPR30 in myocardial hypertrophy. The model of cardiac hypertrophy was induced by infusion of angiotensin (Ang) II in mice, and was induced by Ang II treatment in neonatal rat cardiomyocyte (NRCM). The mouse model of myocardial hypertrophy was induced by angiotensin (Ang) Ⅱ, and the neonatal rat cardiomyocyte (NRCM) was induced by Ang Ⅱ treatment. GPR30 agonist G1 reduced cardiac hypertrophy induced by Ang II in mice, and reduced cardiac atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) induced by Ang II. Ang Ⅱ treatment of myocardial fibrosis in mice was suppressed after administration of G1. GPR30 deficiency produced the opposite results. Oxidative stress and apoptosis were enhanced in the mice heart induced by Ang II, which were suppressed by G1 administration, but were further exacerbated after GPR30 deficiency. The outcomes demonstrated that GPR30 participated in the regulation of cardiac hypertrophy and fibrosis. Activation of GPR30 ameliorated cardiac hypertrophy and fibrosis by reducing oxidative stress and apoptosis.

Topics: Angiotensin II; Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Female; Fibrosis; GTP-Binding Proteins; Mice; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Oxidative Stress; Peptide Hormones; Rats; Receptors, Estrogen; Receptors, G-Protein-Coupled

A-kinase anchoring protein 5 (AKAP5) is involved in ventricular remodeling in rats with heart failure after myocardial infarction; however, the specific mechanism is not clear. This study investigated whether AKAP5 anchors calcineurin (CaN) to regulate the remodeling of H9c2 cardiomyocytes.. H9c2 cells were subjected to hypoxia stress for 3 h and reoxygenation for 24 h to create a hypoxia-reoxygenation (H/R) model. These cells were divided into three groups: H/R (model), empty vector +H/R (NC), and siRNA-AKAP5+H/R (siRNA-AKAP5) groups. The non-H/R H9c2 cells were used as normal controls. Western blotting was used to detect cardiac hypertrophy-related protein expression in the cells, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta myosin heavy chain (β-MHC), and phosphorylated nuclear factor of activated T-cell 3 (p-NFATc3). Phalloidin staining was used to label the cytoskeleton and the cell area in different groups was measured. Immunofluorescence staining and coimmunoprecipitation were used to study the relationship between AKAP5 and CaN. H9c2 cells pretreated with the CaN inhibitor FK506 were used to further verify the relationship between AKAP5 and CaN.. In the siRNA-AKAP5+H/R group, the expression level of cardiac hypertrophy-related proteins (ANP, BNP, and β-MHC) and CaN and the area of cardiomyocytes were significantly increased, while the p-NFATc3/NFATc3 ratio was decreased in H9c2H/R cells. AKAP5 and CaN proteins were colocalized and interacted in the cells. The CaN inhibitor significantly suppressed the expression of CaN, increased the p-NFATc3/NFATc3 ratio, and reduced the expression levels of ANP, BNP, and β-MHC proteins in the cells with low AKAP5 expression.. AKAP5 downregulation aggravated the remodeling of cardiomyocytes after H/R. AKAP5 may anchor CaN to form a complex, which in turn activates NFATc3 dephosphorylation and expression of hypertrophy-related proteins.

Topics: A Kinase Anchor Proteins; Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Hypoxia; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phalloidine; Rats; RNA, Small Interfering; Tacrolimus

Auraptene derived from the peel of Citrus hassaku possesses anti-tumor, anti-inflammatory, and neuroprotective activities. Thus, it could be a valuable pharmacological alternative to treat some diseases. However, the therapeutic value of auraptene for heart failure (HF) is unknown.. In cultured cardiomyocytes from neonatal rats, the effect of auraptene on phenylephrine-induced hypertrophic responses and peroxisome proliferator-activated receptor-alpha (PPARα)-dependent gene transcriptions. To investigate whether auraptene prevents the development of heart failure after myocardial infarction (MI) in vivo, Sprague-Dawley rats with moderate MI (fractional shortening < 40%) were randomly assigned for treatment with low- or high-dose auraptene (5 or 50 mg/kg/day, respectively) or vehicle for 6 weeks. The effects of auraptene were evaluated by echocardiography, histological analysis, and the measurement of mRNA levels of hypertrophy, fibrosis, and PPARα-associated genes.. In cultured cardiomyocytes, auraptene repressed phenylephrine-induced hypertrophic responses, such as increases in cell size and activities of atrial natriuretic factor and endothelin-1 promoters. Auraptene induced PPARα-dependent gene activation by enhancing cardiomyocyte peroxisome proliferator-responsive element reporter activity. The inhibition of PPARα abrogated the protective effect of auraptene on phenylephrine-induced hypertrophic responses. In rats with MI, auraptene significantly improved MI-induced systolic dysfunction and increased posterior wall thickness compared to the vehicle. Auraptene treatment also suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, and expression of hypertrophy and fibrosis response markers at the mRNA level compared with vehicle treatment. MI-induced decreases in the expression of PPARα-dependent genes were improved by auraptene treatment.. Auraptene has beneficial effects on MI-induced cardiac hypertrophy and left ventricular systolic dysfunction in rats, at least partly due to PPARα activation. Further clinical studies are required to evaluate the efficacy of auraptene in patients with HF.

Topics: Animals; Atrial Natriuretic Factor; Biological Products; Cardiomegaly; Citrus; Coumarins; Endothelin-1; Fibrosis; Heart Failure; Myocardial Infarction; Peroxisome Proliferators; Phenylephrine; PPAR alpha; Rats; Rats, Sprague-Dawley; RNA, Messenger

Atrial natriuretic peptide (ANP)-mediated natriuresis is known as a cardiac endocrine function in sodium and body fluid homeostasis. Corin is a protease essential for ANP activation. Here, we studied the role of renal corin in regulating salt excretion and blood pressure. We created corin conditional knockout (cKO), in which the

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Homeostasis; Hypertension; Kidney; Mice; Serine Endopeptidases; Sodium; Sodium Chloride; Sodium Chloride, Dietary

Modified citrus pectin (MCP) is a specific inhibitor of galectin-3 (Gal-3) that is regarded as a new biomarker of cardiac hypertrophy, but its effect is unclear. The aim of this study is to investigate the role and mechanism of MCP in isoproterenol (ISO)-induced cardiac hypertrophy. Rats were injected with ISO to induce cardiac hypertrophy and treated with MCP. Cardiac function was detected by ECG and echocardiography. Pathomorphological changes were evaluated by the haematoxylin eosin (H&E) and wheat germ agglutinin (WGA) staining. The hypertrophy-related genes for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC), and the associated signal molecules were analysed by qRT-PCR and western blotting. The results show that MCP prevented cardiac hypertrophy and ameliorated cardiac dysfunction and structural disorder. MCP also decreased the levels of ANP, BNP, and β-MHC and inhibited the expression of Gal-3 and Toll-like receptor 4 (TLR4). Additionally, MCP blocked the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), but it promoted the phosphorylation of p38. Thus, MCP prevented ISO-induced cardiac hypertrophy by activating p38 signalling and inhibiting the Gal-3/TLR4/JAK2/STAT3 pathway.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiovascular Agents; Disease Models, Animal; Galectin 3; Isoproterenol; Janus Kinase 2; Male; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; p38 Mitogen-Activated Protein Kinases; Pectins; Phosphorylation; Rats, Wistar; Signal Transduction; STAT3 Transcription Factor; Toll-Like Receptor 4; Ventricular Function, Left; Ventricular Remodeling

Isoprenaline-induced cardiac hypertrophy can deteriorate to heart failure, which is a leading cause of mortality. Endogenous vasonatrin peptide (VNP) has been reported to be cardioprotective against myocardial ischemia/reperfusion injury in diabetic rats. However, little is known about the effect of exogenous VNP on cardiac hypertrophy. We further explored whether VNP attenuated isoprenaline-induced cardiomyocyte hypertrophy by examining the levels and activities of cGMP and PKG. In this study, we found that VNP significantly attenuated isoprenaline-induced myocardial hypertrophy and cardiac fibroblast activation in vivo. Moreover, VNP effectively halted the activation of apoptosis and oxidative stress in the isoprenaline-treated myocardium. VNP promoted superoxide dismutase (SOD) activity. Further study revealed that the protective effects of VNP might be mediated by the activity of the cGMP-PKG signaling pathway in vivo or in vitro, while the use of agonists and antagonists confirmed these results. Therefore, we demonstrated that the antiapoptosis and antioxidative stress effects of VNP depends on elevated cGMP-PKG signaling activity both in vivo and in vitro. These results suggest that VNP may be used in the treatment of myocardial hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cyclic GMP; Humans; Isoproterenol; Mice; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Oxidative Stress; Primary Cell Culture; Rats; Signal Transduction; Superoxide Dismutase

Si-Miao-Yong-An decoction (SMYAD), a classical traditional Chinese medicine (TCM) formula, has been used to treat various cardiovascular diseases in clinics.. The aim of this study is to investigate the effective combinatorial components from SMYAD and its mechanism regarding the intervention on myocardial hypertrophy.. SMYAD constituents absorbed in rat plasma and heart were identified using UHPLC Q-Exactive-Orbitrap MS/MS. The identified constituents in SMYAD were further analyzed using ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. The effective constituents were identified using isoproterenol (ISO)-induced H9c2 cardiomyocyte hypertrophy, and neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid C (ICAC), angoroside C (AGDC), isochlorogenic acid A (ICAA), sweroside (SRD), and harpagide (HPD) in SMYAD extract were quantified by HPLC for compatibility. Finally, anti-hypertrophic activities of candidate effective combinatorial components, which were prepared according to the determined molar concentration ratio of effective constituents using reference substance solution, were analyzed using immunofluorescence staining and Quantitative real-time PCR. The expression levels of PI3Kα, p-ERK, p-Akt, Akt, p-mTOR, mTOR and HIF-1α were measured using Western blot.. 32 prototypes of SMYAD were identified from plasma and heart tissue of rat. Combining with ADMET prediction, 31 dominant constituents were focused. Based on HIF-1 pathway identified in preliminary result, 17 targets were focused, which were used to dock with 31 constituents. 27 constituents were therefore hit as the potential effective constituents of SMYAD in inhibiting myocardial hypertrophy. Bioactivity evaluation showed that NCA, CA, CCA, ICAC, AGDC, ICAA, SRD, and HPD significantly inhibited the increase of H9c2 cell surface area induced by ISO. Except for ICAA and AGDC, the remaining 6 effective constituents, showing a certain inhibitory effect on ISO-induced ANP mRNA overexpression at high and low concentrations, participated in compatibility based on the molar concentration ratio determined by HPLC. Effective combinatorial components composed of the 6 effective constituents (effective combinatorial components ABC) showed significant inhibitory effect on the increase of cell surface area, and the overexpression of ANP and β-MHC mRNA in H9c2 cells induced by ISO. Moreover, effective combinatorial components ABC significantly inhibited the protein overexpressions of p-Akt, p-mTOR and HIF-1α. Based on the results, we put forward the strategy of "Focusing constituents" and "Focusing targets" for the effective constituents research of TCM formula.. Effective combinatorial components ABC composed of NCA, CA, CCA, ICAC, SRD and HPD from SMYAD inhibited ISO-induced cardiomyocyte hypertrophy and down-regulated expression of ANP and β-MHC mRNA through the inactivation of Akt/mTOR/HIF-1α pathway.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Hypoxia-Inducible Factor 1, alpha Subunit; Isoproterenol; Male; Medicine, Chinese Traditional; Molecular Docking Simulation; Myocytes, Cardiac; Myosin Heavy Chains; Phosphatidylinositol 3-Kinase; Phytochemicals; Plasma; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; TOR Serine-Threonine Kinases

Exercise can induce physiological myocardial hypertrophy (PMH), and former athletes can live 5 to 6 years longer than nonathletic controls, suggesting a benefit after regression of PMH. We previously reported that regression of pathological myocardial hypertrophy has antihypertrophic effects. Accordingly, we hypothesized that antihypertrophic memory exists even after PMH has regressed, increasing myocardial resistance to subsequent pathological hypertrophic stress.. C57BL/6 mice were submitted to 21 days of swimming training to develop PMH. After termination of exercise, PMH regressed within 1 week. PMH regression mice (exercise hypertrophic preconditioning [EHP] group) and sedentary mice (control group) then underwent transverse aortic constriction or a sham operation for 4 weeks. Cardiac remodeling and function were evaluated with echocardiography, invasive left ventricular hemodynamic measurement, and histological analysis. LncRNA sequencing, chromatin immunoprecipitation assay, and comprehensive identification of RNA-binding proteins by mass spectrometry and Western blot were used to investigate the role of

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Echocardiography; Glycogen Synthase Kinase 3 beta; Hemodynamics; Histone Deacetylase 2; Mice; Mice, Inbred C57BL; Physical Conditioning, Animal; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Up-Regulation; Ventricular Function, Left; Ventricular Remodeling

Mitochondrial dysfunction has been suggested to be the key factor in the development and progression of cardiac hypertrophy. The onset of mitochondrial dysfunction and the mechanisms underlying the development of cardiac hypertrophy (CH) are incompletely understood. The present study is based on the use of multiple bioinformatics analyses for the organization and analysis of scRNA-seq and microarray datasets from a transverse aortic constriction (TAC) model to examine the potential role of mitochondrial dysfunction in the pathophysiology of CH. The results showed that NADH:ubiquinone oxidoreductase core subunit S1- (Ndufs1-) dependent mitochondrial dysfunction plays a key role in pressure overload-induced CH. Furthermore,

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Constriction, Pathologic; Down-Regulation; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; NADH Dehydrogenase; Natriuretic Peptide, Brain; Pressure; Rats; RNA-Seq; Single-Cell Analysis

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3'UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3'UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression Regulation; MAP Kinase Kinase 4; MicroRNAs; Myoblasts, Cardiac; NFATC Transcription Factors; Paxillin; Proto-Oncogene Proteins c-jun; rac1 GTP-Binding Protein; Rats; Sirtuin 1; Vasoconstrictor Agents

Ginkgolide B (GB) is an active ingredient extracted from Ginkgo biloba leaves. However, the effects of GB on cardiac hypertrophy remain unclear. The study is aimed at determining whether GB could alleviate cardiac hypertrophy and exploring its underlying molecular mechanism. Rat cardiomyocyte cell line H9c2 cells were pretreated with GB and incubated with angiotensin II (Ang II) to simulate an in vitro cardiac hypertrophy model. Cell viability, cell size, hypertrophy markers, and autophagy were determined in H9c2 cells after Ang II treatment. Proteins involved in autophagy and the SIRT1 pathway were determined by western blot. Our data demonstrated that GB attenuated Ang II-induced cardiac hypertrophy and reduced the mRNA expressions of hypertrophy marker, atrial natriuretic peptide (ANP), and

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Cell Line; Ginkgolides; Lactones; Myocytes, Cardiac; Nerve Tissue Proteins; Protective Agents; Rats; Signal Transduction; Sirtuin 1; Transcription, Genetic; Ventricular Myosins

Arbutin is a glycoside reported for its anti-oxidant, anti-inflammatory and anti-tumor properties. However, the cardioprotective effect of Arbutin is not well established. The study aims to understand the effect of arbutin on isoproterenol (ISO)-induced cardiac hypertrophy in mice. The animals were pretreated with Arbutin for a week and ISO was administered for 10 days and then sacrificed. Cardiac injury markers such as creatinine kinase and lactate dehydrogenase concentrations were measured in the serum. The mRNA expression of cardiac hypertrophy markers namely atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured using qRT-PCR. The levels of pro-inflammatory cytokines TNF-α and IL-6 were quantified by ELISA in isolated tissues and serum. Other tissue anti-oxidant parameters such as GST, GSH, SOD and TBARS were also measured. TUNEL assay was performed to detect apoptosis. Histology studies were performed using H & E and Masson trichome staining. Immunoblot analysis was used to quantify the protein expression of TLR-4 and NF-κB. ISO-alone-treated group showed significant increase in CK-MB, LDH along with increase in hypertrophic markers ANP and BNP, TNF-α and IL-6 levels in serum and tissues and increased cardiomyocyte apoptosis. Anti-oxidant parameters were significantly decreased and TLR-4 and NF-κB protein expression was found to be upregulated in comparison to the control group. Pretreatment with Arbutin-exhibited significant inhibition of TLR-4/NF-κB pathway with decreased levels of pro-inflammatory cytokines and enhanced myocardial anti-oxidant status. Our study demonstrated that pretreatment with Arbutin exhibits marked protective effects on ISO-induced cardiac hypertrophy in mice. Thus, Arbutin may be used as potential pharmacological interventions in the management of cardiac hypertrophy.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Arbutin; Atrial Natriuretic Factor; Cardiomegaly; Cardiotoxicity; Disease Models, Animal; Interleukin-6; Isoproterenol; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B; Oxidative Stress; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Ventricular Remodeling

The maternal circulatory system and hormone balance both change dynamically during pregnancy, delivery, and the postpartum period. Although atrial natriuretic peptides and brain natriuretic peptides produced in the heart control circulatory homeostasis through their common receptor, NPR1, the physiologic and pathophysiologic roles of endogenous atrial natriuretic peptide/brain natriuretic peptide in the perinatal period are not fully understood.. To clarify the physiologic and pathophysiologic roles of the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system during the perinatal period, the phenotype of female wild-type and conventional or tissue-specific Npr1-knockout mice during the perinatal period was examined, especially focusing on maternal heart weight, blood pressure, and cardiac function.. In wild-type mice, lactation but not pregnancy induced reversible cardiac hypertrophy accompanied by increases in fetal cardiac gene mRNAs and ERK1/2 (extracellular signaling-regulated kinase) phosphorylation. Npr1-knockout mice exhibited significantly higher plasma aldosterone level than did wild-type mice, severe cardiac hypertrophy accompanied by fibrosis, and left ventricular dysfunction in the lactation period. Npr1-knockout mice showed a high mortality rate over consecutive pregnancy-lactation cycles. In the hearts of Npr1-knockout mice during or after the lactation period, an increase in interleukin-6 mRNA expression, phosphorylation of signal transducer and activator of transcription 3, and activation of the calcineurin-nuclear factor of the activated T cells pathway were observed. Pharmacologic inhibition of the mineralocorticoid receptor or neuron-specific deletion of the mineralocorticoid receptor gene significantly ameliorated cardiac hypertrophy in lactating Npr1-knockout mice. Anti-interleukin-6 receptor antibody administration tended to reduce cardiac hypertrophy in lactating Npr1-knockout mice.. These results suggest that the characteristics of lactation-induced cardiac hypertrophy in wild-type mice are different from exercise-induced cardiac hypertrophy, and that the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system plays an important role in protecting the maternal heart from interleukin-6-induced inflammation and remodeling in the lactation period, a condition mimicking peripartum cardiomyopathy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; Lactation; MAP Kinase Signaling System; Mice; Mice, Knockout; Peripartum Period; Receptors, Atrial Natriuretic Factor

Circular RNA (circRNA) is a novel class of noncoding RNAs, and the roles of circRNAs in the development of cardiac hypertrophy remain to be explored. Here, we investigate the potential roles of circRNAs in cardiac hypertrophy. By circRNA sequencing in left ventricular specimens collected from 8-week-old mice with isoproterenol hydrochloride-induced cardiac hypertrophy, we found 401 out of 3323 total circRNAs were dysregulated in the hypertrophic hearts compared with the controls. Of these, 303 circRNAs were upregulated and 98 were downregulated. Moreover, the GO and KEGG analyses revealed that the majority of parental gene of differentially expressed circRNAs were not only related to biological process such as metabolic process and response to stimulus, but also related to pathway such as circulatory system and cardiovascular diseases. On the other hand, total 1974 miRNAs were predicted to binding to these differentially expressed circRNAs, and the possible target mRNAs of those miRNAs were also predicted and analyzed in terms of functional annotation. Finally, we identified that ANF and miR-23a are downstream targets of circRNA wwp1, suggesting that circRNA wwp1 exerts inhibitory roles of cardiac hypertrophy via down-regulation of ANF and miR-23a, which underlying the potential mechanisms whereby circRNA regulates cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression Profiling; Gene Expression Regulation; Isoproterenol; Mice; MicroRNAs; RNA, Circular; Ubiquitin-Protein Ligases

Sirtuin 3 (SIRT3) is a type III histone deacetylase that inhibits cardiac hypertrophy. It is mainly localized in the mitochondria and is thus implicated in mitochondrial metabolism. Recent studies have shown that SIRT3 can also accumulate in the nuclear under stressed conditions, and participated in histone deacetylation of target proteins. Poly [ADP-ribose] polymerase 1 (PARP-1) functions as an important PARP isoform that was involved in cardiac hypertrophy. Our experiments showed that SIRT3 accumulated in the nuclear of cardiomyocytes treated with isoproterenol or SIRT3 overexpression. Moreover, overexpression of SIRT3 by adenovirus inhibited the expression of cardiac hypertrophic genes-ANF and BNP, as well as abrogating PARP-1 activation induced by isoproterenol or phenylephrine. In addition, co-immunoprecipitation experiments revealed that SIRT3 could interact with PARP-1, and overexpression of SIRT3 could decrease the acetylation level of PARP-1. Our results indicate that SIRT3 exerts protective effects against cardiac hypertrophy by reducing the level of acetylation and activity of PARP-1, thus providing novel mechanistic insights into SIRT3-mediated cardiprotective actions.

Topics: Acetylation; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cell Line; Isoproterenol; Male; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phenylephrine; Poly (ADP-Ribose) Polymerase-1; Rats; Rats, Sprague-Dawley; Sirtuin 3

Topics: Albuminuria; Animals; Atrial Natriuretic Factor; Atrial Remodeling; Blood Pressure; Cardiomegaly; Cardiotonic Agents; Dinoprostone; Drug Evaluation, Preclinical; Fibrosis; Glomerular Filtration Rate; Heart; Hypertension; Kidney; Kidney Diseases; Male; Myocytes, Cardiac; Natriuresis; Peptide Fragments; Potassium; Rats; Rats, Inbred Dahl; Smad2 Protein; Sodium Chloride, Dietary; Ventricular Remodeling

Cardiac hypertrophy is an independent risk factor of many cardiovascular diseases. Several cardiovascular protective properties of

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Collagen Type I; Collagen Type III; Cymbopogon; Fibrosis; Gas Chromatography-Mass Spectrometry; Heart; Injections, Intraperitoneal; Isoproterenol; Male; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Oils, Volatile; Protective Agents; Rats

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiac hypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and β-myosin heavy chain. Taurine also reduced ROS, intracellular Ca

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Calcium; Calcium-Binding Proteins; Calpain; Cardiomegaly; Caspase 3; Caspase 9; Cell Line; Cytochromes c; Isoproterenol; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; Natriuretic Peptides; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Taurine; Ventricular Myosins

This study aimed to explore the role of miR-130a-3p in cardiomyocyte hypertrophy and its underlying mechanisms. Pressure-overload induced myocardial hypertrophy mice model was constructed by thoracic aortic constriction (TAC).. 本研究旨在探索 miR-130a-3p 对心肌细胞肥大的作用及其可能机制。通过胸主动脉缩窄法（TAC）构建压力超负荷所致心肌肥厚小鼠模型。使用去甲肾上腺素（NE）刺激 SD 乳鼠原代心肌细胞（NRCMs）及 H9c2 大鼠心肌细胞系，诱导这两种心肌细胞发生肥大表型转变。检测 miR-130a-3p 的表达变化，并进一步探索其对心肌细胞肥大是否有调控作用。结果表明，miR-130a-3p 在肥厚心肌组织、肥大 NRCMs 及 H9c2 细胞中的表达均明显降低。给予 miR-130a-3p mimics 使其过表达后，H9c2 细胞中肥大标志基因心房利钠肽（ANP）、脑利钠肽（BNP）和肌球蛋白重链 β（β-MHC）的表达较对照组（mimics N.C.+NE 组）明显下调，且细胞面积明显减小。而给予 miR-130a-3p inhibitor 抑制其表达后，肥大心肌细胞中 ANP、BNP、β-MHC 的表达进一步上升，且细胞面积进一步增加。Western blot 检测发现，过表达 miR-130a-3p 后心肌细胞中磷酸化 Akt 和磷酸化 mTOR 的表达水平下调。以上结果提示，miR-130a-3p mimics 可缓解心肌细胞肥大的程度；其 inhibitor 则可使心肌细胞肥大进一步加剧。过表达 miR-130a-3p 可能通过影响 Akt 通路来缓解 H9c2 心肌细胞肥大的程度。.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Mice; MicroRNAs; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Nonmuscle Myosin Type IIB; Proto-Oncogene Proteins c-akt; Rats

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.

Topics: Aging; Animals; Atrial Natriuretic Factor; Base Sequence; Cardiomegaly; Gene Expression Regulation, Developmental; Gene Ontology; Isoproterenol; Male; MicroRNAs; Models, Cardiovascular; Myocardial Infarction; Myocardium; Rats, Sprague-Dawley; Reproducibility of Results; RNA, Long Noncoding; Signal Transduction

Cardiac hypertrophy causes heart failure and is associated with hyperglycemia in patients with diabetes mellitus. Mibefradil, which acts as a T-type calcium channel blocker, exerts beneficial effects in patients with heart failure. In this study, we explored the effects and mechanism of mibefradil on high-glucose-induced cardiac hypertrophy in H9c2 cells. H9c2 cells were incubated in a high-glucose medium and then treated with different concentrations of mibefradil in the presence or absence of the Akt inhibitor MK2206 or mTOR inhibitor rapamycin. Cell size was evaluated through immunofluorescence, and mRNA expression of cardiac hypertrophy markers (atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain) was assessed by using quantitative real-time polymerase chain reaction. Changes in the expression of p-PI3K, p-Akt, and p-mTOR were evaluated using Western blotting, and autophagosome formation was detected using transmission electron microscopy. Our results indicate that mibefradil reduced the size of H9c2 cells, decreased mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain, and decreased the level of autophagic flux. However, MK2206 and rapamycin induced autophagy and reversed the effects of mibefradil on high-glucose-induced H9c2 cells. In conclusion, mibefradil ameliorated high-glucose-induced cardiac hypertrophy by activating the PI3K/Akt/mTOR pathway and inhibiting excessive autophagy. Our study shows that mibefradil can be used therapeutically to ameliorate cardiac hypertrophy in patients with diabetes mellitus.

Topics: Animals; Atrial Natriuretic Factor; Autophagy; Calcium Channel Blockers; Cardiomegaly; Cell Line; Cell Size; Glucose; Mibefradil; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; TOR Serine-Threonine Kinases

The aim of the present study was to examine the role of sirtuin 3 (Sirt3)‑autophagy in regulating myocardial energy metabolism and inhibiting myocardial hypertrophy in angiotensin (Ang) II‑induced myocardial cell hypertrophy. The primary cultured myocardial cells of neonatal Sprague Dawley rats were used to construct a myocardial hypertrophy model induced with Ang II. Following the activation of Sirt3 by resveratrol (Res), Sirt3 was silenced using small interfering (si)RNA‑Sirt3, and the morphology of the myocardial cells was observed under an optical microscope. Reverse transcription‑polymerase chain reaction was used to detect the mRNA expression of the following myocardial hypertrophy markers; atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), Sirt3, medium‑chain acyl‑CoA dehydrogenase (MCAD) and pyruvate kinase (PK). Western blot analysis was used to detect the protein expression of Sirt3, light chain 3 (LC3) and Beclin1. Ang II may inhibit the protein expression of Sirt3, LC3 and Beclin1. Res, an agonist of Sirt3, may promote the protein expression of Sirt3, LC3 and Beclin1. Res inhibited the mRNA expression of ANP and BNP, and reversed the Ang II‑induced myocardial cell hypertrophy. The addition of siRNA‑Sirt3 decreased the protein expression of Sirt3, LC3 and Beclin1, increased the mRNA expression of ANP and BNP, and weakened the inhibitory effect of Res on myocardial cell hypertrophy. Res promoted the mRNA expression of MCAD, inhibited the mRNA expression of PK, and reversed the influence of Ang II on myocardial energy metabolism. siRNA‑Sirt3 intervention significantly decreased the effect of Res in eliminating abnormal myocardial energy metabolism. In conclusion, Sirt3 may inhibit Ang II‑induced myocardial hypertrophy and reverse the Ang II‑caused abnormal myocardial energy metabolism through activation of autophagy.

Topics: Acyl-CoA Dehydrogenase; Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Autophagy; Beclin-1; Cardiomegaly; Cells, Cultured; Energy Metabolism; Female; Gene Silencing; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Natriuretic Peptide, Brain; Pyruvate Kinase; Rats, Sprague-Dawley; Resveratrol; Sirtuins

Several reports demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy. Resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. Nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. Since resveratrol is a well-known CYP1B1 inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin II (Ang II)-induced cellular hypertrophy through inhibition of CYP1B1/mid-chain HETEs mechanism. RL-14 and H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM resveratrol for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography-mass spectrometry (LC/MS). Hypertrophic markers and CYP1B1 gene expression and protein levels were measured using real-time PCR and Western blot analysis, respectively. Our results demonstrated that resveratrol, at concentrations of 10 and 50 μM, was able to attenuate Ang-II-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (MHC)/α-MHC and atrial natriuretic peptide. Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid-chain HETEs. Interestingly, the protective effect of resveratrol was associated with a significant decrease of CYP1B1 protein expression and mid-chain HETEs. Our results provided the first evidence that resveratrol protects against Ang II-induced cellular hypertrophy, at least in part, through CYP1B1/mid-chain HETEs-dependent mechanism.

Topics: Angiotensin II; Antioxidants; Atrial Natriuretic Factor; Cardiomegaly; Cardiotoxicity; Cell Line; Cytochrome P-450 CYP1B1; Humans; Hydroxyeicosatetraenoic Acids; Myosin Heavy Chains; Protective Agents; Resveratrol; Vasoconstrictor Agents

Di-(2-ethylhexyl) phthalate (DEHP) is a prevalent environmental contaminant that severely impacts the health of human and animals. Taxifolin (TAX), a plant flavonoid isolated from yew, exerts protective effects on cardiac diseases. Nevertheless, whether DEHP could induce cardiomyocyte hypertrophy and its mechanism remains unclear. This study aimed to highlight the specific molecular mechanisms of DEHP-induced cardiomyocyte hypertrophy and the protective potential of TAX against it. Chicken primary cardiomyocytes were treated with DEHP (500 μM) and/or TAX (0.5 μM) for 24 h. The levels of glucose and adenosine triphosphate (ATP) were detected, and cardiac hypertrophy-related genes were validated by real-time quantitative PCR (qRT-PCR) and Western blot (WB) in vitro. The results showed that DEHP-induced cardiac hypertrophy was ameliorated by TAX, as indicated by the increased cardiomyocyte area and expression of atrial natriuretic peptide (ANP), natriuretic peptides A-like (BNP) and β-myosin heavy cardiac muscle (β-MHC). Furthermore, DEHP induced cardiac hypertrophy via the interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in vitro. In addition, DEHP disrupted mitochondrial function and glycometabolism by activating the insulin-like growth factor 1 (IGF1)/phosphatidylinositol 3-kinase (PI3K) pathway and the peroxisome proliferator activated receptors (PPARs)/PPARG coactivator 1 alpha (PGC-1α) pathway to induce cardiac hypertrophy in vitro. Intriguingly, those DEHP-induced changes were obviously alleviated by TAX treatment. Taken together, cardiac hypertrophy was induced by DEHP via activating the IL-6/JAK/STAT3 signaling pathway, triggering glycometabolism disorder and mitochondrial dysfunction in vitro, can be ameliorated by TAX. Our findings may provide a feasible molecular mechanism for the treatment of cardiomyocyte hypertrophy induced by DEHP.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Chickens; Diethylhexyl Phthalate; Mitochondria; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Quercetin; Ventricular Myosins

Long-term high salt intake leads to cardiac hypertrophy, but the mechanism remains elusive. Transient receptor potential channel, canonical 3(TRPC3), located in mitochondria, regulates mitochondrial calcium and reactive oxygen species(ROS) production. Herein, we investigated whether TRPC3 participates in high salt-induced cardiac hypertrophy by impairing cardiac mitochondrial function. High salt treatment increased the expression of mitochondrial TRPC3 in cardiomyocytes, accompanied by enhanced mitochondrial calcium uptake and elevated ROS production. Inhibition of TRPC3 significantly reduced high salt-induced ROS generation, promoted ATP production by stimulating oxidative phosphorylation, and increased enzyme activity in mitochondria in cardiomyocytes. Additionally, TRPC3 deficiency inhibited high salt-induced cardiac hypertrophy in vivo. A long-term high salt diet increased cardiac mitochondrial TRPC3 expression, elevated expression of cardiac hypertrophic markers atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) and decreased ATP production and mitochondrial complex I and II enzyme activity in a TRPC3-dependent manner. TRPC3 deficiency antagonises high salt diet-mediated cardiac hypertrophy by ameliorating TRPC3-mediated cardiac mitochondrial dysfunction. TRPC3 may therefore represent a novel target for preventing high salt-induced cardiac damage.

Topics: Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Cell Line; Electron Transport Complex I; Electron Transport Complex II; Mice, Knockout; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats; Reactive Oxygen Species; Sodium Chloride, Dietary; TRPC Cation Channels

Sex hormones contribute to sex differences in blood pressure. Inappropriate activation of the renin-angiotensin system is involved in vascular dysfunction and hypertension. This study evaluated the role of androgens (testosterone) in angiotensin II (Ang II)-induced increase in blood pressure, vascular reactivity, and cardiac hypertrophy. Eight-week-old male Wistar rats underwent sham operation, castration, or castration with testosterone replacement. After 12 weeks of chronic changes in androgen status, Ang II (120 ng/kg per minute) or saline was infused for 28 days via subcutaneous miniosmotic pump, and changes in blood pressure was measured. Vascular reactivity and Ang II receptor levels were examined in mesenteric arteries. Heart weight, cardiac ANP mRNA levels, and fibrosis were also assessed. Ang II infusion increased arterial pressure in intact males. The Ang II-induced increase in hypertensive response was prevented in castrated males. Testosterone replacement in castrated males restored Ang II-induced hypertensive responses. Castration reduced vascular AT1R/AT2R ratio, an effect that was reversed by testosterone replacement. Ang II-induced hypertension was associated with increased contractile response of mesenteric arteries to Ang II and phenylephrine in intact and testosterone-replaced castrated males; these increases were prevented in castrated males. Ang II infusion induced increased left ventricle-to-body weight ratio and ANP mRNA expression, indicators of left ventricular hypertrophy, and fibrosis in intact and testosterone-replaced castrated males, and castration prevented the increase in these parameters caused by Ang II. This study demonstrates that testosterone plays a permissive role in development and maintenance of Ang II-induced vascular dysfunction, hypertension, and cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Hypertension; Male; Mesenteric Arteries; Orchiectomy; Rats; Rats, Wistar; Receptors, Androgen; Receptors, Angiotensin; Renin-Angiotensin System; Testosterone

Cardiac hypertrophy is a key pathological process in the context of diabetic cardiomyopathy. Naringenin exhibits multiple pharmacological activities, but the effect of naringenin on cardiomyocyte hypertrophy under diabetic conditions is still far from clear.. Cardiomyocyte hypertrophy was induced by high glucose (HG, glucose at 25.5 mmol/L) in H9c2 cells, which was determined by cell surface area, protein content and atrial natriuretic factor (ANF) mRNA expression. The effect of naringenin on cardiomyocyte hypertrophy was observed and its mechanisms were investigated by administration with various inhibitors on epoxyeicosatrienoic acids (EETs)/peroxisome proliferator-activated receptors (PPARs). The level of 14,15-EET was measured by ELISA. The mRNA and protein expressions were detected by qRT-PCR or Western blot, respectively.. Naringenin (0.1, 1, 10 μmol/L) inhibited cardiomyocyte hypertrophy in a concentration-dependent manner (P < 0.05), up-regulated the expressions of PPARα, PPARβ, PPARγ and CYP2J3 (P < 0.05), and increased the level of 14,15-EET (P < 0.05). PPOH, a CYP2J3 inhibitor, blocked the naringenin-mediated improvement of myocardial hypertrophy (P < 0.01), and abolished the up-regulation of PPARs expressions (P < 0.01). Meanwhile, MK886, a PPARα antagonist, GSK0660, a PPARβ antagonist, and GW9662, a PPARγ antagonist, reversed the protection of naringenin on cardiomyocytes (P < 0.05), and abrogated the up-regulation of CYP2J3-EET produced by naringenin (P < 0.05).. Activation of EETs and PPARs function together may be contributed to the anti-hypertrophic effect of naringenin in H9c2 cells under high glucose condition.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Diabetic Cardiomyopathies; Flavanones; Glucose; Myocytes, Cardiac; PPAR gamma; Rats; RNA, Messenger; Signal Transduction; Up-Regulation

Cardiac hypertrophy is a risk factor which can intrigue heart failure. In the present study, we explored whether AdipoRon attenuates isoprenaline (ISO) or l-thyroxine-induced cardiac hypertrophy in Sprague-Dawley (SD) rats and whether the anti-hypertrophy effect is mediated by AMPK-related pathway. Here, cardiac hypertrophy was induced by injection of l-thyroxine or ISO in SD rats. In the treatment group, AdipoRon was co-administered. We examined the effects of AdipoRon on cardiac hypertrophy and hypertrophy signaling pathway. The weight of SD rats was recorded every day. Rats were killed for collection of blood and heart under anesthesia. The left heart weight and heart weight were weighed. Paraffin-embedded heart tissue regions (4 μm) were stained with hematoxylin and eosin or Masson to detect left heart hypertrophy and myocardial fibrosis. The serum BNP levels were determined by using an enzyme-linked immunosorbent assay. The mRNA levels of ANP, BNP, PGC-1α, and ERRα were evaluated by real-time PCR analysis. The protein expression levels of PGC-1α, ERRα, and pAMPK/AMPK were determined by western blot analysis. The results showed that AdipoRon significantly reversed heart weight (HW)/body weight (BW) ratio, left ventricular (LV)/BW ratio, serum BNP level and the mRNA level of ANP and BNP induced by ISO or l-thyroxine. ISO or l-thyroxine reduced both the mRNA level and protein level of ERRα and PGC-1α, and also reduced the protein level of pAMPK/AMPK. However, AdipoRon reversed ISO or l-thyroxine-induced changes of pAMPK/AMPK, ERRα, and PGC-1α. Our data indicated that the effects of AdipoRon are mediated partly by activating AMPK-related pathway, and AdipoRon plays a potential role in the prevention of cardiac hypertrophy.

Topics: AMP-Activated Protein Kinases; Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Gene Expression; Isoproterenol; Male; Natriuretic Peptide, Brain; Organ Size; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Piperidines; Rats, Sprague-Dawley; Signal Transduction; Thyroxine

Although cardiac hypertrophy is widely recognized as a risk factor that leads to cardiac dysfunction and, ultimately, heart failure, the complex mechanisms underlying cardiac hypertrophy remain incompletely characterized. The nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) is involved in the regulation of cardiac lipid metabolism. Here, we describe a novel PPARδ-dependent molecular cascade involving microRNA-29a (miR-29a) and atrial natriuretic factor (ANF), which is reactivated in cardiac hypertrophy. In addition, we identify a novel role of miR-29a, in which it has a cardioprotective function in isoproterenol hydrochloride-induced cardiac hypertrophy by targeting PPARδ and downregulating ANF. Finally, we provide evidence that miR-29a reduces the isoproterenol hydrochloride-induced cardiac hypertrophy response, thereby underlining the potential clinical relevance of miR-29a in which it may serve as a potent therapeutic target for heart hypertrophy treatment.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Down-Regulation; Gene Expression Regulation; Mice; Mice, Inbred ICR; MicroRNAs; Myocytes, Cardiac; Receptors, Cytoplasmic and Nuclear

The antihypertrophic effect of nebivolol over cardioselective beta-blockers (β-blockers) is attributed to the activation of cardiac nitric oxide signaling. However, the precise role of nebivolol on hypertrophied cardiomyocytes remains unclear. In the current study, in vitro cardiomyocyte hypertrophy model was induced with isoprenaline (10 μM), angiotensin II (1 μM), and phenylephrine (20 μM) in neonatal cardiomyocytes isolated from 0- to 2-day-old Sprague-Dawley rats. In addition to hypertrophic agents, cardiomyocytes were treated with nebivolol (1 μM), metoprolol (10 μM), N(ω)-nitro-L-arginine methyl ester (L-NAME) (100 μM), KT5823 (1 μM), DETA-NONOate (1-10 μM), and BAY412272 (10 μM). After 24 hours of treatment, cardiomyocyte size and transcriptional changes in cardiac hypertrophy markers were evaluated. Cardiomyocyte size increased equally in response to all hypertrophic agents. Nebivolol reduced the enhancement in cell size in response to both isoprenaline and angiotensin II; metoprolol did not. The antihypertrophic effect of nebivolol was prevented with L-NAME blockage indicating the role of NOS signaling on cardiomyocyte hypertrophy. The increased mRNA levels of atrial natriuretic peptide induced by isoprenaline decreased with nebivolol, but both β-blockers reduced the angiotensin II-induced increase in atrial natriuretic peptide expression. Combined, these results reveal that by activating NOS signaling, nebivolol exerts antihypertrophic effects on neonatal cardiomyocytes independent from the action mechanism of hypertrophic stimulus.

Topics: Adrenergic beta-1 Receptor Antagonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nebivolol; Nitric Oxide; Nitric Oxide Synthase; Rats, Sprague-Dawley; Signal Transduction

Cardiac hypertrophy is widely diagnosed in clinical cardiac disorders. The pathophysiology of hypertrophy is complex and multifactorial, a series of molecular and cellular changes are participated, such as activation of different signaling pathways, a switch of fetal gene program in the myocardium, and apoptosis. Some biomarkers have been applied to assess cardiac hypertrophy including atrial natriuretic peptides (ANP), brain/B-type natriuretic peptides (BNP), and α- or β- Myosin Heavy Chain (MHC) in addition to others. Recently, ubiquitin-protein ligase E3A (UBE3A) has been observed to increase in cardiac hypertrophy. Therefore, UBE3A as a new biomarker seems valuable in the clinic. The cardiac hypertrophy is induced in rat-derived heart cell line H9c2 cells by potassium bromate (KBrO3), high glucose (HG), or isoproterenol (Iso), respectively. As an oxidizing agent, KBrO3 increased cell size at concentrations less than 250 μM. Similarly, HG and Iso also induced cardiac hypertrophy in H9c2 cells. Interestingly, each kind of the cell models promoted the gene expression of the well-known biomarkers of cardiac hypertrophy including atrial natriuretic peptides (ANP) and brain/B-type natriuretic peptides (BNP). Additionally, UBE3A is also raised with the signals involved in cardiac hypertrophy such as calcineurin and nuclear factor of activated T-cells (NFAT) determined using Western blots. KBrO3 increased the protein levels of these signals and the specific inhibitor, such as cyclosporine A and tacrolimus, attenuated the signaling in H9c2 cells at concentrations sufficient to inhibit calcineurin in addition to the reduction of mRNA levels of UBE3A, similar to ANP or BNP. Moreover, HG or Iso also significantly increased protein levels of UBE3A in H9c2 cells. Taken together, we provided a new view that UBE3A is markedly raised in cardiac hypertrophy using various cell models, mainly through the activation of the calcineurin/NFAT signaling pathway in H9c2 cells. Therefore, UBE3A could be developed as a new biomarker in the diagnosis of cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Calcineurin; Cardiomegaly; Cell Line; Humans; Models, Biological; Myocytes, Cardiac; Natriuretic Peptide, Brain; NFATC Transcription Factors; Rats; Ubiquitin-Protein Ligases

To investigate the effect of acupuncture at PC6 on cardiac hypertrophy in isoproterenol (ISO)-treated mice.. 48 male C57BL/6 mice underwent subcutaneous injection of ISO for 14 days and were randomly divided into four groups (n=12 each) that remained untreated (ISO group), received verum manual acupuncture (MA) treatment at PC6 (ISO+MA(PC6) group), sham MA at location on the tail not corresponding to any traditional acupuncture point (ISO+MA(tail) group), or propranolol (ISO+PR group). An additional 12 mice were given an injection of phosphate-buffered saline (PBS) and formed a healthy control (Normal) group. After performing echocardiography and measuring the ratio of heart weight (HW)/tibia length (TL) at 14 days, all mice were euthanased. Morphological examination was performed following haematoxylin and eosin and Masson's staining of heart tissues. Ultrastructural changes were observed by electron microscopy. Cardiac protein expression of atrial natriuretic peptide (ANP) and tumour necrosis factor α (TNFα) were measured by immunohistochemical (IHC) staining and Western blotting.. Compared with the untreated model group, acupuncture at PC6 lowered the heart rate, reduced the ratio of HW/TL, improved the left ventricular (LV) anterior wall thickness (LVAWd), LV end-diastolic anterior wall thickness (LVAWs), LV end-systolic posterior wall thickness (LVPWd), LV end-diastolic posterior wall thickness (LVPWs), and fractional shortening (FS) as observed by echocardiography (ISO+MA(PC6) vs. ISO groups: P<0.05). Moreover, evidence from morphological studies demonstrated that acupuncture at PC6 inhibited myocardial hypertrophy and collagen deposition, and normalised the ultrastructural changes. In addition, ANP and TNFα expression were attenuated in the verum acupuncture group compared with the untreated model group (ISO+MA(PC6) vs. ISO groups: P<0.05).. The results demonstrated that acupuncture at PC6 attenuates sympathetic overactivity. Additionally, it may improve cardiac performance by reversing adverse cardiac remodelling. Acupuncture has potential as a treatment for sympathetic hypertension.

Topics: Acupuncture Points; Acupuncture Therapy; Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Humans; Injections, Subcutaneous; Isoproterenol; Male; Mice; Mice, Inbred C57BL; Propranolol; Tumor Necrosis Factor-alpha

Angiotensin II (AII) has been well known to induce cardiomyocyte hypertrophy. Epigallocatechin-3-gallate (EGCG) is the main active component of green tea and it has been shown to exhibit strong cardioprotective potential, although the underlying molecular mechanisms remain unclear. In this study, we investigated the role and mechanism of EGCG in preventing AII-induced cardiomyocyte hypertrophy using rat H9c2 cardiomyocytes cells. Reactive oxygen species assay, cell size, and mRNA expression of cardiac hypertrophy markers ANP and BNP were assessed in response to AII treatment. In addition, expression of proteins involved in Hippo signaling pathway were determined by western blot analysis. We found that AII treatment resulted in significant upregulation of ANP and BNP expression levels and increase in H9c2 cell size, which were markedly attenuated by EGCG treatment. Furthermore, our results suggested that EGCG inhibited AII-induced cardiac hypertrophy via regulating the Hippo signaling pathway. Therefore, EGCG may be an effective agent for preventing cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Catechin; Cell Line; Gene Expression Regulation; Molecular Structure; Myocytes, Cardiac; Natriuretic Peptide, Brain; Protein Serine-Threonine Kinases; Rats; Reactive Oxygen Species; Signal Transduction

Nppa is a cardiac hormone which plays critical roles in regulating salt-water balance. Its expression is restricted to the atria of the healthy post-natal heart. During heart development, spatio-temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re-expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking-in a tagBFP reporter cassette into 3'-UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa-tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa-tagBFP reporter knock-in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Gene Knock-In Techniques; Genes, Reporter; Heart; Heart Ventricles; Mice; Mice, Inbred C57BL

Apigenin, a natural flavonoid compound, can improve the myocardial abnormal glucolipid metabolism and down-regulate the myocardial hypoxia inducible factor-1α (HIF-1α) in hypertensive cardiac hypertrophic rats. However, whether or not the ameliorative effect of glucolipid metabolism is from the reduction of HIF-1α expression remains uncertain.. This study aimed to investigate the exact relationship between them in angiotensin Ⅱ (Ang Ⅱ)/hypoxia-stimulated or HIF-1α overexpressed H9c2 cells.. Two cell models with Ang Ⅱ/hypoxia-induced hypertrophy and HIF-1α overexpression were established. After treatment of the cells with different concentrations of apigenin, the levels of total protein, free fatty acids (FFA), and glucose were detected by the colorimetric method, the level of atrial natriuretic peptide (ANP) was detected by the ELISA method, and the expressions of HIF-1α, peroxisome proliferator-activated receptor α/γ (PPARα/γ), carnitine palmitoyltmnsferase-1 (CPT-1), pyruvate dehydrogenase kinase-4 (PDK-4), glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter-4 (GLUT-4) proteins were detected by the Western blot assay.. Following treatment of the both model cells with apigenin 1-10 μM for 24 h, the levels of intracellular total protein, ANP, and FFA were decreased, while the level of cultured supernatant glucose was increased. Importantly, apigenin treatment could inhibit the expressions of HIF-1α, PPARγ, GPAT, and GLUT-4 proteins, and increase the expressions of PPARα, CPT-1, and PDK-4 proteins.. Apigenin could exert an ameliorative effect on abnormal glucolipid metabolism in AngⅡ/hypoxia-stimulated or HIF-1α-overexpressed H9c2 cells, and its mechanisms were associated with the inhibition of HIF-1α expression and subsequent upregulation of PPARα-mediated CPT-1 and PDK-4 expressions and downregulation of PPARγ-mediated GPAT and GLUT-4 expressions.

Topics: Angiotensin II; Animals; Apigenin; Atrial Natriuretic Factor; Cardiomegaly; Cell Hypoxia; Cell Line; Down-Regulation; Glucose; Glucose Transporter Type 4; Hypoxia-Inducible Factor 1, alpha Subunit; Myocytes, Cardiac; PPAR alpha; PPAR gamma; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Rats; Up-Regulation

This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.

Topics: Aconitine; Actins; Angiotensin II; Atrial Natriuretic Factor; Cardiac Myosins; Cardiomegaly; Cells, Cultured; Humans; Hypertrophy; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain

Stress-induced cardiac hypertrophy leads to heart failure. Our previous studies demonstrate that insulin-like growth factor-II receptor (IGF-IIR) signaling is pivotal to hypertrophy regulation. In this study, we show a novel IGF-IIR alternative spliced transcript, IGF-IIRα (150 kDa) play a key role in high-salt induced hypertrophy mechanisms. Cardiac overexpression of IGF-IIRα and high-salt diet influenced cardiac dysfunction by increasing pathophysiological changes with up-regulation of hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). We found that, cardiac hypertrophy under high-salt conditions were amplified in the presence of IGF-IIRα overexpression. Importantly, high-salt induced angiotensin II type I receptor (AT1R) up regulation mediated IGF-IIR expressions via upstream mitogen activated protein kinase (MAPK)/silent mating type information regulation 2 homolog 1 (SIRT1)/heat shock factor 1 (HSF1) pathway. Further, G-coupled receptors (Gαq) activated calcineurin/nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3)/protein kinase C (PKC) signaling was significantly up regulated under high-salt conditions. All these effects were observed to be dramatically over-regulated in IGF-IIRα transgenic rats fed with a high-salt diet. Altogether, from the findings, we demonstrate that IGF-IIRα plays a crucial role during high-salt conditions leading to synergistic cardiac hypertrophy.

Topics: Alternative Splicing; Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; Male; MAP Kinase Signaling System; Natriuretic Peptide, Brain; Organ Specificity; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Receptor, IGF Type 2; Sodium Chloride, Dietary

Dioxins are a group of structurally related chemicals that persist in the environment. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener, is a suspected risk factor for cardiac diseases in humans. TCDD induces signs of cardiotoxicity in various animals. Mouse models of TCDD exposure suggest cardiotoxicity phenotypes develop differently depending on the timing and time-course of exposure. In order to clarify and characterize the TCDD-induced cardiotoxicity in the developing period, we utilized mouse pups exposed to TCDD. One day after delivery, groups of nursing C57BL/6J dams were orally administered TCDD at a dose of 0 (Control), 20 (TCDD-20), or 80 μg/kg (TCDD-80) body weight (BW). On postnatal days (PNDs) 7 and 21, pups' hearts were examined by histological and gene expression analyses. The TCDD-80 group was found to have a left ventricular remodeling on PND 7, and to develop heart hypertrophy on PND 21. It was accompanied by fibrosis and increased expression of associated genes, such as those for atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), and endothelin-1 (ET-1). These results revealed that TCDD directly induces cardiotoxicity in the postnatal period represented by progressive hypertrophy in which ANP, β-MHC, and ET-1 have potentials to mediate the cardiac hypertrophy and heart failure.

Topics: Administration, Oral; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cardiotoxicity; Endothelin-1; Environmental Pollutants; Female; Gene Expression; Heart Failure; Humans; Lactation; Mice, Inbred C57BL; Models, Animal; Myosin Heavy Chains; Polychlorinated Dibenzodioxins; Pregnancy

Topics: Animals; Aspartate Aminotransferases; Atrial Natriuretic Factor; Biomarkers; Bone Morphogenetic Proteins; Cardiomegaly; Creatine Kinase; DNA Methylation; Female; Gene Expression Regulation; Homeobox Protein Nkx-2.5; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; L-Lactate Dehydrogenase; Male; Maternal Exposure; Mice; Natriuretic Peptide, Brain; Nitrogen Dioxide; Pregnancy; Reactive Oxygen Species; Ventricular Myosins

Molecular hydrogen has been shown to have antioxidant effect and have been used to prevent oxidative stress-related diseases. The goal of this study was to explore if hydrogen-rich saline (HRS) plays a cardioprotective effect on abdominal aortic constriction (AAC) induced cardiac hypertrophy in rats. 60adult Sprague-Dawley rats received surgically the AAC for 6-week. After the surgery, the rats were randomly divided into 4 groups (15 for each):1: sham-operated (sham); 2: AAC-model; 3: AAC + Low HRS (LHRS); and 4: AAC + High HRS (HHRS). The rats in sham and AAC-model groups were treated with normal saline intraperitoneally, while rats in LHRS and HHRS groups were intraperitoneally treated with 3 or 6 mL/kg HRS daily, respectively, for 6-week.. The ratios of HW/BW and LVW/BW were shown in an order of Model > LHRS > HHRS > SHAM groups. The cardiac hypertrophy was also manifested with increased expressions of atrial natriuretic peptide (ANP), brain natriuretic peptides (BNP) and fibrosis of cardiac tissues in AAC-model group, which could likewise be restrained in LHRS and HHRS groups. Moreover, the JAK-STAT (Janus Kinase-Signal transducers and activators of transcription) signaling molecule expressions were decreased with HRS treatment.. Our results showed a protective effect of HRS on pressure overload-induced cardiac hypertrophy in rats, which may be associated to a decreasing in JAK-STAT signaling pathway.

Topics: Animals; Aorta, Abdominal; Apoptosis; Arterial Pressure; Atrial Natriuretic Factor; Cardiomegaly; Constriction; Disease Models, Animal; Fibrosis; Fluid Therapy; Hydrogen; Janus Kinases; Male; Myocardium; Natriuretic Peptide, Brain; Rats, Sprague-Dawley; Signal Transduction; Sodium Chloride; STAT Transcription Factors

Neonatal cardiomyocytes cultured on flat surfaces are commonly used as a model to study cardiac failure of diverse origin. A major drawback of such a system is that the cardiomyocytes do not exhibit alignment, organization and calcium transients, similar to the native heart. Therefore, there is a need to develop in vitro platforms that recapitulate the cellular microenvironment of the murine heart as organotypic models to study cardiovascular diseases. In this study, we report an engineered platform that mimics cardiac cell organization and function of the heart. For this purpose, microscale ridges were fabricated on silicon using ultraviolet lithography and reactive ion etching techniques. Physical characterization of the microstructures was done using scanning electron microscopy and atomic force microscopy. Cardiomyocytes grown on these micro-ridges showed global parallel alignment and elliptical nuclear morphology as observed in the heart. Interestingly, calcium currents traversed the engineered cardiomyocytes in a coordinated and directional manner. Moreover, the cardiomyocytes on the engineered substrates were found to be responsive to hypertrophic stimuli, as observed by the expression of a fetal gene, atrial natriuretic peptide and increase in calcium transients upon agonist treatment. Taken together, our work demonstrates that micro-ridges can be used to obtain cardiomyocyte response in vitro, which closely resembles mammalian heart.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Connectin; Gene Expression; Heart; Models, Biological; Myocytes, Cardiac; Phenylephrine; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Rats, Wistar; Silicon; Surface Properties; Tissue Engineering; Tissue Scaffolds; Ultraviolet Rays

Pregnancy evokes many challenges on the maternal cardiovascular system that may unmask predispositions for future disease. This is particularly evident for women who develop pregnancy-related disorders, for example, pre-eclampsia and gestational diabetes or hypertension. Such pregnancy-related syndromes increase the risk for cardiovascular disease (CVD) postpartum. As a result, pregnancy has been termed as a cardiovascular stress test and an indicator or marker to predict the development of CVD later in life. In addition, pregnancy-related disorders impact the development of offspring also placing them at a higher risk for disease. Utilizing pregnancy as a physiological stressor, the current investigation sought to determine whether the cardiovascular system of offspring exposed to gestational hypertension in utero would respond adversely to the stress of pregnancy. Heterozygous atrial natriuretic peptide gene-disrupted (ANP

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Female; Male; Mice; Mice, Knockout; Pregnancy; Pregnancy Complications, Cardiovascular

Activation of stromal interaction molecule 1 (STIM1) and Orai1 participates in the development of cardiac hypertrophy. Store-operated Ca2+ entry-associated regulatory factor (SARAF) is an intrinsic inhibitor of STIM1-Orai1 interaction. Thus, we hypothesized that SARAF could prevent cardiac hypertrophy.. Male C57BL/6 mice, aged 8 weeks, were randomly divided into sham and abdominal aortic constriction surgery groups and were infected with lentiviruses expressing SARAF and GFP (Lenti-SARAF) or GFP alone (Lenti-GFP) via intramyocardial injection. At 4 weeks after aortic constriction, left ventricular structure and function were assessed by echocardiography and hemodynamic assays. The gene and protein expressions of SARAF, STIM1, and Orai1 were measured by quantitative PCR and Western blot, respectively.. Gene and protein expressions of SARAF were significantly decreased, while STIM1 and Orai1 were increased in the heart tissue compared with sham group. Overexpression of SARAF in the heart prevented the upregulation of STIM1 and Orai1, and importantly, attenuated aortic constriction-induced decrease in maximal rate of left ventricular pressure decay and increases in thickness of interventricular septum and left ventricular posterior wall, heart weight/body weight ratio, and size of cardiomyocytes. Blood pressure detected through the carotid artery and left ventricular systolic function were not affected by SARAF overexpression. In addition, overexpression of SARAF also attenuated angiotensin II-induced upregulation of STIM1 and Orai1 and hypertrophy of cultured cardiomyocytes.. Overexpression of SARAF in the heart prevents cardiac hypertrophy, probably through suppressing the upregulation of STIM1/Orai1.

Topics: Angiotensin II; Animals; Aorta, Abdominal; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Echocardiography; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; ORAI1 Protein; Pressure; Stromal Interaction Molecule 1; Up-Regulation; Ventricular Function, Left

In cardiac myocytes activation of an exchange factor activated by cAMP (Epac) leads to activation of phospholipase Cε (PLCε)-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus a process critical for development of cardiac hypertrophy. Here we show that β-adrenergic receptor (βAR) stimulation does not stimulate this pathway in the presence of the broad spectrum phosphodiesterase (PDE) inhibitor IBMX, but selective PDE3 inhibition revealed βAR-dependent PI4P depletion. On the other hand, selective inhibition of PDE2 or PDE9A blocked endothelin-1 (ET-1) and cAMP-dependent PI4P hydrolysis by PLCε. Direct activation of protein kinase A (PKA), protein kinase G (PKG), or the atrial natriuretic factor (ANF) receptor abolished PI4P hydrolysis in response to multiple upstream stimuli. These results reveal distinct pools of cyclic nucleotides that either inhibit PLCε at the Golgi through PKA/PKG, or activate PLCε at the Golgi through Epac. These data together reveal a new mechanism by which ANF and selective PDE inhibitors can protect against cardiac hypertrophy.

Topics: 1-Methyl-3-isobutylxanthine; A Kinase Anchor Proteins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Hypertrophic; Cell Compartmentation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Golgi Apparatus; Humans; Myocytes, Cardiac; Nucleotides; Phosphatidylinositol Phosphates; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Signal Transduction

Atrial myocyte hypertrophy is one of the most important substrates in the development of atrial fibrillation (AF). The TWEAK/Fn14 axis is a positive regulator of cardiac hypertrophy in cardiomyopathy. This study therefore investigated the effects of Fn14 on atrial hypertrophy and underlying cellular mechanisms using HL-1 atrial myocytes. In patients with AF, Fn14 protein levels were higher in atrial myocytes from atrial appendages, and expression of TWEAK was increased in peripheral blood mononuclear cells, while TWEAK serum levels were decreased. In vitro, Fn14 expression was up-regulated in response to TWEAK treatment in HL-1 atrial myocytes. TWEAK increased the expression of ANP and Troponin T, and Fn14 knockdown counteracted the effect. Inhibition of JAK2, STAT3 by specific siRNA attenuated TWEAK-induced HL-1 atrial myocytes hypertrophy. In conclusion, TWEAK/Fn14 axis mediates HL-1 atrial myocytes hypertrophy partly through activation of the JAK2/STAT3 pathway.

Topics: Aged; Animals; Atrial Fibrillation; Atrial Natriuretic Factor; Cardiomegaly; Case-Control Studies; Cytokine TWEAK; Disease Models, Animal; Female; Gene Expression Regulation; Heart Atria; Humans; Janus Kinase 2; Leukocytes, Mononuclear; Male; Mice; Middle Aged; Myocytes, Cardiac; Primary Cell Culture; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Troponin T; TWEAK Receptor

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a cytoplasmic protein, involved in autoimmune diseases, mammalian reproduction, neuronal cell death, and stroke. However, the role of NLRP1 in cardiac hypertrophy remains unclear. We used in vivo and in vitro models to investigate the effects of NLRP1 on cardiac hypertrophy.. We used NLRP1-deficient mice and cultured neonatal rat cardiomyocytes with gain and loss of NLRP1 function. Cardiac hypertrophy was estimated by echocardiographic and hemodynamic measurements, and by pathological and molecular analysis.. Eight weeks after aortic banding (AB), NLRP1 deficiency significantly inhibited aortic banding-induced cardiac hypertrophy, inflammation, and fibrosis. Activation of MAPK, NF-κB, and TGF-β/Smad pathways was reduced in NLRP1-knockout (KO) mice compared with that in wild-type (WT) mice. Consistent with these results, in vitro studies, performed using cultured neonatal mouse cardiomyocytes, confirmed that NLRP1 deficiency protects against cardiomyocyte hypertrophy induced by isoproterenol (PE); this protective activity was associated with the arrest of MAPK and NF-κB signaling.. Our data illustrates that NLRP1 plays a crucial role in the development of cardiac hypertrophy via positive regulation of the MAPK, NF-κB, and TGF-β/Smad signaling pathways.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B; Pressure; Rats; RNA Interference; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction

BACKGROUND Dracocephalum heterophyllum Benth flavonoid (DHBF) is a Tibetan and Uighur traditional medicine used to treat various disorders such as hypertension, lung heat, cough, and bronchitis; it has good antioxidant activity. Previous studies have shown that DHBF can reduce blood pressure in renovascular hypertensive rats, improve left ventricular systolic and diastolic function, and improve myocardial contractility. Therefore, we aimed to study the effect of DHBF on cardiomyocyte hypertrophy in cultured cells. MATERIAL AND METHODS Neonatal rat cardiomyocytes were cultured, and hypertrophy was induced by angiotensin II (Ang II), with or without varying concentrations of the DHBF extract. Cell Counting Kit-8 assay was used to assess cell viability, RT-qPCR was used to determine mRNA levels, confocal laser scanning microscopy was used to measure cell surface area and intracellular Ca2+ concentrations ([Ca2+]i), and colorimetric assays were used to assess nitric oxide (NO) levels and nitric oxide synthase (NOS) activity. RESULTS Ang II treatment of cardiomyocytes reduced cell viability to ~75% that of controls. Ang II treatment also increased cell surface area; increased mRNA expression of c-jun, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC); increased [Ca2+]i; and reduced NOS activity and NO production. DHBF treatment could reverse these effects in a concentration-dependent manner. CONCLUSIONS These results showed that DHBF can ameliorate cardiomyocyte hypertrophy induced by Ang II, as indicated by the downregulation of cardiac hypertrophy genes (ANP, BNP, and β-MHC) and reduction in cell surface area. The mechanism may be related to NO release and [Ca2+]I regulation.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Hypertrophic; Cells, Cultured; China; Flavonoids; Heart Ventricles; Medicine, Tibetan Traditional; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Signal Transduction

Bakuchiol (Bak), a monoterpene phenol isolated from the seeds of Psoralea corylifolia, has been widely used to treat a large variety of diseases in both Indian and Chinese folkloric medicine. However, the effects of Bak on cardiac hypertrophy remain unclear. Therefore, the present study was designed to determine whether Bak could alleviate cardiac hypertrophy. Mice were subjected to aortic banding (AB) to induce cardiac hypertrophy model. Bak of 1 ml/100 g body weight was given by oral gavage once a day from 1 to 8 weeks after surgery. Our data demonstrated for the first time that Bak could attenuate pressure overload-induced cardiac hypertrophy and could attenuate fibrosis and the inflammatory response induced by AB. The results further revealed that the effect of Bak on cardiac hypertrophy was mediated by blocking the activation of the NF-κB signaling pathway.

Topics: Administration, Oral; Angiotensin II; Animals; Aorta; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Collagen; Connective Tissue Growth Factor; Constriction, Pathologic; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B; Phenols; Plant Extracts; Primary Cell Culture; Psoralea; Signal Transduction

Our previous studies have shown that Src homology 2 (SH2) B adaptor protein 1 (SH2B1) plays an important role in cardiac hypertrophy, but the specific mechanism remains to be studied. Through bioinformatics and related research, it is found that miR-14 2-3 p is closely related to SH2B1. Exploring the relationship between miR-14 2-3 p and gene SH2B1 expression is beneficial for the treatment of cardiac hypertrophy. SH2B1 is a key factor regulating energy metabolism, mitochondria are the main organelles of energy metabolism and cardiac hypertrophy are closely related to mitochondrial dysfunction. So it is particularly important to explore the relationship between miR-14 2-3 p and SH2B1 and myocardial mitochondrial function. In this study, we investigated whether overexpression of miR-14 2-3 p can inhibit the expression of gene SH2B1, ameliorate cardiac mitochondrial dysfunction and cardiac hypertrophy.. We first constructed a pressure overload myocardial hypertrophy model by ligation of the abdominal aorta(AB) of rats. After 4 weeks of modeling, echocardiographic examination showed that the heart volume of the model group became larger, and Hematoxylin and Eosin Staining Kit (HE) staining showed that the cross-sectional area of the heart tissue became larger. The expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-Myosin Heavy Chain (β-MHC) messenger RNA (mRNA) increased by real‑time polymerase chain reaction (PCR), which proved that the model of cardiac hypertrophy was successfully constructed. Then, miR-14 2-3 p agomir was injected into the tail vein of rats 2 weeks and 4 weeks respectively. The expression of miR-4 2-3 p mRNA was increased by PCR, suggesting that the miR-14 2-3 p plasmid was successfully transfected. At 4 weeks of pressure overload myocardial hypertrophy model, echocardiography was used to detect cardiac function. HE staining of heart tissue and the expression of ANP, BNP, β-MHC mRNA were used to detect cardiac hypertrophy. Flow cytometry was used to detect changes in mitochondrial membrane potential. Secondly, we observed the effect of miR-14 2-3 p on cardiomyocyte hypertrophy and mitochondrial function in vitro by culture neonatal rat cardiomyocytes. Afterwards, using angiotensin (Ang)II-, miRNA mimic- and miRNA mimic nc- treated cardiomyocytes for a given time. α-actin staining found that the myocardial cells became larger, The expression of ANP, BNP, β-MHC mRNA increased by PCR, which proved that AngII-induced cardiac hypertrophy was successfully constructed. Then, the mitochondrial density was measured using mitochondrial Mito-Red staining by Confocal microscope, the mitochondrial membrane potential was evaluated using flow cytometry, Mitochondrial respiration oxygen consumption rate (OCR) was measured by a Seahorse Extracellular Flux Analyzer XF96, and the expression levels of miR-14 2-3 p, ANP, BNP, β-MHC mRNA, SH2B1 in the cardiomyocytes of different groups were measured by RT-PCR and Western blotting. Finally, we used luciferase assay and transfected miR-14 2-3 p agomir in rats, transfected miR-14 2-3 p mimic in Cardiomyocytes, it is found that myocardial SH2B1 mRNA and protein expression both were reduced.. When the pressure overload myocardial hypertrophy model was constructed for four weeks, echocardiography revealed that the heart volume, Left ventricular end diastolic diameter(LVIDd), Left ventricular end systolic diameter (LVIDs), Left ventricular posterior wall thickness (LVPWd), Systolic left ventricular posterior wall (LVPWs), Left ventricle (LV) Mass increased, Ejection fraction (EF) % decreased of AB group increased, but transfected with miR-14 2-3 p agomir of AB, these increase was not significant, EF% reduction was not obvious. HE staining showed that the myocardial cross-sectional area of AB group increased significantly, but the miR-14 2-3 p agomir treatment of AB group did not increase significantly. PCR analysis showed that the expression of ANP, BNP,β-MHC mRNA was significantly increased in AB group, but the miR-14 2-3 p agomir treatment of AB group was not significantly increased. Flow cytometry showed that the mitochondrial membrane potential of AB group was significantly reduced, and the miR-14 2-3 p agomir treatment of AB group was not significantly decreased. During AngII-induced cardiomyocyte hypertrophy, ANP, BNP,β-MHC mRNA expression was increased, while these factors was not significantly increased in miR-14 2-3 p mimic treatment group; mitochondrial membrane potential, mitochondrial density and OCR was significantly decreased in AngII treated group, and these were not significantly reduced in miR-14 2-3 p mimic treatment group; CONCLUSIONS: miR-14 2-3 p not only mitigate cardiac hypertrophy by directly inhibit the expression of gene SH2B1, but also can protect mitochondrial function in cardiac hypertrophy of vitro and vivo.

Topics: Angiotensin II; Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Carrier Proteins; Cells, Cultured; Heart Failure; Intracellular Signaling Peptides and Proteins; Male; MicroRNAs; Mitochondria, Heart; Myocardium; Natriuretic Peptide, Brain; Rats; Rats, Sprague-Dawley

Cardiac hypertrophy is a major predisposing factor for heart failure and sudden cardiac death. Hyperoside (Hyp), a flavonoid isolated from Rhododendron ponticum L., is a primary component of Chinese traditional patent medicines. Numerous studies have shown that Hyp exerts marked anti-viral, anti-inflammatory, anti-oxidant, anti-cancer, anti-ischemic, and particularly cardio-protective effects. However, the effects of Hyp on cardiac hypertrophy have not been explored. The aims of this study were to determine whether Hyp could protect against cardiac remodeling and to clarify the potential molecular mechanisms.. Neonatal rat cardiac myocytes were isolated and treated with different concentrations of Hyp, then cultured with angiotensin II for 48 h. Mice were subjected to either aortic banding or sham surgery (control group). One week after surgery, the mice were treated with Hyp (20 mg/kg/day) or vehicle by oral gavage for 7 weeks. Hypertrophy was evaluated by assessing morphological changes, echocardiographic parameters, histology, and biomarkers.. Hyp pretreatment suppressed angiotensin II-induced hypertrophy in cardiomyocytes. Hyp exerted no basal effects but attenuated cardiac hypertrophy and dysfunction, fibrosis, inflammation, and oxidative stress induced by pressure overload. Both in vivo and in vitro experiments demonstrated that the effect of Hyp on cardiac hypertrophy was mediated by blocking activation of the AKT signaling pathway.. Hyp improves cardiac function and prevents the development of cardiac hypertrophy via AKT signaling. Our results suggest a protective effect of Hyp on pressure overload-induced cardiac remodeling. Taken together, Hyp may have a role in the pharmacological therapy of cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Interleukin-1beta; Male; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-akt; Quercetin; Rats; Signal Transduction; Superoxide Dismutase; Ventricular Remodeling

Cardiac hypertrophy is a one of common type of CHD, responsible for cardiac mortality worldwide. The present study designed to investigate the effect of muscarinic receptors agonist in the rat model of cardiac hypertrophy. A total of 30 male adult Wistar rats having body weight 300-400 gram were equally distributed in two groups (Test group: Rats with Angiotensin II + M3 receptor agonist [acetylcholine]; Reference group: Rats with cardiac hypertrophy induced by Angiotensin II). Rat model of cardiac hypertrophy were induced by Angiotensin II. Effect of M3 receptor agonist on cardiac hypertrophy was evaluated by electrocardiography, hemodynamic and histological assessment. Also, expression of M3 receptor was analyzed using by real-time-PCR and Western blot analysis. Also, vital signs such as pulse rate, and blood pressure were measured. Echocardiographic related variable including ejection fraction were also assessed in both the groups. The results of this study showed acetylcholine attenuates the hypertrophic response triggered by Angiotensin II, by upregulation of M3 receptor. Upregulation of M3 receptor after administration of acetylcholine ameliorates hypertrophic responses induced by angiotensin II. Also acetylcholine treatment prevents Angiotensin II induced increase in level of ANP and β-myosin, which are responsible for inducing cardiac hypertrophic responses. Moreover, acetylcholine ameliorates Angiotensin II induced cell enlargement by reducing the surface area of cells. Overall finding suggested that acetylcholine improves left ventricle hypertrophy and ejection fraction by activating M3 receptor in heart. The finding of this study gives the new vision to cardiovascular researchers to develop anti- hypertrophy therapy based on M3 receptor.

Topics: Acetylcholine; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Electrocardiography; Hemodynamics; Hypertrophy; Male; Muscarinic Agonists; Rats; Receptor, Muscarinic M3; Ventricular Function, Left; Ventricular Myosins

Growth differentiation factor 11 (GDF11) decreases with age, and increased C-C motif chemokine 11 (CCL11) is involved in aging. However, the effects of GDF11 on Angiotensin II (ANG II)-induced hypertrophic cardiomyopathy and expression of markers for volume overload and hypertrophy such as ANP, BNP and beta-MHC, as well as the relationship between GDF11 and CCL11 in hypertrophic cardiomyopathy are unclear. Therefore, the current study aimed to examine the effects of GDF11 on ANG II-induced hypertrophic cardiomyopathy and expression of ANP, BNP and beta-MHC in mice, and explore possible molecular mechanisms.. Vectors were constructed and viruses were packaged. Mouse cardiomyocytes were treated with ANG II for 24 h. Meanwhile, mouse cardiomyocytes were divided into 4 groups: (1) control; (2) ANG II; (3) ANG II+GDF11; and (4) ANG II+CCL11. Furthermore, mouse cardiomyocytes were treated with GDF11 and CCL11 proteins for 48 h, respectively. The thickness of IVS and LVPS during systole and diastole were measured by cardiac ultrasound in the mouse model of hypertrophic cardiomyopathy. The relative expression of ANP, BNP, beta-MHC, CCL11 and GDF11 in cardiomyocytes or heart tissue of mice was detected by qPCR or Western blot. 3'- UTR luciferase reporter assay was utilized to examine the relationship between GDF11 and the expression of CCL11.. The expression of ANP, BNP, and beta-MHC in mouse cardiomyocytes was significantly increased after the cells were treated with 800 nM ANG II, which was utilized in the following cell experiments. After ANG II treatment, 0.2 ng/ml GDF11 group displayed the highest inhibition of expression of ANP, BNP and beta-MHC in mouse cardiomyocytes, whereas 50 ng/ml CCL11 group displayed the highest stimulation of the expression. GDF11 at 10 ng/ml significantly decreased the expression of CCL11 in mouse cardiomyocytes as compared to the control group. Mice treated with ANG II had increased thickness of IVS and LVPS during both systole and diastole, which was significantly attenuated by GDF11 overexpression. GDF11 overexpression attenuated the increase in expression of ANP, BNP and beta-MHC in the mice model of hypertrophic cardiomyopathy. The relative serum concentration of GDF11 was markedly decreased, and CCL11 was dramatically increased in mice with hypertrophic cardiomyopathy. GDF11 overexpression restored the serum concentration of GDF11 and CCL11 in the mice model of hypertrophic cardiomyopathy. In addition, GDF11 interference group had markedly increased expression of CCL11, whereas GDF11 overexpression group had significantly decreased expression of CCL11 in luciferase reporter assay.. GDF11 attenuated ANG II-induced hypertrophic cardiomyopathy and expression of ANP, BNP and beta-MHC through down-regulating CCL11 in mice.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Bone Morphogenetic Proteins; Cardiomegaly; Chemokine CCL11; Down-Regulation; Growth Differentiation Factors; Mice; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain

Telmisartan, a type of angiotensin II (Ang II) receptor inhibitor, is a common agent used to treat hypertension in the clinic. Hypertension increases cardiac afterload and promotes cardiac hypertrophy. However, the ventricular Ang II receptor may be activated in the absence of hypertension. Therefore, telmisartan may reduce cardiac hypertrophy by indirectly ameliorating hypertensive symptoms and directly inhibiting the cardiac Ang II receptor. Nuclear factor of activated T‑cells (NFAT) contributes to cardiac hypertrophy via nuclear translocation, which induces a cascade of atrial natriuretic peptide (ANP) and brain/B‑type natriuretic peptide (BNP) expression and cardiomyocyte apoptosis. However, NFAT-mediated inhibition of cardiac hypertrophy by telmisartan remains poorly understood. The present study demonstrated that telmisartan suppressed cardiomyocyte hypertrophy in a mouse model of cardiac afterload and in cultured cardiomyocytes by inhibiting NFAT nuclear translocation, as well as by inhibiting ANP and BNP expression and cardiomyocyte apoptosis, in a dose‑dependent manner. The present study provides a novel insight into the potential underlying mechanisms of telmisartan-induced inhibition of cardiomyocyte hypertrophy, which involves inhibition of NFAT activation, nuclear translocation and the ANP/BNP cascade.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Atrial Natriuretic Factor; Benzimidazoles; Benzoates; Cardiomegaly; Cell Nucleus; Male; Mice; Myocytes, Cardiac; Natriuretic Peptide, Brain; NFATC Transcription Factors; Signal Transduction; Telmisartan

Cardiac hypertrophy is a crucial predictor of heart failure and is regulated by microRNAs. MicroRNA-124 (miR-124) is regarded as a prognostic indicator for outcomes after cardiac arrest. However, whether miR-124 participates in cardiac hypertrophy remains unclear. Therefore, our study aimed to determine the role of miR-124 in angiotensin II(AngII)-induced myocardial hypertrophy and the possible mechanism. Primary cultured rat neonatal cardiomyocytes(NCMs) were transfected with miR-124 mimics or inhibitor, followed by AngII stimulation. Quantitative RT-PCR, western blot analysis and determination of cell surface area of NCMs were used to detect the hypertrophic phenotypes. We observed that miR-124 was elevated in AngII-induced hypertrophic cardiomyocytes. Cell surface area of NCMs and mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), indicators of myocardial hypertrophy, were higher in NCMs transfected with miR-124 mimics in the presence of AngII. On the contrary, knockdown of miR-124 by its specific inhibitor could restore these courses. Furthermore, downregulation of miR-124 alleviated the increased protein level of endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (Grp78) and calreticulin(CRT) in AngII-induced NCMs. In conclusion, our study shows that inhibition of miR-124 effectively suppresses AngII-induced myocardial hypertrophy, which is associated with attenuation of ER stress.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calreticulin; Cardiomegaly; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Expression Regulation; Heat-Shock Proteins; Humans; MicroRNAs; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats; Ventricular Myosins

Nicotinamide phosphoribosyltransferase (Nampt) is involved in the development of cardiac hypertrophy. Transient receptor potential canonical channel 1 (TRPC1) and endoplasmic reticulum stress (ER stress) are regarded as critical pathways in cardiac hypertrophy. Therefore, we hypothesizedthat TRPC1 might be associated with ER stress in Nampt-induced cardiac hypertrophy. CulturedH9c2cardiomyocyteswereexposed to Namptfor different timesand the expression of markers of cardiomyocyte hypertrophy and ER stress, as well as TRPC1 were detected. Moreover, specific TRPC1-shRNA (short hairpin RNA) expressing plasmid was transfected to knockdown TRPC1 expression before Nampt stimulation. Thapsigargin was used as an agonist and pravastatin was employed as an inhibitor of ER stress. The results demonstrated that exposure of H9c2 cells to 100 ng/mL Nampt for 24h, 48h or 72h significantly increased the expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), markers of ER stress and TRPC1. The Nampt-induced expression of TRPC1 was attenuated by pre-treatment with pravastatin, whereas promoted by pre-treatment with thapsigargin. Furthermore, transfection of TRPC1-shRNA for 48h partially inhibited Nampt-induced expression of ER stress markers and BNP in H9c2 cells. Our data suggest that TRPC1 might play an important role in cardiomyocyte hypertrophy induced by Namptinan ER stress-dependent way.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Endoplasmic Reticulum Stress; Gene Expression Regulation; Humans; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nicotinamide Phosphoribosyltransferase; Pravastatin; Rats; RNA, Small Interfering; Transfection; TRPC Cation Channels

We aimed to investigate the effect of etanercept, a tumor necrosis factor-α (TNF-α) inhibitor, on rat cardiomyocyte hypertrophy and its underlying mechanism. Primary neonatal rat cardiomyocytes were isolated from Sprague-Dawley rats. The model of rat cardiomyocyte hypertrophy was induced by endothelin, and then treated with different concentrations of etanercept (1, 10, and 50 μM). After treatment, cell counts, viability and cell apoptosis were evaluated. The mRNA levels of myocardial hypertrophy marker genes, including atrial natriuretic factor (ANF), matrix metalloproteinase (MMP)-9 and MMP-13, were detected by qRT-PCR, and the expressions of apoptosis-related proteins (Bcl-2 and Bax) were measured by western blotting. The protein levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) were determined using enzyme linked immunosorbent assay (ELISA) kits. In the present study, TNF-α level in cardiomyocytes with hypertrophy was significantly enhanced (P<0.05). Compared to the model group, cell number and viability were significantly increased and ratio of apoptotic cells was reduced by etanercept (P<0.05, P<0.01, or P<0.001). In addition, etanercept remarkably reduced the mRNA levels of ANF, MMP-9 and MMP-13, inhibited the expression of Bax, and increased the expression of Bcl-2 compared to the model group (P<0.05). ELISA results further showed that etanercept lowered the levels of IL-1β, IL-6, LIF and CT-1 but not TGF-β1 compared to the model group (P<0.05). Etanercept may protect rat cardiomyocytes from hypertrophy by inhibiting inflammatory cytokines secretion and cell apoptosis.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Disease Models, Animal; Etanercept; Inflammation; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Myocytes, Cardiac; Protective Agents; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Adult cardiomyocytes can re-enter cell cycle as stimulated by prohypertrophic factors although they withdraw from cell cycle soon after birth. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, has been implicated in cardiac hypertrophy, however, its precise contribution to this process remains largely unclear.. The gene expression profile in left ventricle (LV) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was determined using quantitative PCR array and verified by real-time PCR and Western blotting. Hypertrophic response of H9c2 cells and neonatal rat ventricular myocytes (NRVM) were induced by angiotensin II (1 µmol/L). Cardiac hypertrophy of mice was elicited by isoproterenol (ISO) infusion (40 mg/kg per day for 14 days). p21-adenovirus and p21-siRNA were employed to transfect NRVM, and sterigmatocystin (STE, 3 mg/kg, ip, qd) was used to inhibit p21 activity. mRNA and protein expression levels of α- and β-myosin heavy chain (MHC), p21WAF1/CIP1, calcineurin (CaN) and atrial natriuretic peptide (ANP) were assayed by realtime PCR and WB, respectively.. Sixteen genes showed two-fold or greater changes between SHR and WKY rats, in which the expression of p21WAF1/CIP1 was upregulated by 4.15-fold (P=0.002) and reversed by losartan. Surface area, protein content, mRNA and protein expressions of β-MHC, ANP and p21WAF1/CIP1 in H9c2 cells treated with AngII elevated significantly compared with control group. p21-Ad transfection markedly increased the surface area and β-MHC mRNA expression of normal NRVMs, and p21-siRNA transfection decreased them in AngII-treated NRVMs. STE treatment decreased HW/BW and cross-sectional area, expression levels of β-MHC, ANP and p21 significantly in ISO-treated mice.. Our findings suggest that p21 facilitates the development of cardiac hypertrophy, and regulating the expression of p21 may be an approach to attenuate hypertrophic growth of cardiomyocytes.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Heart Ventricles; Isoproterenol; Losartan; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sterigmatocystin; Ventricular Myosins

Rosiglitazone is an anti-diabetic agent that raised a major controversy over its cardiovascular adverse effects. There is in vivo evidence that Rosiglitazone promotes cardiac hypertrophy by PPAR-γ-independent mechanisms. However, whether Rosiglitazone directly alters hypertrophic growth in cardiac cells is unknown. Chromatin remodeling by histone post-translational modifications has emerged as critical for many cardiomyopathies. Based on these observations, this study was initiated to investigate the cardiac hypertrophic effect of Rosiglitazone in a cellular model of primary neonatal rat cardiomyocytes (NRCM). We assessed whether the drug alters cardiac hypertrophy and its relationship with histone H3 phosphorylation. Our study showed that Rosiglitazone is a mild pro-hypertrophic agent. Rosiglitazone caused a significant increase in the release of brain natriuretic peptide (BNP) into the cell media and also increased cardiomyocytes surface area and atrial natriuretic peptide (ANP) protein expression significantly. These changes correlated with increased cardiac phosphorylation of p38 MAPK and enhanced phosphorylation of H3 at serine 10 globally and at one cardiac hypertrophic gene locus. These results demonstrate that Rosiglitazone causes direct cardiac hypertrophy in NRCM and alters H3 phosphorylation status. They suggest a new mechanism of Rosiglitazone cardiotoxicity implicating chromatin remodeling secondary to H3 phosphorylation, which activate the fetal cardiac gene program.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Female; Fibrinolytic Agents; Gene Expression Regulation; Histones; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Rosiglitazone; Thiazolidinediones

Previous studies have demonstrated that lercanidipine, a calcium channel blocker, may protect against cardiac hypertrophy; however, the underlying mechanisms remain unclear. In the present study, the effects of lercanidipine on hypertrophy and the mechanisms involved were investigated. Cardiomyocytes isolated from neonatal rats were cultured and treated with angiotensin II (Ang II) in the presence or absence of lercanidipine or tacrolimus (FK506, a calcineurin inhibitor). Reverse transcription‑quantitative polymerase chain reaction was used to assess the mRNA expression of genes of interest, whereas the protein expression of calcium‑dependent signaling molecules was detected using western blot analysis. In addition, the cell surface area and the nuclear translocation of target proteins were evaluated using immunofluorescence. The results of the present study demonstrated that lercanidipine and FK506 inhibited Ang II‑induced cardiomyocyte hypertrophy, as evidenced by decreases in fetal gene (atrial natriuretic peptide and brain natriuretic peptide) expression levels and cell surface area. Notably, lercanidipine suppressed Ang II‑induced activation of calcineurin A (CnA) and nuclear factor of activated T cells 3 (NFAT3). In addition, calcium/calmodulin‑dependent kinase II (CaMKII)‑histone deacetylase 4 (HDAC4) signaling was also inhibited by lercanidipine. In conclusion, the present study demonstrated that lercanidipine may ameliorate cardiomyocyte hypertrophy, possibly partially by blocking Cn-NFAT3 and CaMKII-HDAC4 signaling.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calcineurin; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cells, Cultured; Dihydropyridines; Gene Expression Regulation; Histone Deacetylases; Myocytes, Cardiac; Natriuretic Peptide, Brain; NFATC Transcription Factors; Rats; Signal Transduction

Endothelial-to-mesenchymal transition (EndMT) is a mechanism that promotes cardiac fibrosis induced by Angiotensin II (AngII). Kaempferol (KAE) is a monomer component mainly derived from the rhizome of Kaempferia galanga L. It shows anti-inflammatory, anti-oxidative, anti-microbial and anti-cancer properties, which can be used in the treatment of cancer, cardiovascular diseases, infection, etc. But, its effects on the development of cardiac remodelling remain completely unknown. The aim of the present study was to determine whether KAE attenuates cardiac hypertrophy induced by angiotensin II (Ang II) in cultured neonatal rat cardiac myocytes in vitro and cardiac hypertrophy induced by AngII infusion in mice in vivo.. Male wild-type mice aged 8-10 weeks with or without KAE were subjected to AngII or saline, to induce fibrosis or as a control, respectively. Morphological changes, echocardiographic parameters, histological analyses, and hypertrophic markers were also used to evaluate hypertrophy.. KAE prevented and reversed cardiac remodelling induced by AngII. The KAE in this model exerted no basal effects but attenuated cardiac fibrosis, hypertrophy and dysfunction induced by AngII. Both in vivo and in vitro experiments demonstrated that Ang II infusion or TGF-β induced EndMT can be reduced by KAE and the proliferation and activation of cardiac fibroblasts (CFs) can be inhibited by KAE.. The results suggest that KAE prevents and reverses ventricular fibrosis and cardiac dysfunction, providing an experimental basis for clinical treatment on ventricular fibrosis.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Survival; Cells, Cultured; Collagen Type I; Collagen Type II; Echocardiography; Fibroblasts; Fibrosis; Heart Ventricles; Human Umbilical Vein Endothelial Cells; Humans; Kaempferols; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats; Signal Transduction; Transforming Growth Factor beta1; Ventricular Remodeling

This study aimed to exploit the potential therapeutic value of palmatine in treatment of cardiac hypertrophy and the underlying molecular mechanism. Rat hypertrophy model was established by intraperitoneal isoproterenol (ISO) injection. The hypertrophy was evaluated with cardiac hypertrophic parameters, hemodynamic parameters, lipid profile, and non-specific cardiac markers. The animals were intraperitoneally administrated with either palmatine or vehicle. The relative expressions of ANP, BNP, HDAC2, HDAC5, KLF4, and INPP5F transcripts were determined by real-time polymerase chain reaction (PCR). The relative protein levels of HDAC2, HDAC5, KLF4, and INPP5F were analyzed by immunoblotting. Palmatine treatment significantly attenuated ISO-induced hypertrophy in rats and elicited remarkable repressions in ANP, BNP, and HDAC2 transcriptions but not HDAC5. The downstream effector genes KLF4 and INPP5F were greatly restored in a dose-dependent manner in response to palmatine treatment. Our data demonstrated that palmatine possessed promising therapeutic potential against hypertrophy, which was mediated by modulation of HDAC2-KLF4/INPP5F pathway.

Topics: Animals; Atrial Natriuretic Factor; Berberine Alkaloids; Cardiomegaly; Cardiotonic Agents; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Inositol Polyphosphate 5-Phosphatases; Isoproterenol; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Male; Natriuretic Peptide, Brain; Rats, Wistar; RNA, Messenger

Previous animal studies have shown that the administration of probiotic Lactobacillus rhamnosus can provide a protective effect against ischemia/reperfusion and necrotic injury to the intestine, liver, and heart, as well as a therapeutic effect to the outcome of ischemic injury to the heart, including cardiac hypertrophy and heart failure. We hypothesized that L. rhamnosus GR-1 major secreted protein 1 (MSP-1), also known as p75, plays a major role in this phenomenon. Experiments using neonatal rat ventricular cardiomyocytes showed that live and dead GR-1 bacteria, probiotic-conditioned media, and other probiotic species and strains inhibited the α1-adrenergic receptor agonist phenylephrine-induced hypertrophy as assessed by markers atrial natriuretic peptide and α-skeletal actin. However, using a mutant strain, we showed that this MSP-1 was not required for the inhibition. The ability of factors produced by lactobacilli to improve cardiac function warrants further study for the management of cardiac hypertrophy and heart failure.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Bacterial Proteins; Cardiomegaly; Disease Models, Animal; Heart Failure; Lacticaseibacillus rhamnosus; Myocytes, Cardiac; Phenylephrine; Probiotics; Rats; Rats, Sprague-Dawley

We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro.. Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4.. Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~10. We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Western; Calcium; Cardiomegaly; Cells, Cultured; Culture Media, Conditioned; Cytosol; Disease Models, Animal; DNA; Gene Expression Regulation, Developmental; Humans; Keratins, Hair-Specific; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Microscopy, Atomic Force; Myocytes, Cardiac; Nanoparticles; Rats; Rats, Sprague-Dawley; Rats, Wistar; Signal Transduction

Topics: 3' Untranslated Regions; Angiotensin II; Animals; Atrial Natriuretic Factor; Calmodulin; Cardiomegaly; Cells, Cultured; MicroRNAs; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats; Real-Time Polymerase Chain Reaction; RNA, Messenger

Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-қB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-қB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-қB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-қB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-қB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated.. Inflammation-related signaling is activated by T3 in cardiomyocytes. S100A8 and MyD88 have a crucial role in cardiomyocyte hypertrophy by T3. S100A8 and MyD88 mediate NF-қB activation by T3. NF-қB contributes to T3-induced cardiac hypertrophy in vitro and in vivo.

Topics: Animals; Atrial Natriuretic Factor; Calgranulin A; Cardiomegaly; Cells, Cultured; Male; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Differentiation Factor 88; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B; Rats, Wistar; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 4; Triiodothyronine

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (HETEs) in the development of cardiovascular disease. Mid-chain HETEs have been reported to have vasoconstrictive and pro-inflammatory effects. However, whether mid-chain HETEs can induce cardiac hypertrophy remains unclear. Therefore, the overall objective of the present study was to elucidate the potential hypertrophic effect of mid-chain HETEs in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanisms involved. For this purpose, RL-14 cells were treated with increasing concentrations of mid-chain HETEs (2.5, 5, 10 and 20 µM). Thereafter, the cardiac hypertrophy markers and cell size were determined using real-time polymerase chain reaction and phase contrast imaging, respectively. Phosphorylated mitogen-activated protein kinase (MAPK) level and nuclear factor kappa B (NF-κB) binding activity were determined. Our results showed that mid-chain HETEs induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers, α- and β-myocin heavy chain and atrial and brain natriuretic peptide as well as the increase in cell size. Mechanistically, all mid-chain HETEs were able to induce the binding activity of NF-κB to its responsive element in a HETE-dependent manner, and they significantly induced the phosphorylation of ERK 1/2. The induction of cellular hypertrophy was associated with proportional increase in the formation of dihydroxyeicosatrienoic acids parallel to the increase of soluble epoxide hydrolase enzyme activity. In conclusion, our study provides the first evidence that mid-chain HETEs induce cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Cell Size; Cell Survival; Dose-Response Relationship, Drug; Heart Ventricles; Humans; Hydroxyeicosatetraenoic Acids; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B

DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.

Topics: Animals; Atrial Natriuretic Factor; Basic Helix-Loop-Helix Transcription Factors; Cardiomegaly; Casein Kinase II; Cell Differentiation; Chromatin; Chromatin Assembly and Disassembly; DNA Helicases; DNA-Binding Proteins; GATA4 Transcription Factor; Gene Expression Regulation; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Mice; Myoblasts; Myocytes, Cardiac; Nuclear Proteins; Phosphorylation; Protein Isoforms; Rats; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transcription, Genetic

Cardiovascular (CV) toxicity is an important cause of failure during drug development. Blood-based biomarkers can be used to detect CV toxicity during preclinical development and prioritize compounds at lower risk of causing such toxicities. Evidence of myocardial degeneration can be detected by measuring concentrations of biomarkers such as cardiac troponin I and creatine kinase in blood; however, detection of functional changes in the CV system, such as blood pressure, generally requires studies in animals with surgically implanted pressure transducers. This is a significant limitation because sustained changes in blood pressure are often accompanied by changes in heart rate and together can lead to cardiac hypertrophy and myocardial degeneration in animals, and major adverse cardiovascular events (MACE) in humans. Increased concentrations of NPs in blood correlate with higher risk of cardiac mortality, all-cause mortality, and MACE in humans. Their utility as biomarkers of CV function and toxicity in rodents was investigated by exploring the relationships between plasma concentrations of NTproANP and NTproBNP, blood pressure, heart rate, and heart weight in Sprague Dawley rats administered compounds that caused hypotension or hypertension, including nifedipine, fluprostenol, minoxidil, L-NAME, L-thyroxine, or sunitinib for 1-2 weeks. Changes in NTproANP and/or NTproBNP concentrations were inversely correlated with changes in blood pressure. NTproANP and NTproBNP concentrations were inconsistently correlated with relative heart weights. In addition, increased heart rate was associated with increased heart weights. These studies support the use of natriuretic peptides and heart rate to detect changes in blood pressure and cardiac hypertrophy in short-duration rat studies.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Blood Pressure; Cardiomegaly; Heart Rate; Indoles; Male; Minoxidil; Natriuretic Peptide, Brain; NG-Nitroarginine Methyl Ester; Nifedipine; Organ Size; Peptide Fragments; Prostaglandins F, Synthetic; Pyrroles; Rats; Rats, Sprague-Dawley; Sunitinib; Thyroxine

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; DNA, Mitochondrial; Electron Transport Complex I; Electron Transport Complex II; Enzyme Inhibitors; Gene Expression; Heart; Heart Failure; Hydrogen Peroxide; Mice; Mitochondria, Heart; Myocardium; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Protein Precursors; Pyruvate Dehydrogenase Complex; Reverse Transcriptase Polymerase Chain Reaction; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Vasoconstrictor Agents

Cardiomyocyte hypertrophy is an important structural feature of diabetic cardiomyopathy. Calcineurin/nuclear factor of activated T-cell (NFAT) pathway plays a central role in the pathogenesis of cardiac hypertrophy. The purpose of this study was to investigate the effects of tropisetron, a novel calcineurin inhibitor, on high glucose (HG)-induced cardiomyocyte hypertrophy and its underlying mechanism.. H9c2 myocardial cells were treated with tropisetron or cyclosporine A 1 h before exposure to HG for 48 h.. Exposure to HG resulted in enhanced cell size, protein content and atrial natriuretic peptide (ANP) protein expression. HG significantly increased Ca(2+) level, calcineurin expression and nuclear translocation of NFATc4. Both tropisetron and cyclosporine A markedly prevented the hypertrophic characteristic features, calcineurin overexpression and nuclear localization of NFATc4 while intracellular Ca(2+) was not affected.. Our results showed that tropisetron may have protective effects against HG-induced cardiomyocyte hypertrophy. The mechanism responsible for this beneficial effect seems to be, at least in part, blockade of calcineurin/NFAT signalling pathway.

Topics: Active Transport, Cell Nucleus; Animals; Atrial Natriuretic Factor; Calcineurin; Calcineurin Inhibitors; Calcium; Cardiomegaly; Cell Line; Cell Size; Cytoprotection; Diabetic Cardiomyopathies; Glucose; Indoles; Myocytes, Cardiac; Nerve Tissue Proteins; NFATC Transcription Factors; Rats; Signal Transduction; Tropisetron

Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear.. Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis.. DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine.. The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Topics: Acetyltransferases; Animals; Antineoplastic Agents; Apoptosis; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Cardiomegaly; Caspase 12; Down-Regulation; Eflornithine; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Isoproterenol; Male; Myocytes, Cardiac; Ornithine Decarboxylase; Polyamines; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Transcription Factor CHOP

Backgroud: Myocardial fibrosis results in myocardial remodelling and dysfunction. Celastrol, a traditional oriental medicine, has been suggested to have cardioprotective effects. However, its underlying mechanism is unknown. This study investigated the ability of celastrol to prevent cardiac fibrosis and dysfunction and explored the underlying mechanisms.. Animal and cell models of cardiac fibrosis were used in this study. Myocardial fibrosis was induced by transverse aortic constriction (TAC) in mice. Cardiac hypertrophy and fibrosis were evaluated based on histological and biochemical measurements. Cardiac function was evaluated by echocardiography. The levels of transforming growth factor beta 1 (TGF-β1), extracellular signal regulated kinases 1/2 (ERK1/2) signalling were measured using Western blotting, while the expression of miR-21was analyzed by real-time qRT-PCR in vitro and in vivo. In vitro studies, cultured cardiac fibroblasts (CFs) were treated with TGF-β1 and transfected with microRNA-21(miR21).. Celastrol treatment reduced the increased collagen deposition and down-regulated α-smooth muscle actin (α-SMA), atrial natriuretic peptide (ANP), brain natriuretic peptides (BNP), beta-myosin heavy chain (β-MHC), miR-21 and p-ERK/ERK. Cardiac dysfunction was significantly attenuated by celastrol treatment in the TAC mice model. Celastrol treatment reduced myocardial fibroblast viability and collagen content and down-regulated α-SMA in cultured CFs in vitro. Celastrol also inhibited the miR-21/ERK signalling pathway. Celastrol attenuated miR-21 up-regulation by TGF-β1 and decreased elevated p-ERK/ERK levels in CFs transfected with miR-21.. MiR-21/ERK signalling could be a potential therapeutic pathway for the prevention of myocardial fibrosis. Celastrol ameliorates myocardial fibrosis and cardiac dysfunction, these probably related to miR-21/ERK signaling pathways in vitro and in vivo.

Topics: Actins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Survival; Collagen; Disease Models, Animal; Down-Regulation; Fibrosis; Heart Ventricles; Male; MAP Kinase Signaling System; Mice; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardium; Natriuretic Peptide, Brain; Pentacyclic Triterpenes; Phosphorylation; Transforming Growth Factor beta1; Triterpenes; Up-Regulation

Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA.. The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes.. PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes.. QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.

Topics: Actinin; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Down-Regulation; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Mice; Mice, Inbred C57BL; MicroRNAs; Myocardial Infarction; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Oligonucleotides, Antisense; Phenylephrine; Rats; Rats, Sprague-Dawley

Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5'-AMP-activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p-Akt1), and stimulated nuclear translocation of p-Akt1, to exert their antihypertrophic effects. AMPKα2(-/-) mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2(-/-) mice. The CYP2J2 metabolites, 11,12-EET, activated AMPKα2 to induce nuclear translocation of p-Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co-immunoprecipitation analysis, we found that AMPKα2β2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12-EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure.

Topics: 8,11,14-Eicosatrienoic Acid; AMP-Activated Protein Kinases; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Enzyme Activation; HEK293 Cells; Humans; Mice, Knockout; Myocardium; Myocytes, Cardiac; Organ Specificity; Phosphorylation; Protein Binding; Protein Domains; Protein Subunits; Protein Transport; Proto-Oncogene Proteins c-akt

Resveratrol (Res), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. This study was undertaken to investigate whether Res can protect the heart suffering from hypertrophy injuries induced by isoproterenol (ISO), and whether the protective effect is mediated by endoplasmic reticulum (ER) stress.. Cardiomyocytes were randomly assigned to the control group, ISO group (100 nM ISO for 48 h), Res + ISO group (50 μM Res and 100 nM ISO for 48 h) and Res group (50 μM Res for 48h only). Hypertrophy was estimated by measuring the cell surface area and the atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using Hoechst 33258 staining and transmission electron microscopy. Protein expression of ER stress and apoptosis factors was analyzed using Western Blot analysis.. Res effectively suppress the cardiomyocytes hypertrophy and apoptosis induced by ISO, characterized by the reduction of the myocardial cell surface area, the ANP gene expression, the LDH and MDA leakage amount and the rate of cell apoptosis, while decrease of the protein expression of GRP78, GRP94 and CHOP, and reverse the expression of Bcl-2 and Bax.. In summary, Res treatment effectively suppressed myocardial hypertrophy and apoptosis at least partially via inhibiting ER stress.

Topics: Animals; Animals, Newborn; Antioxidants; Apoptosis; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Blotting, Western; Cardiomegaly; Cardiotonic Agents; Cell Size; Cells, Cultured; Endoplasmic Reticulum Stress; Gene Expression; Heat-Shock Proteins; Isoproterenol; Microscopy, Electron, Transmission; Myocytes, Cardiac; Proto-Oncogene Proteins c-bcl-2; Rats, Wistar; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes

The RNA binding protein Human antigen R (HuR) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes. Specifically, HuR cytoplasmic translocation is significantly increased in NRVMs (neonatal rat ventricular myocytes) following treatment with phenylephrine or angiotensin II, agonists of two independent Gαq-coupled GPCRs known to induce hypertrophy. This Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, we show that HuR activation is necessary for Gq-mediated hypertrophic growth of NRVMs as siRNA-mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). Additionally, HuR overexpression is sufficient to induce hypertrophic cell growth. To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT-luciferase reporter assay, we show an acute inhibition of NFAT transcriptional activity following pharmacological inhibition of HuR. In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Cell Proliferation; ELAV-Like Protein 1; Gene Knockdown Techniques; Myocytes, Cardiac; NFATC Transcription Factors; p38 Mitogen-Activated Protein Kinases; Rats, Sprague-Dawley; Transcriptional Activation

Hydrogen sulfide (H2S) is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel-like factor 5 (KLF5) exerts diverse functions in the cardiovascular system. Whether and how H2S regulates KLF5 in myocardial hypertrophy is unknown.. In our study, hypertrophic myocardial samples in the clinic were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats and neonatal rat cardiomyocytes were studied for functional and signaling responses to GYY4137, an H2S-releasing compound. Expression of cystathionine γ-lyase, a principal enzyme for H2S generation in heart, decreased in human hypertrophic myocardium, whereas KLF5 expression increased. After GYY4137 administration for 4 weeks, myocardial hypertrophy was inhibited in spontaneously hypertensive rats, as demonstrated by improvement in cardiac structural parameters, heart mass, size of cardiac myocytes, and expression of atrial natriuretic peptide. H2S diminished expression of KLF5 in myocardium of spontaneously hypertensive rats and in hypertrophic cardiomyocytes. H2S also inhibits platelet-derived growth factor A promoter activity, decreased recruitment of KLF5 to the platelet-derived growth factor A promoter, and reduced atrial natriuretic peptide expression in angiotensin II-stimulated cardiomyocytes, and these effects are suppressed by KLF5 knockdown. KLF5 promoter activity and KLF5 expression was also reversed by H2S. H2S increased the S-sulfhydration on specificity protein 1 in cardiomyocytes. Moreover, H2S decreased KLF5 promoter activity; reduced KLF5 mRNA expression; attenuated specificity protein 1 binding activity with KLF5 promoter; and inhibited hypertrophy after specificity protein 1 mutated at Cys659, Cys689, and Cys692 but not Cys664 overexpression.. These findings suggest that H2S regulates KLF5 transcription activity via specificity protein 1 S-sulfhydration at Cys664 to prevent myocardial hypertrophy.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Case-Control Studies; Cystathionine gamma-Lyase; Gene Expression Regulation; Humans; Hydrogen Sulfide; Kruppel-Like Transcription Factors; Male; Morpholines; Myocardium; Myocytes, Cardiac; Organothiophosphorus Compounds; Platelet-Derived Growth Factor; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; RNA, Messenger; Sp1 Transcription Factor; Transcriptional Activation

The role of microRNA-214-3p (miR-214-3p) in cardiac hypertrophy was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced mouse cardiac hypertrophy. In mice with either Ang-II infusion or transverse aortic constriction (TAC) model, miR-214-3p expression was markedly decreased in the hypertrophic myocardium. Down-regulation of miR-214-3p was observed in the myocardium of patients with cardiac hypertrophy. Expression of miR-214-3p was upregulated in Ang-II-induced hypertrophic neonatal mouse ventricular cardiomyocytes. Cardiac hypertrophy was attenuated in Ang-II-infused mice by tail vein injection of miR-214-3p. Moreover, miR-214-3p inhibited the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) in Ang-II-treated mouse cardiomyocytes in vitro. Myocyte-specific enhancer factor 2C (MEF2C), which was increased in Ang-II-induced hypertrophic mouse myocardium and cardiomyocytes, was identified as a target gene of miR-214-3p. Functionally, miR-214-3p mimic, consistent with MEF2C siRNA, inhibited cell size increase and protein expression of ANP and β-MHC in Ang-II-treated mouse cardiomyocytes. The NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in cardiomyocytes. Taken together, our results revealed that MEF2C is a novel target of miR-214-3p, and attenuation of miR-214-3p expression may contribute to MEF2Cexpressionin cardiac hypertrophy.

Topics: Angiotensin II; Animals; Antagomirs; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Heart Ventricles; Male; MEF2 Transcription Factors; Mice; Mice, Inbred C57BL; MicroRNAs; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; NF-kappa B; RNA Interference; Signal Transduction; Up-Regulation

Cucurbitacin-I, a natural triterpenoids initially identified in medicinal plants, shows a potent anticancer effect on a variety of cancer cell types. Nevertheless, the cardiotoxicity of cucurbitacin-I has not heretofore been reported. In this study, the mechanisms of cucurbitacin-I-induced cardiotoxicity were examined by investigating the role of MAPK-autophagy-dependent pathways. After being treated with 0.1-0.3μM cucurbitacin-I for 48h, H9c2 cells showed a gradual decrease in the cell viabilities, a gradual increase in cell size, and mRNA expression of ANP and BNP (cardiac hypertrophic markers). Cucurbitacin-I concentration-dependent apoptosis of H9c2 cells was also observed. The increased apoptosis of H9c2 cells was paralleling with the gradually strong autophagy levels. Furthermore, an autophagy inhibitor, 3-MA, was used to block the cucurbitacin-I-stirred autophagy, and then the hypertrophy and apoptosis induced by 0.3μM cucurbitacin-I were significantly attenuated. In addition, cucurbitacin-I exposure also activated the MAPK signaling pathways, including ERK1/2, JNK, and p38 kinases. Interestingly, only the ERK inhibitor U0126, but not the JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580, weakened the induction of 0.3μM cucurbitacin-I in hypertrophy, autophagy and apoptosis. Our findings suggest that cucurbitacin-I can increase the autophagy levels of H9c2 cells, most likely, through the activation of an ERK-autophagy dependent pathway, which results in the hypertrophy and apoptosis of cardiomyocytes.

Topics: Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Cell Size; Cell Survival; Humans; Imidazoles; MAP Kinase Kinase 4; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Myoblasts, Cardiac; Natriuretic Peptide, Brain; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Triterpenes

Recent evidence suggests that polydatin (PD), a resveratrol glucoside, may have beneficial actions on the cardiac hypertrophy. Therefore, the current study focused on the underlying mechanism of the PD anti-hypertrophic effect in cultured cardiomyocytes and in progression from cardiac hypertrophy to heart failure in vivo. Experiments were performed on cultured neonatal rat, ventricular myocytes as well as adult mice subjected to transverse aortic constriction (TAC). Treatment of cardiomyocytes with phenylephrine for three days produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and atrial natriuretic peptide (ANP) protein expression. These effects were attenuated by PD in a concentration-dependent manner with a complete inhibition of hypertrophy at the concentration of 50 µM. Phenylephrine increased ROCK activity, as well as intracellular reactive oxygen species production and lipid peroxidation. The oxidizing agent DTDP similarly increased Rho kinase (ROCK) activity and induced hypertrophic remodeling. PD treatment inhibited phenylephrine-induced oxidative stress and consequently suppressed ROCK activation in cardiomyocytes. Hypertrophic remodeling and heart failure were demonstrated in mice subjected to 13 weeks of TAC. Upregulation of ROCK signaling pathway was also evident in TAC mice. PD treatment significantly attenuated the increased ROCK activity, associated with a markedly reduced hypertrophic response and improved cardiac function. Our results demonstrated a robust anti-hypertrophic remodeling effect of polydatin, which is mediated by inhibition of reactive oxygen species dependent ROCK activation.

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cell Size; Cells, Cultured; Disease Models, Animal; Drugs, Chinese Herbal; Glucosides; Heart Failure; Heart Ventricles; Male; Mice, Inbred C57BL; Oxidative Stress; Phenylephrine; Rats; rho-Associated Kinases; Stilbenes; Ventricular Remodeling

Hypertrophic growth of cardiomyocytes in response to pressure overload is an important stage during the development of many cardiac diseases. Ca(2+) overload as well as subsequent activation of Ca(2+) signaling pathways has been reported to induce cardiac hypertrophy. Myocardin, a transcription cofactor of serum response factor (SRF), is a key transducer of hypertrophic signals. However, the direct role of myocardin in Ca(2+) signal-induced cardiomyocyte hypertrophy has not been explained clearly. In the present study, we discovered that embryonic rat heart-derived H9c2 cells responded to the stimulation of calcium ionophore A23187 with a cell surface area enlargement and an increased expression of cardiac hypertrophy marker genes. Increased Ca(2+) also induces an organization of sarcomeres in neonatal rat cardiomyocytes, as revealed by α-actinin staining. Increased Ca(2+) could upregulate the expression of myocardin. Knockdown of myocardin by shRNA attenuates hypertrophic responses triggered by increased intracellular Ca(2+), suggesting that Ca(2+) signals induce cardiomyocyte hypertrophy partly through activation of myocardin. Furthermore, A23187 treatment directly activates myocardin promoter, chelation of Ca(2+) by EGTA inhibits this activation and knockdown of myocardin expression using shRNA also abrogates A23187-induced ANF and SK-α-actin promoter activity. CSA (calcineurin inhibitor) and KN93 (CaMKII inhibitor) inhibit A23187-induced the increase in myocardin expression. These results suggest that myocardin plays a critical role in Ca(2+) signal-induced cardiomyocyte hypertrophy, which may serve as a novel mechanism that is important for cardiac hypertrophy.

Topics: Actins; Animals; Atrial Natriuretic Factor; Calcimycin; Calcineurin Inhibitors; Calcium; Calcium Chelating Agents; Calcium Ionophores; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cell Line; Egtazic Acid; Enzyme Activation; Myocytes, Cardiac; Nuclear Proteins; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Sarcomeres; Trans-Activators; Transcriptional Activation

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [(3)H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Cells, Cultured; Glucosides; Male; Myocardium; Myocytes, Cardiac; NADPH Oxidases; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Resveratrol; RNA, Messenger; Signal Transduction; Stilbenes; Superoxides; Ventricular Myosins

Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.

Topics: Animals; Antioxidants; Atrial Natriuretic Factor; Butadienes; Buthionine Sulfoximine; Cardiomegaly; Cell Line; Cell Survival; Enzyme Activation; Epoxide Hydrolases; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; Glutathione; Heart Failure; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Myoblasts, Cardiac; Natriuretic Peptide, Brain; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proline; Rats; RNA, Messenger; Thiocarbamates; Transcription Factor RelA; Up-Regulation

The orphan nuclear receptor NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, and is involved in glucose and fat metabolism. However, its potential contribution to cardiovascular diseases remains to be assessed. Here, the roles of NOR1 in cardiac hypertrophy induced by isoprenaline and the underlying molecular mechanisms were investigated.. NOR1 was expressed in cardiomyocytes treated with isoprenaline. After NOR1 overexpression or knockdown in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA of hypertrophic biomarkers. Interactions between NOR1 and PARP-1 were investigated by co-immunoprecipitation. NOR1 expression and PARP-1 activity were measured in rats with cardiac hypertrophy induced by isoprenaline.. Treatment with isoprenaline significantly up-regulated NOR1 expression and PARP-1 activity both in vivo and in vitro. Specific gene silencing of NOR1 attenuated isoprenaline-induced cardiomyocyte hypertrophy, whereas NOR1 overexpression exacerbated cardiac hypertrophy. We identified a physical interaction between NOR1 and PARP-1, which was enhanced by NOR1 transfection and thereby led to PARP-1 activation. Overexpression of NOR1, but not C293Y, a NOR1 mutant lacking the PARP-1 binding activity, increased cellular surface area and the mRNA levels of atrial natriuretic factor and brain natriuretic polypeptide, effects blocked by the PARP-1 inhibitor 3-aminobenzamide or siRNA for PARP-1.. This is the first evidence that NOR1 was involved in isoprenaline-induced cardiac hypertrophy. The pro-hypertrophic effect of NOR1 can be partly attributed to its regulation of PARP-1 enzymic activity.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; DNA-Binding Proteins; Gene Knock-In Techniques; Gene Knockdown Techniques; Immunoprecipitation; In Vitro Techniques; Isoproterenol; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; RNA, Messenger; Sympathomimetics; Up-Regulation

Hydrogen peroxide (H2O2) causes cell damage via oxidative stress. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that can protect cardiomyocytes against oxidative stress. In this study, we investigated whether the heme precursor 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) could protect cardiomyocytes from H2O2-induced hypertrophy via modulation of HO-1 expression. HL-1 cells pretreated with/without 5-ALA and SFC were exposed to H2O2 to induce a cardiomyocyte hypertrophy model. Hypertrophy was evaluated by planar morphometry, (3)H-leucine incorporation, and RT-PCR analysis of hypertrophy-related gene expressions. Reactive oxygen species (ROS) production was assessed by 5/6-chloromethyl-2',7'-ichlorodihydrofluorescein diacetate acetylester. HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expressions were analyzed by Western blot. In our experiments, HL-1 cells were transfected with Nrf2 siRNA or treated with a signal pathway inhibitor. We found several results. 1) ROS production, cell surface area, protein synthesis, and expressions of hypertrophic marker genes, including atrial natriuretic peptide, brain natriuretic peptide, atrial natriuretic factor, and β-myosin heavy chain, were decreased in HL-1 cells pretreated with 5-ALA and SFC. 2) 5-ALA and SFC increased HO-1 expression in a dose- and time-dependent manner, associated with upregulation of Nrf2. Notably, Nrf2 siRNA dramatically reduced HO-1 expression in HL-1 cells. 3) ERK1/2, p38, and SAPK/JNK signaling pathways were activated and modulate 5-ALA- and SFC-enhanced HO-1 expression. SB203580 (p38 kinase), PD98059 (ERK), or SP600125 (JNK) inhibitors significantly reduced this effect. In conclusion, our data suggest that 5-ALA and SFC protect HL-1 cells from H2O2-induced cardiac hypertrophy via activation of the MAPK/Nrf2/HO-1 signaling pathway.

Topics: Aminolevulinic Acid; Animals; Antioxidants; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Citric Acid; Ferrous Compounds; Heme Oxygenase-1; Hydrogen Peroxide; Membrane Proteins; Mice; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; NF-E2-Related Factor 2; Oxidative Stress; RNA Interference; RNA, Small Interfering; Signal Transduction; Ventricular Myosins

Cyclic nucleotides are second messengers that regulate cardiomyocyte function through compartmentalized signaling in discrete subcellular microdomains. However, the role of different microdomains and their changes in cardiac disease are not well understood.. To directly visualize alterations in β-adrenergic receptor-associated cAMP and cGMP microdomain signaling in early cardiac disease.. Unexpectedly, measurements of cell shortening revealed augmented β-adrenergic receptor-stimulated cardiomyocyte contractility by atrial natriuretic peptide/cGMP signaling in early cardiac hypertrophy after transverse aortic constriction, which was in sharp contrast to well-documented β-adrenergic and natriuretic peptide signaling desensitization during chronic disease. Real-time cAMP analysis in β1- and β2-adrenergic receptor-associated membrane microdomains using a novel membrane-targeted Förster resonance energy transfer-based biosensor transgenically expressed in mice revealed that this unexpected atrial natriuretic peptide effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between both receptor compartments. Functionally, this led to a significant shift in cGMP/cAMP cross-talk and, in particular, to cGMP-driven augmentation of contractility in vitro and in vivo.. Redistribution of cGMP-regulated phosphodiesterases and functional reorganization of receptor-associated microdomains occurs in early cardiac hypertrophy, affects cGMP-mediated contractility, and might represent a previously not recognized therapeutically relevant compensatory mechanism to sustain normal heart function.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Biosensing Techniques; Cardiomegaly; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide Phosphodiesterases, Type 3; Disease Models, Animal; Enzyme Activation; Female; Fluorescence Resonance Energy Transfer; Guanine Nucleotide Exchange Factors; Isoproterenol; Membrane Microdomains; Mice; Mice, Transgenic; Myocardial Contraction; Myocytes, Cardiac; Protein Transport; Receptor Cross-Talk; Receptors, Adrenergic, beta; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Second Messenger Systems; Time Factors

We previously reported that Astragaloside IV (ASIV), a major active constituent of Astragalus membranaceus (Fisch) Bge protects against cardiac hypertrophy in rats induced by isoproterenol (Iso), however the mechanism underlying the protection remains unknown. Dysfunction of cardiac energy biosynthesis contributes to the hypertrophy and Nuclear Factor κB (NF-κB)/Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α (PGC-1α) signaling gets involved in the dysfunction. The present study was designed to investigate the mechanism by which ASIV improves the cardiac hypertrophy with focuses on the NF-κB/PGC-1α signaling mediated energy biosynthesis. Sprague-Dawley (SD) rats or Neonatal Rat Ventricular Myocytes (NRVMs) were treated with Iso alone or in combination with ASIV. The results showed that combination with ASIV significantly attenuated the pathological changes, reduced the ratios of heart weight/body weight and Left ventricular weight/body weight, improved the cardiac hemodynamics, down-regulated mRNA expression of Atrial Natriuretic Peptide (ANP) and Brain Natriuretic Peptide (BNP), increased the ratio of ATP/AMP, and decreased the content of Free Fat Acid (FFA) in heart tissue of rats compared with Iso alone. In addition, pretreatment with ASIV significantly decreased the surface area and protein content, down-regulated mRNA expression of ANP and BNP, increased the ratio of ATP/AMP, and decreased the content of FFA in NRVMs compared with Iso alone. Furthermore, ASIV increased the protein expression of ATP5D, subunit of ATP synthase and PGC-1α, inhibited translocation of p65, subunit of NF-κB into nuclear fraction in both rats and NRVMs compared with Iso alone. Parthenolide (Par), the specific inhibitor of p65, exerted similar effects as ASIV in NRVMs. Knockdown of p65 with siRNA decreased the surface areas and increased PGC-1α expression of NRVMs compared with Iso alone. The results suggested that ASIV protects against Iso-induced cardiac hypertrophy through regulating NF-κB/PGC-1α signaling mediated energy biosynthesis.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Down-Regulation; Energy Metabolism; Gene Knockdown Techniques; Heart Ventricles; Hemodynamics; Isoproterenol; Male; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats; Rats, Sprague-Dawley; RNA, Messenger; RNA, Small Interfering; Saponins; Signal Transduction; Transcription Factor RelA; Transcription Factors; Triterpenes

Adipose stem cells (ASCs) are a source of regenerative cells available for autologous transplantation to hearts. We compared protective actions of ASC sheets on rat myocardial infarction (MI) in comparison with those of skeletal myoblast cell sheets. Their effects on infarcted hearts were evaluated by biological, histochemical as well as physiological analyses. ASC sheets secreted higher concentrations of angiogenic factors (HGF, VEGF, and bFGF; P < 0.05) under normoxic and hypoxic conditions than those of myoblast cell sheets, associated with reduction of cell apoptosis (P < 0.05). Like myoblast cell sheets, ASC sheets improved cardiac function (P < 0.05) and decreased the plasma level of ANP (P < 0.05) in MI hearts. ASC sheets restored cardiac remodeling characterized by fibrosis, cardiac hypertrophy and impaired angiogenesis (P < 0.05), which was associated with increases in angiogenic factors (P < 0.05). In isolated perfused rat hearts, ASC sheets improved both systolic and diastolic functions, which was comparable to cardiac functions of myoblast cell sheets, while both cell sheets failed to restore cardiac contractile response to either isoproterenol, pimobendan or dibutyryl cAMP. These results indicated that ASC sheets improved cardiac function and remodeling of MI hearts mediated by their paracrine action and this improvement was comparable to those by myoblast cell sheets.

Topics: Adipocytes; Animals; Atrial Natriuretic Factor; Atrial Remodeling; Bucladesine; Cardiomegaly; Cell Differentiation; Diastole; Fibroblast Growth Factors; Hepatocyte Growth Factor; Isoproterenol; Male; Myoblasts, Skeletal; Myocardial Contraction; Myocardial Infarction; Myocardium; Organ Culture Techniques; Paracrine Communication; Pyridazines; Rats; Rats, Inbred Lew; Stem Cell Transplantation; Stem Cells; Systole; Vascular Endothelial Growth Factor A

Pathological cardiac hypertrophy, a common response of the heart to a variety of cardiovascular diseases, is typically associated with myocytes remodeling and fibrotic replacement, cardiac dysfunction. Exercise preconditioning (EP) increases the myocardial mechanical load and enhances tolerance of cardiac ischemia-reperfusion injury (IRI), however, is less reported in pathological cardiac hypertrophy. To determine the effect of EP in pathological cardiac hypertrophy, Male 10-wk-old Sprague-Dawley rats (n=30) were subjected to 4 weeks of EP followed by 4-8 weeks of pressure overload (transverse aortic constriction, TAC) to induce pathological remodeling. TAC in untrained controls (n=30) led to pathological cardiac hypertrophy, depressed systolic function. We observed that left ventricular wall thickness in end diastole, heart size, heart weight-to-body weight ratio, heart weight-to-tibia length ratio, cross-sectional area of cardiomyocytes and the reactivation of fetal genes (atrial natriuretic peptide and brain natriuretic peptide) were markedly increased, meanwhile left ventricular internal dimension at end-diastole, systolic function were significantly decreased by TAC at 4 wks after operation (P < 0.01), all of which were effectively inhibited by EP treatment (P < 0.05), but the differences of these parameters were decreased at 8 wks after operation. Furthermore, EP treatment inhibited degradation of IκBα, and decreased NF-κB p65 subunit levels in the nuclear fraction, and then reduced IL2 levels in the myocardium of rats subject to TAC. EP can effectively attenuate pathological cardiac hypertrophic responses induced by TAC possibly through inhibition of degradation of IκB and blockade of the NF-κB signaling pathway in the early stage of pathological cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; I-kappa B Proteins; Male; Myocardium; Natriuretic Peptide, Brain; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; Signal Transduction

Proteinuria is a hallmark of chronic kidney disease (CKD) and cardiovascular disease (CVD), and a good predictor of clinical outcome. Selective endothelin A (ETA) receptor antagonist used with renin-angiotensin system (RAS) inhibitors prevents development of proteinuria in CKD. However, whether the improvement in proteinuria would have beneficial effects on CVD, independent of RAS inhibition, is not well understood. In this study, we investigated whether atrasentan, an ETA receptor antagonist, has renal and cardiovascular effects independent of RAS inhibition. Male Dahl salt sensitive (DSS) rats, at six weeks of age, received water with or without different doses of atrasentan and/or enalapril under high salt (HS) diet or normal diet (ND) for 6 weeks. At the end of 12th week, atrasentan at a moderate dose significantly attenuated proteinuria and serum creatinine without reducing mean arterial pressure (MAP), thereby preventing cardiac hypertrophy and improving cardiac function. ACE inhibitor enalapril at a dose that did not significantly lowered BP, attenuated cardiac hypertrophy while moderately improving cardiac function without reducing proteinuria and serum creatinine level. Nonetheless, combined therapy of atrasentan and enalapril that does not altering BP exerted additional cardioprotective effect. Based on these findings, we conclude that BP independent monotherapy of ETA receptor antagonist attenuates the progression of CKD and significantly mitigates CVD independent of RAS inhibition.

Topics: Animals; Atrasentan; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Disease Models, Animal; Echocardiography; Endothelin A Receptor Antagonists; Gene Expression; Heart Diseases; Hemodynamics; Hypertension; Hypertrophy; Kidney Function Tests; Male; Myocytes, Cardiac; Phenylephrine; Pyrrolidines; Rats; Receptor, Endothelin A; Renal Insufficiency, Chronic

Cardiac hypertrophy and myocardial fibrosis significantly contribute to the pathogenesis of diabetic cardiomyopathy (DCM). Altered expression of several genes and their regulation by microRNAs has been reported in hypertrophied failing hearts. This study aims to examine the role of Cdc42, Pak1, and miR-30c in the pathogenesis of cardiac hypertrophy in DCM.. DCM was induced in Wistar rats by low-dose streptozotocin-high-fat diet for 12 weeks. Cardiac expression of Cdc42, Pak1 and miR-30c, and hypertrophy markers (ANP and β-MHC) was studied in DCM vs control rats and in high-glucose (HG)-treated H9c2 cardiomyocytes.. Diabetic rats showed cardiomyocyte hypertrophy, increased heart-to-body weight ratio, and an increased expression of ANP and β-MHC. Cardiac expression of Cdc42 and Pak1 genes was increased in diabetic hearts and in HG-treated cardiomyocytes. miR-30c was identified to target Cdc42 and Pak1 genes, and cardiac miR-30c expression was found to be decreased in DCM rats, patients with DCM, and in HG-treated cardiomyocytes. miR-30c overexpression decreased Cdc42 and Pak1 genes and attenuated HG-induced cardiomyocyte hypertrophy, whereas miR-30c inhibition increased Cdc42 and Pak1 gene expression and myocyte hypertrophy in HG-treated cardiomyocytes.. Downregulation of miR-30c mediates prohypertrophic effects of hyperglycemia in DCM by upregulation of Cdc42 and Pak1 genes.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Myosins; Cardiomegaly; cdc42 GTP-Binding Protein; Cell Line; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; MicroRNAs; Myocytes, Cardiac; Myosin Heavy Chains; p21-Activated Kinases; Rats; Rats, Wistar; Streptozocin; Up-Regulation

3,3'-Diindolylmethane (DIM) is the major product of the acid-catalyzed condensation of indole-3-carbinol (I3C), a component of extracts of Brassica food plants. Numerous studies have suggested that DIM has several beneficial biological activities, including elimination of free radicals, antioxidant and anti-angiogenic effects and activation of apoptosis of various tumor cells. In the present study, an in vitro model was established, using 1 µM angiotensin II (Ang II) in cultured rat cardiac H9c2 cells, to observe the effects of DIM on cardiac hypertrophy. Following 24 h stimulation with DIM (1, 5, and 10 µM) with or without Ang II, cells were characterized by immunofluorescence to analyze cardiac α-actinin expression. Cardiomyocyte hypertrophy and molecular markers of cardiac hypertrophy were assessed by quantitative polymerase chain reaction. Atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain β mRNA expression were induced by Ang II in H9c2 cells treated with the optimal concentration of DIM for 6, 12, and 24 h. The levels of phosphorylated and total proteins of the 5' AMP-activated protein kinase α (AMPKα)/mitogen-activated protein kinase (MAPK)/mechanistic target of rapamycin (mTOR) signaling pathways in H9c2 cells treated with DIM for 0, 15, 30, and 60 min induced by Ang II were determined by western blot analysis. The results showed that DIM attenuated cellular hypertrophy in vitro, enhanced the phosphorylation of AMPKα and inhibited the MAPK‑mTOR signaling pathway in response to hypertrophic stimuli.

Topics: AMP-Activated Protein Kinases; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cell Line; Gene Expression Regulation; Indoles; Models, Biological; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phosphorylation; Rats; Signal Transduction; TOR Serine-Threonine Kinases; Vasoconstrictor Agents

Angiotensin II (Ang II) is an important inflammatory mediator. Ang II induces cyclooxygenase-2 (COX-2) expression and prostaglandin F2α release followed by cardiac hypertrophy. Inhibition of COX-2 may modulate high blood pressure but controversy still exists. The aim of this study was to determine the role of COX-2 in the regulation of blood pressure and to define the mechanisms in two kidney one-clip hypertensive (2K1C) rats. Chronic treatment with nimesulide or NS-398 (5 mg/kg/day) for 3 weeks lowered high blood pressure and cardiac hypertrophy with decreased expression levels of cardiac hypertrophy markers [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP)], Ang type 1 receptor, urotensin II, and urotensin II receptor in 2K1C rats. Plasma level of ANP was markedly increased and plasma levels of Ang II and aldosterone were decreased by treatment with nimesulide or NS-398. In both in vitro and in vivo experiments, nimesulide or NS-398 augmented ANP release in 2K1C rats. The inhibitory effect of NS-398 on blood pressure was attenuated by the pretreatment with natriuretic peptide receptor-A (NPR-A) antagonist (A71915, 30 μg/kg/day). These results suggest that chronic treatment with nimesulide or NS-398 attenuated hypertension and cardiac hypertrophy partly through ANP release in 2K1C rats.

Topics: Aldosterone; Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Hypertension, Renovascular; Natriuretic Peptide, Brain; Nitrobenzenes; Rats; Sulfonamides

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Female; Isoproterenol; Membrane Microdomains; Myocardial Contraction; Myocytes, Cardiac; Receptors, Adrenergic, beta

Intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is involved in maintaining circulatory homeostasis and is a protective factor of heart and vessel. Here, we investigated the effects of IMD on cardiac hypertrophy in vivo and in vitro and explored the mechanisms involved.. IMD1-53 (100 ng/kg/h) was systemically administered to rats with cardiac hypertrophy induced by abdominal aortic constriction (AAC) by a mini-osmotic pump the next day after surgery continuously for 4 weeks. The AAC-treated rats before IMD infusion showed increased IMD content and expression of its receptors in the hearts. In-vivo administration of IMD1-53 greatly attenuated the cardiac hypertrophy as shown by heart weight to body weight ratio (HW/BW), haemodynamics, echocardiography, histological analyses and expression of hypertrophic markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) induced by AAC. IMD1-53 treatment significantly reduced the myocardial protein expression of endoplasmic reticulum stress (ERS) markers such as glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-12, whereas the protein level of phosphorylated AMP-activated protein kinase (p-AMPK) was upregulated with IMD1-53 treatment, which was further confirmed in cultured cardiomyocytes. Concurrently, cardiomyocyte apoptosis in vivo and in vitro was ameliorated by IMD1-53 treatment. The inhibitory effects of IMD1-53 on ERS and apoptosis were eliminated on pretreatment with compound C, an AMPK inhibitor.. IMD1-53 could exert its cardioprotective effect on cardiac hypertrophy by inhibiting myocardial ERS and apoptosis, possibly via activation of AMPK signalling.

Topics: Adrenomedullin; AMP-Activated Protein Kinases; Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Caspase 12; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Echocardiography; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Male; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Neuropeptides; Organ Size; Phosphorylation; Rats

ApoE-/- mice fed a high fat and high cholesterol (HFHC) diet (20% fat and 1.25% cholesterol) from 12 weeks of age to 36 weeks revealed an age-dependent increase in the left ventricular mass (LV mass) and decline in fractional shortening (FS%), which worsened with HFHC diet. These traits are indicative of maladaptive pathological cardiac hypertrophy and dysfunction. This was accompanied by loading of glycosphingolipids and increased gene expression of ANP, BNP in myocardial tissue. Masson's trichrome staining revealed a significant increase in cardiomyocyte size and fibrosis. In contrast, treatment with 5 and 10 μM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, dose-dependently decreased the load of glycosphingolipids and preserved fractional shortening and maintained left ventricular mass to normal 12-week-old control levels over a 6 month treatment period. Our mechanistic studies showed that D-PDMP inhibited cardiac hypertrophy by inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK). We propose that associating increased glycosphingolipid synthesis with cardiac hypertrophy could serve as a novel approach to prevent this phenotype in experimental animal models of diet -induced atherosclerotic heart disease.

Topics: Animals; Apolipoproteins E; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cholesterol; Diet, High-Fat; Enzyme Inhibitors; Galactosyltransferases; Gene Expression; Glucosyltransferases; Glycosphingolipids; Heart Ventricles; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Morpholines; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phosphorylation

A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25mM glucose, but when the concentration of glucose was increased to 35mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.

Topics: Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression Regulation; Gene Knockdown Techniques; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Myocytes, Cardiac; RNA Interference; SOX9 Transcription Factor; Up-Regulation

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes.

Topics: Actins; Atrial Natriuretic Factor; Carbon Dioxide; Cardiomegaly; Cells, Cultured; Cyclooxygenase 2; Cytoprotection; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme Inhibitors; Heme Oxygenase-1; Humans; Myocytes, Cardiac; Natriuretic Peptide, Brain; Organometallic Compounds; Primary Cell Culture; RNA Interference; Signal Transduction; Thrombin; Time Factors; Transfection

To investigate the effect of peroxisiome proliferator activated receptor-α (PPAR-α) on the regulation of cardiomyocyte hypertrophy and the relationship between the effect of PPAR-α with PI3K/Akt//mTOR signal pathway.. Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and PPAR-α mRNA were detected by qRT-PCR. The protein expressions of Akt, mTOR and P70S6K were detected by Western blot. The expression of PPAR-α was suppressed by RNAi.. (1) The expression of PPAR-α was significantly reduced in cardiomyocyte hypertrophy. PPAR-α activator Fenofibrate (Feno) increased the expression of PPAR-α and suppressed cardiomyocyte hypertrophy. The inhibitory effect of Feno on cardiomyocyte hypertrophy was reversed by PPAR-α RNAi. (2) Feno significantly inhibited the increase of the protein expressions of p-Akt, p-mTOR and p-p70S6K in ISO induced cardiomyocyte hypertrophy, which could be blocked by PPAR-α RNAi. (3) PI3K antagonist LY294002 (LY) or mTOR antagonist rapamycin (RAPA) markedly-inhibited cardiomyocyte hypertrophy. The inhibitory effects of LY or RAPA on cardiomyocyte hypertrophy were reversed by PPAR-α RNAi.. PPAR-α can negatively regulate cardiomyocyte hypertrophy. The effect might be associated with PPAR-α inhiting PI3K/ Akt/mTOR signal pathway.

Topics: Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Fenofibrate; Humans; Isoproterenol; Myocytes, Cardiac; Myosin Heavy Chains; Phosphatidylinositol 3-Kinases; PPAR alpha; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; TOR Serine-Threonine Kinases

Oxytocin and its receptor are synthesised in the heart and blood vessels but effects of chronic activation of this peripheral oxytocinergic system on cardiovascular function are not known. In acute studies, systemic administration of low dose oxytocin exerted a protective, preconditioning effect in experimental models of myocardial ischemia and infarction. In this study, we investigated the effects of chronic administration of low dose oxytocin following angiotensin II-induced hypertension, cardiac hypertrophy and renal damage. Angiotensin II (40 μg/Kg/h) only, oxytocin only (20 or 100 ng/Kg/h), or angiotensin II combined with oxytocin (20 or 100 ng/Kg/h) were infused subcutaneously in adult male Sprague-Dawley rats for 28 days. At day 7, oxytocin or angiotensin-II only did not change hemodynamic parameters, but animals that received a combination of oxytocin and angiotensin-II had significantly elevated systolic, diastolic and mean arterial pressure compared to controls (P < 0.01). Hemodynamic changes were accompanied by significant left ventricular cardiac hypertrophy and renal damage at day 28 in animals treated with angiotensin II (P < 0.05) or both oxytocin and angiotensin II, compared to controls (P < 0.01). Prolonged oxytocin administration did not affect plasma concentrations of renin and atrial natriuretic peptide, but was associated with the activation of calcium-dependent protein phosphatase calcineurin, a canonical signalling mechanism in pressure overload-induced cardiovascular disease. These data demonstrate that oxytocin accelerated angiotensin-II induced hypertension and end-organ renal damage, suggesting caution should be exercised in the chronic use of oxytocin in individuals with hypertension.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Hypertension; Injections, Subcutaneous; Kidney; Male; Oxytocin; Rats; Rats, Sprague-Dawley; Renin

Pregnancy induces cardiovascular adaptations in response to increased volume overload. Aside from the hemodynamic changes that occur during pregnancy, the maternal heart also undergoes structural changes. However, cardiac modulation in pregnancies complicated by gestational hypertension is incompletely understood. The objectives of the current investigation were to determine the role of the natriuretic peptide (NP) system in pregnancy and to assess alterations in pregnancy-induced cardiac hypertrophy between gestationally hypertensive and normotensive dams. Previously we have shown that mice lacking the expression of atrial NP (ANP; ANP(-/-)) exhibit a gestational hypertensive phenotype. In the current study, female ANP(+/+) and ANP(-/-) mice were mated with ANP(+/+) males. Changes in cardiac size and weight were evaluated across pregnancy at Gestational Days 15.5 and 17.5 and Postnatal Days 7, 14, and 28. Nonpregnant mice were used as controls. Physical measurement recordings and histological analyses demonstrated peak cardiac hypertrophy occurring at 14 days postpartum in both ANP(+/+) and ANP(-/-) dams with little to no change during pregnancy. Additionally, left ventricular expression of the renin-angiotensin system (RAS) and NP system was quantified by real-time quantitative polymerase chain reaction. Up-regulation of Agt and AT(1a) genes was observed late in pregnancy, while Nppa and Nppb genes were significantly up-regulated postpartum. Our data suggest that pregnancy-induced cardiac hypertrophy may be influenced by the RAS throughout gestation and by the NP system postpartum. Further investigations are required to gain a complete understanding of the mechanistic aspects of pregnancy-induced cardiac hypertrophy.

Topics: Angiotensins; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Disease Models, Animal; Female; Hypertension, Pregnancy-Induced; Mice; Mice, Knockout; Pregnancy; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Up-Regulation

The expression of Nppa (ANF) and Nppb (BNP) marks the chamber myocardium in the embryo, and both genes serve as early and accurate markers for hypertrophy and heart failure. Non-invasive visualization of Nppa-Nppb expression in living mice would enable to evaluate the disease state during the course of time in heart disease models. We sought to develop a method to assess the pattern and level of Nppa and Nppb expression within living mice.. A modified bacterial artificial chromosome containing a genomic segment spanning the Nppa-Nppb locus was randomly integrated into the mouse genome. Firefly Luciferase was inserted into Nppa and the red fluorescent protein gene Katushka into Nppb. Both reporters precisely recapitulated the spatio-temporal patterns of Nppa and Nppb, respectively. In a hypertrophy model (transverse aortic constriction) and myocardial infarction model (left anterior descending coronary artery occlusion), the non-invasively measured bioluminescent signal from Luciferase correlated with Nppa expression, and the intensity of red fluorescence with levels of the expression of Katushka and Nppb. After myocardial infarction, the border zone of the infarct area was readily identified by an increased intensity of Katushka fluorescence.. A genomic region sufficient to regulate the developmental pattern and stress response of Nppa and Nppb has been defined. The double reporter mice can be used for the functional imaging and investigation of cardiac hypertrophy and myocardial infarction in vivo.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Chromosomes, Artificial, Bacterial; Disease Models, Animal; Genes, Reporter; Heart Ventricles; Luminescent Proteins; Mice; Mice, Transgenic; Myocardial Infarction; Natriuretic Peptide, Brain; Up-Regulation

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Genes, Reporter; Myocardial Infarction; Natriuretic Peptide, Brain

Astragaloside IV is widely used for the treatment of cardiovascular diseases in China. However, its role in cardiac hypertrophy remains unclear. In this study, we aim to determine the protective effects of astragaloside IV on myocardial hypertrophy induced by lipopolysaccharide and to identify their precise molecular and cellular mechanisms. Cell size, reorganization of actin filaments, and ANP and BNP mRNA expression were used as indices of hypertrophy; CaN and GATA-4 expression and the distribution of NFAT-3 in both cytoplasm and nucleus were determined by Western blot analysis; Ca2+ transient in Fura-2/AM-loaded cells was measured by Till image system. Our data demonstrated that lipopolysaccharide challenge induced cardiac hypertrophy, increased resting Ca2+ transient level, promoted activation of CaN and GATA-4, and enhanced nuclear translocation of NFAT-3. Administration of astragaloside IV (16, 32, and 64 µM) 1 h prior to lipopolysaccharide stimulation dose-dependently attenuated cardiac hypertrophy induced by lipopolysaccharide. Further studies demonstrated that astragaloside IV inhibited the increment of the resting intracellular free Ca2+, and its effect was similar to verapamil. Moreover, astragaloside IV also inhibited the activation of CaN and GATA-4, and the nuclear translocation of NFAT-3 induced by lipopolysaccharide. In conclusion, our results revealed that astragaloside IV had the potential to protect against cardiac hypertrophy through Ca2+-mediated CaN signaling pathways.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Calcium Signaling; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Drugs, Chinese Herbal; Lipopolysaccharides; Myocytes, Cardiac; Natriuretic Peptide, Brain; NFATC Transcription Factors; Rats; Saponins; Signal Transduction; Triterpenes

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca(2+)) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6(ΔC)). Furthermore, stable overexpression of Anxa6(T356D), a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.

Topics: Animals; Annexin A6; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Cell Line; Cytosol; Mutation; Myocytes, Cardiac; Protein Transport; Rats; Secretory Vesicles

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg(2+)-mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp-epoxygenases and Cyp-ω-hydroxylases and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5 mg/kg of mercuric chloride (HgCl₂). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg(2+) significantly increased sEH protein expression and activity levels in hearts of mercury-treated mice, with a consequent decrease in 14,15-, and 11,12-epoxyeicosatrienoic acids (EETs) levels. Whereas the formation of 14,15-, 11,12-, 8,9-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg(2+) toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg(2+)-induced hypertrophy.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cytochrome P-450 Enzyme System; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Heart; Injections, Intraperitoneal; Isoenzymes; Male; Mercuric Chloride; Mice; Mice, Inbred C57BL; Myocardium; Natriuretic Peptide, Brain; RNA, Messenger

To understand the role of ANP mRNA transcription regulation in gp130-mediated cardiomyocyte hypertrophy, and the involved mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK, also called p42/p44 MAPK) signaling pathway.. Isolated neonatal ventricular myocytes were treated with different concentrations of CT-1 (10(-9), 10(-8)and 10(-7)mol/L). MTT was used to analyze the viability and RT-PCR was used to detect ANP mRNA levels in cardiomyocyte. To inhibit p42/p44 MAPK activity in hypertrophic cardiomyocytes, the cells were pretreated with a specific MEK1 inhibitor.. CT-1 significantly induced ANP mRNA expression and the viability of cardiomyocytes in a dose- and time-dependent manner. Furthermore, blocking p42/p44 MAPK activity by the special MEK1 inhibitor upregulated the ANP mRNA.. p42/p44 MAPK have an important role in suppressing ANP mRNA transcription and cell activity in gp130-mediated hypertrophic ventricular myocytes.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cytokine Receptor gp130; Cytokines; Heart Ventricles; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of cardiac hypertrophy and myocyte survival. Extracellular signal regulated protein kinase 1 and 2 (ERK1/2) are key components in the MAPK signaling pathways. Dysfunction of ERK1/2 in congenital heart diseases (Noonan syndrome and LEOPARD syndrome) leads to cardiac hypertrophy. ERK2 contributes 70% of protein content to total ERK1/2 content in myocardium; however, the specific role of ERK2 in regulating cardiac hypertrophy is yet to be further defined. To investigate the specific role of ERK2 played in the cardiomyocytes, we generated and examined mice with cardiomyocyte-specific deletion of the erk2 gene (ERK2(cko) mice). Following short-term pathological hypertrophic stresses, the mutant mice showed attenuated hypertrophic remodeling characterized by a blunted increase in the cross-sectional area of individual myocytes, downregulation of hypertrophic foetal gene markers (ANP and BNP), and less interstitial fibrosis. However, increased cardiomyocyte apoptosis was observed. Upon prolonged stimulation, ERK2(cko) mice developed deterioration in cardiac function. However, absence of ERK2 did not affect physiological hypertrophy induced by 4weeks of swimming exercise. These results revealed an essential role for ERK2 in cardiomyocytes in the development of pathological hypertrophic remodeling and resistance to cell death.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Fibrosis; Gene Expression Regulation; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Primary Cell Culture; Stress, Physiological; Swimming

The developmental origins of health and disease refer to the theory that adverse maternal environments influence fetal development and the risk of cardiovascular disease in adulthood. We used the chronically hypertensive atrial natriuretic peptide knockout (ANP-/-) mouse as a model of gestational hypertension, and attempted to determine the effect of gestational hypertension on left ventricular (LV) structure and function in adult offspring. We crossed normotensive ANP+/+ females with ANP-/- males (yielding ANP+/-(WT) offspring) and hypertensive ANP-/- females with ANP+/+ males (yielding ANP+/-(KO) offspring). Cardiac gene expression was measured using real-time quantitative PCR. Cardiac function was assessed using echocardiography. Daily injections of isoproterenol (ISO) were used to induce cardiac stress. Collagen deposition was assessed using picrosirius red staining. All mice were 10 weeks of age. Gestational hypertension resulted in significant LV hypertrophy in offspring, with no change in LV function. Treatment with ISO resulted in significant LV diastolic dysfunction with a restrictive filling pattern (increased E/A ratio and E/e') and interstitial myocardial fibrosis only in ANP+/-(KO) and not ANP+/-(WT) offspring. Gestational hypertension programs adverse LV structural and functional remodeling in offspring. These data suggest that adverse maternal environments may increase the risk of heart failure in offspring later in life.

Topics: Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; Fibrosis; GATA Transcription Factors; Hypertension, Pregnancy-Induced; Isoproterenol; Male; Mice, Knockout; Models, Cardiovascular; Myocardium; Pregnancy; Ultrasonography; Ventricular Dysfunction, Left

Obesity is often associated with the development of cardiac hypertrophy but the hypertrophy-related pathways in obesity remain unknown. The purpose of this study was to evaluate cardiac hypertrophy-related markers, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), tumor necrosis factor-alpha (TNFα) and hypertrophy-related pathways, interleukin (IL)-6-STAT3, IL-6-MEK5-ERK5 and calcineurin-nuclear factor of activated T-cells (NFAT)3 in the excised hearts from obese rats. Twelve obese Zucker rats were studied at 5-6 months of age and twelve age-matched lean Zucker rats served as the control group. The cardiac characteristics, myocardial architecture, ANP, BNP, TNFα levels, IL-6, STAT3, p-STAT3, MEK5, ERK-5, p-ERK-5, calcineurin and NFAT3 in the left ventricle from the rats were measured by heart weight index, echocardiography, vertical cross section, histological analysis, reverse transcription polymerase chain reaction and western blotting. Compared with the lean control, the whole heart weight, the left ventricule weight, the ratio of the whole heart weight to tibia length, echocardiographic interventricular septum, left ventricular posterior wall thickness, myocardial morphological changes and systolic blood pressure were found to increase in the obese rats. The protein levels of ANP, BNP, TNFα, IL-6, STAT3, p-STAT3, MEK-5, ERK-5, p-ERK 5, calcineurin and NFAT3 were also significantly increased in the hearts of the obese rats. The results showed that the hypertrophy-related markers, ANP, BNP and TNFα, the hypertrophy-related pathways IL-6-STAT3 and IL-6-MEK5-ERK5, and the calcineurin-NFAT3 hypertrophy-related pathways were more active in obese Zucker rats, which may provide possible hypertrophic mechanisms for developing cardiac hypertrophy and pathological changes in obesity.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Interleukin-6; Male; MAP Kinase Kinase 5; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 7; Natriuretic Peptide, Brain; Obesity, Morbid; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,β-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.

Topics: 5'-Nucleotidase; Adenosine; Adrenergic alpha-1 Receptor Agonists; Alkaline Phosphatase; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; GPI-Linked Proteins; Myocytes, Cardiac; Phenylephrine; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-1; Signal Transduction; Time Factors; Vascular Calcification

Pathological cardiac hypertrophy induced by increased sympathetic drive can subsequently lead to congestive heart failure, which represents the major cause of morbidity and mortality worldwide. Astragalus polysaccharide (APS) is an active compound extracted from Chinese herb Astragalus membranaceus (AM), a frequently used "Qi-invigorating" herbal medicine in traditional medicine broadly used for the treatment of cardiovascular and other diseases. Currently, little is known about the effect of APS on cardiac hypertrophy. In the present study, we aimed to investigate its effect on cardiac hypertrophy and to clarify its possible mechanisms. In vitro cardiac hypertrophic model induced by isoprenaline (ISO) was employed to explore the anti-hypertrophic action of APS. We found that 10 μM ISO treatment for 48 h caused cultured cardiomyocytes to undergo significant increases in cell surface area, total protein content, protein synthesis as well as the expression of hypertrophic markers, including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which were effectively inhibited by APS in a dose dependent manner. Moreover, we found that APS pretreatment alleviated the augment of intracellular free calcium during cardiac hypertrophy induced by ISO. Our further study revealed that the upregulated expression of calcineurin, translocation of nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) into nucleus and activation of calmodulin kinase II (reflected by p-CaMKII) were dose dependently suppressed by the application of APS. According to this research, APS exerted its anti-hypertrophic action via inhibiting Ca(2+)-mediated calcineurin/NFATc3 and CaMKII signaling cascades, which provided new insights into the application of APS to the therapy of heart diseases.

Topics: Animals; Animals, Newborn; Astragalus Plant; Atrial Natriuretic Factor; Calcineurin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cardiotonic Agents; Cell Size; Cells, Cultured; Isoproterenol; Myocytes, Cardiac; NFATC Transcription Factors; Phytotherapy; Polysaccharides; Rats, Sprague-Dawley; Signal Transduction

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated.. We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy.. The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice.. These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response.

Topics: Animals; Atrial Natriuretic Factor; Binding Sites; Cardiomegaly; Disease Models, Animal; GATA6 Transcription Factor; Gene Expression Regulation; Genotype; HEK293 Cells; Humans; Male; Metformin; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; Phenotype; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; RNA Interference; Signal Transduction; Transfection

Polydatin (PD), a resveratrol glucoside extracted from the perennial herbage Polygonum cuspidatum, has been suggested to have wide cardioprotective effects. This study aimed to explore the direct antihypertrophic role of PD in cultured neonatal rat ventricular myocytes (NRVMs) and its therapeutic effects against pressure overload (PO)-induced hypertrophic remodeling and heart failure. Furthermore, we investigated the mechanisms underlying the actions of PD. Treatment of NRVMs with phenylephrine for 72 h induced myocyte hypertrophy, where the cell surface area and protein levels of atrial natriuretic peptide and β-myosin heavy chain (β-MHC) were significantly increased. The amplitude of systolic Ca(2+) transient was increased, and sarcoplasmic reticulum Ca(2+) recycling was prolonged. Concomitantly, calcineurin activity was increased and NFAT protein was imported into the nucleus. PD treatment restored Ca(2+) handling and inhibited calcineurin-NFAT signaling, thus attenuating the hypertrophic remodeling in NRVMs. PO-induced cardiac hypertrophy was produced by transverse aortic constriction (TAC) in C57BL/6 mice, where the left ventricular posterior wall thickness and heart-to-body weight ratio were significantly increased. The cardiac function was increased at 5 wk of TAC, but significantly decreased at 13 wk of TAC. The amplitude of Ca(2+) transient and calcineurin activity were increased at 5 wk of TAC. PD treatment largely abolished TAC-induced hypertrophic remodeling by inhibiting the Ca(2+)-calcineurin pathway. Surprisingly, PD did not inhibit myocyte contractility despite that the amplitude of Ca(2+) transient was decreased. The cardiac function remained intact at 13 wk of TAC. In conclusion, PD is beneficial against PO-induced cardiac hypertrophy and heart failure largely through inhibiting the Ca(2+)-calcineurin pathway without compromising cardiac contractility.

Topics: Active Transport, Cell Nucleus; Animals; Atrial Natriuretic Factor; Calcineurin; Calcium; Calcium Signaling; Cardiomegaly; Cell Nucleus; Cells, Cultured; Glucosides; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocytes, Cardiac; Myosin Heavy Chains; NFATC Transcription Factors; Phenylephrine; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Stilbenes; Ventricular Remodeling

The precise molecular mechanisms leading to disturbance of Ca(2+)/calmodulin-dependent intracellular signalling in cardiac hypertrophy remains unclear. As an endogenous calmodulin regulator protein, the pathophysiology role of PEP-19 during cardiac hypertrophy was investigated in the present study. We here demonstrated that PEP-19 protein levels are significantly elevated in the aortic banding model in vivo and angiotensin II-induced cardiomyocyte hypertrophy in vitro. Consistent with inhibitory actions of PEP-19 on cardiomyocyte hypertrophy, induction of CaMKII and calcineurin activation as well as hypertrophy-related genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was significantly inhibited by PEP-19 transfection. Moreover, PEP-19 partially ameliorates angiotensin II-induced elevation of phospho-phospholamban (Thr-17) and sarcoplasmic reticulum Ca(2+) release in cardiomyocytes. Together, our results suggest that PEP-19 attenuates angiotensin II-induced cardiomyocyte hypertrophy via suppressing the disturbance of CaMKII and calcineurin signaling.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calcineurin; Calcium; Calcium Signaling; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Calmodulin-Binding Proteins; Cardiomegaly; Cells, Cultured; Gene Expression; Hypertrophy; Male; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats, Sprague-Dawley; Sarcoplasmic Reticulum

Cardiomyocyte hypertrophy requires a source of Ca(2+) distinct from the Ca(2+) that regulates contraction. The canonical transient receptor potential channel (TrpC) family, a family of cation channels regulated by activation of phospholipase C (PLC), has been implicated in this response. Cardiomyocyte hypertrophy downstream of Gq-coupled receptors is mediated specifically by PLCβ1b that is scaffolded onto a SH3 and ankyrin repeat protein 3 (Shank3) complex at the sarcolemma. TrpC4 exists as two splice variants (TrpC4α and TrpC4β) that differ only in an 84-residue sequence that binds to phosphatidylinositol(4,5)bisphosphate (PIP2), the substrate of PLCβ1b. In neonatal rat cardiomyocytes, TrpC4α, but not TrpC4β, coimmunoprecipitated with both PLCβ1b and Shank3. Heightened PLCβ1b expression caused TrpC4α, but not TrpC4β, translocation to the sarcolemma, where it colocalized with PLCβ1b. When overexpressed in cardiomyocytes, TrpC4α, but not TrpC4β, increased cell area (893 ± 18 to 1497 ± 29 mm(2), P < 0.01) and marker gene expression (atrial natriuretic peptide increased by 409 ± 32%, and modulatory calcineurin inhibitory protein 1 by 315 ± 28%, P < 0.01). Dominant-negative TrpC4 reduced hypertrophy initiated by PLCβ1b, or PLCβ1b-coupled receptor activation, by 72 ± 8% and 39 ± 5 %, respectively. We conclude that TrpC4α is selectively involved in mechanisms downstream of PLCβ1b culminating in cardiomyocyte hypertrophy, and that the hypertrophic response is dependent on the TrpC4α splice variant-specific sequence that binds to PIP2.

Topics: Animals; Apoptosis Regulatory Proteins; Atrial Natriuretic Factor; Binding Sites; Cardiomegaly; Carrier Proteins; Female; Male; Mutation; Myocytes, Cardiac; Nerve Tissue Proteins; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C beta; Protein Binding; Protein Isoforms; Protein Transport; Rats; Rats, Sprague-Dawley; Sarcolemma; TRPC Cation Channels

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.

Topics: Adenoviridae; Angiotensin II; Animals; Animals, Newborn; Aorta; Atrial Natriuretic Factor; Calcineurin; Calpain; Cardiomegaly; Constriction, Pathologic; Cyclosporine; Gene Expression Regulation; Genetic Vectors; Glycoproteins; Imidazoles; Mice; Muscle Proteins; Myocytes, Cardiac; Myosin Heavy Chains; Nuclear Proteins; Primary Cell Culture; Protein Transport; Rats; Repressor Proteins; RNA, Small Interfering; Signal Transduction; Tetrazoles

Kruppel-like factor 4 (KLF4) plays an important role in vascular diseases, including atherosclerosis and vascular injury. Although KLF4 is expressed in the heart in addition to vascular cells, the role of KLF4 in cardiac disease has not been fully determined. The goals of this study were to investigate the role of KLF4 in cardiac hypertrophy and to determine the underlying mechanisms. Cardiomyocyte-specific Klf4 knockout (CM Klf4 KO) mice were generated by the Cre/LoxP technique. Cardiac hypertrophy was induced by chronic infusion of the β-adrenoreceptor agonist isoproterenol (ISO). Results showed that ISO-induced cardiac hypertrophy was enhanced in CM Klf4 KO mice compared with control mice. Accelerated cardiac hypertrophy in CM Klf4 KO mice was accompanied by the augmented cellular enlargement of cardiomyocytes as well as the exaggerated expression of fetal cardiac genes, including atrial natriuretic factor (Nppa). Additionally, induction of myocardin, a transcriptional cofactor regulating fetal cardiac genes, was enhanced in CM Klf4 KO mice. Interestingly, KLF4 regulated Nppa expression by modulating the expression and activity of myocardin, providing a mechanical basis for accelerated cardiac hypertrophy in CM Klf4 KO mice. Moreover, we showed that KLF4 mediated the antihypertrophic effect of trichostatin A, a histone deacetylase inhibitor, because ISO-induced cardiac hypertrophy in CM Klf4 KO mice was attenuated by olmesartan, an angiotensin II type 1 antagonist, but not by trichostatin A. These results provide novel evidence that KLF4 is a regulator of cardiac hypertrophy by modulating the expression and the activity of myocardin.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Gene Expression; Gene Expression Regulation; Histone Deacetylase Inhibitors; Hydroxamic Acids; Imidazoles; Isoproterenol; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Myocytes, Cardiac; Natriuretic Peptide, C-Type; Nuclear Proteins; Protein Precursors; Tetrazoles; Trans-Activators

Polyamines (putrescine, spermidine and spermine) play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS) in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy.. Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO). Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO). Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting.. DFMO (0.5 mM and 2 mM) treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) level in the culture medium. In addition, DFMO (0.5 mM) down regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM).. The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Topics: Acetyltransferases; Animals; Apoptosis; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Cardiomegaly; Caspase 12; Down-Regulation; Endoplasmic Reticulum Stress; HSP70 Heat-Shock Proteins; Isoproterenol; L-Lactate Dehydrogenase; Membrane Proteins; Myocytes, Cardiac; Ornithine Decarboxylase; Polyamines; Proto-Oncogene Proteins c-bcl-2; Putrescine; Rats; Rats, Wistar; Spermidine; Spermine; Transcription Factor CHOP; Up-Regulation

Pressure overload induces cardiac hypertrophy through activation of Janus kinase 2 (Jak2), however, the underlying mechanisms remain largely unknown. In the current study, we tested whether histone deacetylase 2 (HDAC2) was involved in the process. We found that angiotensin II (Ang-II)-induced re-expression of fetal genes (Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)) in cultured cardiomyocytes was prevented by the Jak2 inhibitor AG-490 and HDAC2 inhibitor Trichostatin-A (TSA), or by Jak2/HDAC2 siRNA knockdown. On the other hand, myocardial cells with Jak2 or HDAC2 over-expression were hyper-sensitive to Ang-II. In vivo, pressure overload by transverse aorta binding (AB) induced a significant cardiac hypertrophic response as well as re-expression of ANP and BNP in mice heart, which were markedly reduced by AG-490 and TSA. Significantly, AG-490, the Jak2 inhibitor, largely suppressed pressure overload-/Ang-II-induced HDAC2 nuclear exportation in vivo and in vitro. Meanwhile, TSA or HDAC2 siRNA knockdown reduced Ang-II-induced ANP/BNP expression in Jak2 over-expressed H9c2 cardiomyocytes. Together, these results suggest that HDAC2 might be a downstream effector of Jak2 to mediate cardiac hypertrophic response by pressure overload or Ang-II.

Topics: Active Transport, Cell Nucleus; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Cells, Cultured; Histone Deacetylase 2; Hydroxamic Acids; Janus Kinase 2; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Natriuretic Peptide, Brain; Peptide Fragments; Pressure; Rats; Signal Transduction; Tyrphostins

The importance of chronic stimulation of β-adrenoceptors in the development of cardiac dysfunction is the rationale for the use of β-blockers in the treatment of heart failure. Nebivolol is a third-generation β-blocker, which has further properties including stimulation of endothelial nitric oxide synthase and/or β3-adrenoceptors. The aim of this study was to investigate whether nebivolol has additional effects on β-adrenoceptor-mediated functional responses along with morphologic and molecular determinants of cardiac hypertrophy compared with those of metoprolol, a selective β1-adrenoceptor blocker. Rats infused by isoprenaline (100 μg·kg(-1)·day(-1), 14 days) were randomized into three groups according to the treatment with metoprolol (30 mg·kg(-1)·day(-1)), nebivolol (10 mg·kg(-1)·day(-1)), or placebo for 13 days starting on day 1 after implantation of minipump. Both metoprolol and nebivolol caused a similar reduction on heart rate. Nebivolol mediated a significant improvement on cardiac mass, coronary flow, mRNA expression levels of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and atrial natriuretic peptide and phospholamban (PLN)/SERCA2a and phospho-PLN/PLN ratio compared with metoprolol and placebo. Nebivolol prevented the detrimental effects of isoprenaline infusion on isoprenaline (68% of control at 30 μM), BRL37344 (63% of control at 0.1 μM), and forskolin (64% of control at 1 μM) responses compared with metoprolol (isoprenaline, 34% of control; BRL37344, no response; forskolin, 26% of control) and placebo (isoprenaline, 33% of control; BRL37344, 28% of control; forskolin, 12% of control). Both β-blockers improved the changes in mRNA expressions of β1- and β3-adrenoceptors. Our results suggest that nebivolol partially protects the responsiveness of β-adrenoceptor signaling and the development of cardiac hypertrophy independent of its β1-adrenoceptor blocking effect.

Topics: Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Benzopyrans; Calcium-Binding Proteins; Cardiomegaly; Coronary Circulation; Ethanolamines; Heart Rate; In Vitro Techniques; Isoproterenol; Male; Metoprolol; Nebivolol; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Transcription, Genetic

miRNAs play an important role in the pathogenesis of cardiac hypertrophy and dysfunction. However, little is known about how miR-30a regulates cardiomyocyte hypertrophy. In the study, Male C57BL/6 mice were subjected to thoracic aortic constriction, and hearts were harvested at 3 weeks. We assayed miR-30a expression level by real-time PCR and defined the molecular mechanisms of miR-30a-mediated cardiomyocyte hypertrophy. We found that myocardial expression of miR-30a was decreased in mouse models of hypertrophy and in H9c2 cells treated with phenylephrine. MiR-30a inhibition markedly increased mRNA expression of cardiac hypertrophy markers such as atrial natriuretic factor and brain natriuretic peptide in H9c2, and cell size was increased after miR-30a inhibitor treatment. Downregulated miR-30a activated autophagy by inhibiting beclin-1 expression in H9c2 cell. More important, autophagy inhibition suppressed miR-30a inhibitor-induced cardiomyocyte hypertrophy. Together, our data demonstrated that downregulated miR-30a aggravates pressure overload-induced cardiomyocyte hypertrophy by activating autophagy, thus offering a new target for the therapy of cardiomyocyte hypertrophy.

Topics: 3' Untranslated Regions; Animals; Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Cell Line; Cell Size; Down-Regulation; Gene Expression Regulation; HEK293 Cells; Humans; Hypertension; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats

Homers are scaffolding proteins that modulate diverse cell functions being able to assemble signalling complexes. In this study, the presence, sub-cellular distribution and function of Homer 1 was investigated. Homer 1a and Homer 1b/c are constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells, a cardiac cell line. As judged by confocal immunofluorescence microscopy, Homer 1a displays sarcomeric and peri-nuclear localization. In cardiomyocytes and cultured HL-1 cells, the hypertrophic agonist norepinephrine (NE) induces α1-adrenergic specific Homer 1a over-expression, with a two-to-three-fold increase within 1h, and no up-regulation of Homer 1b/c, as judged by Western blot and qPCR. In HL-1 cells, plasmid-driven over-expression of Homer 1a partially antagonizes activation of ERK phosphorylation and ANF up-regulation, two well-established, early markers of hypertrophy. At the morphometric level, NE-induced increase of cell size is likewise and partially counteracted by exogenous Homer 1a. Under the same experimental conditions, Homer 1b/c does not have any effect on ANF up-regulation nor on cell hypertrophy. Thus, Homer 1a up-regulation is associated to early stages of cardiac hypertrophy and appears to play a negative feedback regulation on molecular transducers of hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Carrier Proteins; Cell Line; Cell Nucleus; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Homer Scaffolding Proteins; Mice; Myocytes, Cardiac; Norepinephrine; Phosphorylation; Rats; Rats, Wistar; Sarcomeres; Transcription, Genetic; Up-Regulation

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca²⁺, cardiac hypertrophy, and heart failure in Ryr2ADA/ADA mice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca²⁺, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D). Here we report that in single-channel measurements RyR2-L3591D isolated from Ryr2D/D hearts lost CaM inhibition at diastolic Ca²⁺ only, whereas S100A1 regulation was eliminated at both diastolic and systolic Ca²⁺. In contrast to the ~2-wk life span of Ryr2ADA/ADA mice, Ryr2D/D mice lived longer than 1 yr. Six-month-old Ryr2D/D mice showed a 9% increase in heart weight-to-body weight ratio, modest changes in cardiac morphology, and a twofold increase in atrial natriuretic peptide mRNA levels compared with wild type. After 4-wk pressure overload with transverse aortic constriction, heart weight-to-body weight ratio and atrial natriuretic peptide mRNA levels increased and echocardiography showed changes in heart morphology of Ryr2D/D mice compared with sham-operated mice. Collectively, the findings indicate that the single RyR2-L3591D mutation, which distinguishes the effects of diastolic and systolic Ca²⁺, alters heart size and cardiac function to a lesser extent in Ryr2D/D mice than the triple mutation in Ryr2ADA/ADA mice. They further suggest that CaM inhibition of RyR2 at systolic Ca²⁺ is important for maintaining normal cardiac function.

Topics: Action Potentials; Animals; Atrial Natriuretic Factor; Binding Sites; Calcium; Calcium Signaling; Calmodulin; Cardiomegaly; Heart Failure; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation, Missense; Myocardial Contraction; RNA, Messenger; Ryanodine Receptor Calcium Release Channel; S100 Proteins; Transcription, Genetic

High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload.. Analysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice.. These results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.

Topics: Acetylation; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Endothelin-1; Heart Failure; HMGB1 Protein; Humans; Mice; Myocardium; Protein Transport; Rats

Growing evidence shows that microRNAs (miRNAs) are involved in various cardiac processes including cardiac hypertrophy. However, the modulation of miRNA by pharmacological intervention in cardiomyocyte hypertrophy has not been disclosed yet. methods: We constructed neonatal rat cardiomyocyte hypertrophy induced by angiotensin II stimulation and subjected to cardiomyocyte immunochemistry, qRT-PCR and immunoblotting analysis. In addition, we constructed the mouse cardiac hypertrophy using angomir-22 stimulation and demonstrated the potential antihypertrophic mechnism of atorvastatin.. The results showed that a collection of miRNAs were aberrantly expressed in hypertrophic cardiomyocytes induced by angiotensin II stimulation. In addition, overexpression of miR-22 was found in angiotensin II-induced hypertrophic cardiomyocytes, whereas administration of atorvastatin could reverse the upregulation of miRNA-22 nearly back to the control level. Furthermore, up-regulation of miRNA-22 in cardiomyocytes in vitro and in vivo could induce cardiac hypertrophy, which could suppress the protein level of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Concomitantly, the production of ANP, BNP and β-MHC was enhanced and cardiomyocyte size was increased. However, atorvastatin could markedly knockdown miRNA-22 expression and reverse these changes in cardiomyocytes. These results suggest that the contribution of atrovastatin to cardiomyocyte hypertrophy may be involved in downregulation of miRNA-22 expression, which modulates the activity of PTEN in cardiomyocyte hypertrophy. conclusion: This study demonstrates for the first time the modulation of miRNA-22 can be achieved by pharmacological intervention. The data generated from this study provides a rationale for the development of miRNA-based strategies for antihypertrophic treatment.

Topics: Angiotensin II; Animals; Anticholesteremic Agents; Atorvastatin; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Down-Regulation; Heptanoic Acids; Male; Mice; MicroRNAs; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Oligonucleotides, Antisense; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Pyrroles; Rats

Motorcycle exhaust (ME) is a major source of air pollution and a potential health hazard in urban areas where motorcycles are a popular means of transportation. The main objectives of this study were to determine the ability of ME to cause cardiotoxicity in rats and investigate the possible mechanisms of toxicity. Male rats were exposed to 1:10 diluted ME by inhalation 2 h daily and Monday through Friday for 8 weeks. Exposure to ME increased heart weight and decreased cardiac antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase and glutathione peroxidase activities in a concentration- and time-dependent manner. Analysis of echocardiographic parameters indicated that ME increased left ventricle posterior wall thickness, interventricular septum thickness and left ventricle mass. Histopathological examinations of the hearts revealed that ME exposure caused focal cardial degeneration and necrosis, mononuclear cell infiltration, and fibrosis. The results of reverse transcriptase-polymerase chain reaction studies showed that ME decreased GST-M1 and GST-P1 mRNA expression and increased the expression of proinflammatory cytokine interleukin-1β, hypertrophy marker atrial natriuretic peptide, fibrosis markers type I and III collagen, profibrotic cytokine connective tissue growth factor, and hypertrophy and fibrosis mediator transforming growth factor (TGF)-β1 in the heart. The data of Western blot analysis showed that cardiac TGF-β1 protein was induced by ME. These findings demonstrate that subchronic ME exposure caused hypertrophy and fibrosis, and modulated GST and TGF-β1 expression in rat heart possibly by mechanisms involving oxidative stress and inflammation.

Topics: 7-Alkoxycoumarin O-Dealkylase; Air Pollutants; Animals; Atrial Natriuretic Factor; Carbon Monoxide; Cardiomegaly; Collagen Type I; Collagen Type II; Fibrosis; Glutathione Peroxidase; Glutathione Transferase; Interleukin-1beta; Lipid Peroxidation; Male; Motorcycles; Myocardium; Rats; Rats, Wistar; RNA, Messenger; Superoxide Dismutase; Transforming Growth Factor beta1; Vehicle Emissions

Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor that has biological roles mainly in metabolism and that controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERRγ have not yet been elucidated. In the present study, we aimed to characterize the role of ERRγ in cardiac hypertrophy. The functional roles of ERRγ in the development of cardiac hypertrophy were examined in primary cultured cardiomyocytes and in animal models. ERRγ expression was increased in hearts from human hypertrophic cardiomyopathy patients and in both cellular and animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERRγ in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERRγ blocked agonist-induced hypertrophic phenotypes. ERRγ bound directly to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERRγ-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182, an inverse agonist of ERRγ, completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart. These findings demonstrate a novel ERRγ/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic.

Topics: Adult; Animals; Atrial Natriuretic Factor; Base Sequence; Cardiomegaly; Drug Inverse Agonism; GATA4 Transcription Factor; Gene Knockdown Techniques; Humans; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Myocytes, Cardiac; Phenotype; Protein Binding; Receptors, Estrogen; Response Elements; Tamoxifen; Transcriptional Activation

Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts.. This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts.. L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs. phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 μmol/min per gram of dry weight, mean±SE) vs. PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling.. The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.

Topics: Acetyl-CoA Carboxylase; Animals; Atrial Natriuretic Factor; Carboxy-Lyases; Cardiomegaly; Carnitine O-Palmitoyltransferase; Disease Models, Animal; Energy Metabolism; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Genotype; Liver; Magnetic Resonance Spectroscopy; Male; Malonyl Coenzyme A; Myocardium; Oxidation-Reduction; Palmitic Acid; Phenotype; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Up-Regulation

We have previously shown that isoprenaline-induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 (CYP) and soluble epoxide hydrolase (sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) will protect against isoprenaline-induced cardiac hypertrophy.. Male Sprague-Dawley rats were treated with TUPS (0.65 mg kg(-1) day(-1), p.o.), isoprenaline (5 mg kg(-1) day(-1), i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 μM) in the presence and absence of either TUPS (1 μM) or 11,12 EET (1 μM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR.. Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15-EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid (DHET) and the 20-HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid (AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12-EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2.. TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Cardiotonic Agents; Cell Line; Cytochrome P-450 Enzyme System; Drug Antagonism; Epoxide Hydrolases; Gene Expression Regulation; Heart; Humans; Isoproterenol; Kidney; Liver; Natriuretic Peptide, Brain; Phenylurea Compounds; Piperidines; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction

Sex-specific differences in hormone-mediated gene regulation may influence susceptibility to cardiac hypertrophy, a primary risk factor for cardiovascular disease. Under hormonal influence, natriuretic peptide (NP) and nitric oxide synthase (NOS) systems modulate cardio-protective gene programs through common downstream production of cyclic guanosine 3'-5' monophosphate (cGMP). Ablation of either system can adversely affect cardiac adaptation to stresses and insults. This study elucidates sex-specific differences in cardiac NP and NOS system gene expression and assesses the impact of the estrous cycle on these systems using the atrial natriuretic peptide gene-disrupted (ANP(-/-)) mouse model. Left ventricular expression of the NP and NOS systems was analyzed using real-time quantitative polymerase chain reaction in 13- to 16-week-old male, proestrous and estrous female ANP(+/+) and ANP(-/-) mice. Left ventricular and plasma cGMP levels were measured to assess the convergent downstream effects of the NP and NOS systems. Regardless of genotype, males had higher expression of the NP system while females had higher expression of the NOS system. In females, transition from proestrus to estrus lowered NOS system expression in ANP(+/+) mice while the opposite was observed in ANP(-/-) mice. No significant changes in left ventricular cGMP levels across gender and genotype were observed. Significantly lower plasma cGMP levels were observed in ANP(-/-) mice compared to ANP(+/+) mice. Regardless of genotype, sex-specific differences in cardiac NP and NOS system expression exist, each sex enlisting a predominant system to conserve downstream cGMP. Estrous cycle-mediated alterations in NOS system expression suggests additional hormone-mediated gene regulation in females.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiovascular Diseases; Cyclic GMP; Female; Gene Expression; Heart Ventricles; Male; Mice; Mice, Knockout; Natriuretic Peptides; Nitric Oxide Synthase; RNA, Messenger; Sex Characteristics

Farnesyl pyrophosphate synthase (FPPS), as a key branchpoint of the mevalonate pathway, catalyzes the synthesis of isoprenoid intermediates. The isoprenoid intermediates are needed for protein isoprenylation to participate in cardiac remodeling. We have previously demonstrated that both knockdown of FPPS with small interfering RNA and inhibition of FPPS by alendronate could prevent Ang II-induced hypertrophy in cultured cardiomyocytes. In this study, we evaluated the effects of FPPS inhibition in Ang II-mediated cardiac hypertrophy and fibrosis in vivo. Wild type mice were separately treated with saline, Ang II (2.88 mg/kg per day), FPPS inhibitor alendronate (0.1 mg/kg per day), or the combination of Ang II (2.88 mg/kg per day) and alendronate (0.1 mg/kg per day) for 4 weeks. The results showed that Ang II increased FPPS expression, and the increases of Ang II-induced synthesis of the isoprenoid intermediates, FPP and GGPP, were significantly inhibited by FPPS inhibitor. In the meantime, FPPS inhibition attenuated Ang II-mediated cardiac hypertrophy and fibrosis as indexed by the heart weight to body weight ratio, echocardiographic parameters, histological examinations and expression of ANP and BNP mRNA. Furthermore, it was also found that FPPS inhibitor attenuated Ang II-induced increases of RhoA activity and p-38 MAPK phosphorylation and TGF-β1 mRNA expression. In conclusion, FPPS might play an important role in Ang II-induced cardiac hypertrophy and fibrosis in vivo, at least in part through RhoA, p-38 MAPK and TGF-β1.

Topics: Alendronate; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Contraindications; Fibrosis; Gene Expression Regulation; Geranyltranstransferase; Humans; Male; Mice; Myocytes, Cardiac; Natriuretic Peptide, Brain; p38 Mitogen-Activated Protein Kinases; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Signal Transduction; Terpenes; Transforming Growth Factor beta1

We aimed to investigate whether atrial natriuretic peptide (ANP) attenuates angiotensin II (Ang II)-induced myocardial remodeling and to clarify the possible molecular mechanisms involved. Thirty-five 8-week-old male Wistar-Kyoto rats were divided into control, Ang II, Ang II + ANP, and ANP groups. The Ang II and Ang II + ANP rats received 1 μg/kg/min Ang II for 14 days. The Ang II + ANP and ANP rats also received 0.1 μg/kg/min ANP intravenously. The Ang II and Ang II + ANP rats showed comparable blood pressure. Left ventricular fractional shortening and ejection fraction were lower in the Ang II rats than in controls; these indices were higher (P < 0.001) in the Ang II + ANP rats than in the Ang II rats. In the Ang II rats, the peak velocity of mitral early inflow and its ratio to atrial contraction-related peak flow velocity were lower, and the deceleration time of mitral early inflow was significantly prolonged; these changes were decreased by ANP. Percent fibrosis was higher (P < 0.001) and average myocyte diameters greater (P < 0.01) in the Ang II rats than in controls. ANP decreased both myocardial fibrosis (P < 0.01) and myocyte hypertrophy (P < 0.01). Macrophage infiltration, expression of mRNA levels of collagen types I and III, monocyte chemotactic protein-1, and a profibrotic/proinflammatory molecule, tenascin-C (TN-C) were increased in the Ang II rats; ANP significantly decreased these changes. In vitro, Ang II increased expression of TN-C and endothelin-1 (ET-1) in cardiac fibroblasts, which were reduced by ANP. ET-1 upregulated TN-C expression via endothelin type A receptor. These results suggest that ANP may protect the heart from Ang II-induced remodeling by attenuating inflammation, at least partly through endothelin 1/endothelin receptor A cascade.

Topics: Angiotensin II; Animals; Anti-Inflammatory Agents; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Endothelin-1; Fibrillar Collagens; Fibroblasts; Fibrosis; Heart Diseases; Inflammation; Inflammation Mediators; Infusions, Intravenous; Macrophages; Male; Mitral Valve; Myocardial Contraction; Myocardium; Rats; Rats, Inbred WKY; Receptor, Endothelin A; Signal Transduction; Stroke Volume; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Ghrelin is the endogenous ligand for the growth hormone-secretagogue receptor expressed in various tissues including the heart, blood vessels and kidney. This study sought to determine the effects of long-term treatment with ghrelin (10 nmol/kg, twice a day, intraperitoneally) on the hypertension induced by high salt (8.0% NaCl) diet in Dahl salt-sensitive hypertensive (DS) rats. Systolic blood pressure (SBP) was measured by a tail cuff method. During the treatment period for 3 weeks, high salt diet increased blood pressure compared to normal salt (0.3% NaCl) diet, and this hypertension was partly but significantly (P<0.01) attenuated by simultaneous treatment with ghrelin. Ghrelin significantly increased urine volume and tended to increase urine Na⁺ excretion. Furthermore, ghrelin increased urine nitric oxide (NO) excretion and tended to increase renal neuronal nitric oxide synthase (nNOS) mRNA expression. Ghrelin did not alter the plasma angiotensin II level and renin activity, nor urine catecholamine levels. Furthermore, ghrelin prevented the high salt-induced increases in heart thickness and plasma ANP mRNA expression. These results demonstrate that long-term ghrelin treatment counteracts salt-induced hypertension in DS rats primarily through diuretic action associated with increased renal NO production, thereby exerting cardio-protective effects.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Diuresis; Diuretics; Enzyme Induction; Ghrelin; Heart Ventricles; Hypertension; Injections, Intraperitoneal; Kidney; Male; Nitric Oxide; Nitric Oxide Synthase Type I; Rats; Rats, Inbred Dahl; RNA, Messenger; Sodium; Sodium Chloride, Dietary; Ultrasonography; Up-Regulation

Augmented endothelial nitric oxide (NO) synthase (eNOS) signaling has been reported to be associated with improvements in cardiac remodeling, and NO levels have been shown to be related to cardiac hypertrophy and heart failure. Imperatorin, a dietary furanocoumarin, has been shown to prevent cardiac hypertrophy in the spontaneous hypertension rats (SHR). Thus, we aimed to clarify whether imperatorin attenuates both cardiac hypertrophy and heart failure via the NO-signaling pathway. In neonatal mouse cardiac myocytes, imperatorin inhibited protein synthesis stimulated by either isoproterenol or phenylephrine, which was unchanged by NG-nitro-L-arginine methyl ester (L-NAME). Four weeks after transverse aortic constriction (TAC) on Kunming (KM) male mice, the ratio of heart weight to body weight was lower after imperatorin treatment than in controls (6.60 ± 0.35 mg/g in TAC, 4.54 ± 0.29 mg/g with imperatorin 15 mg kg(-1)d(-1), ig, P<0.01); similar changes in the ratio of lung weight to body weight (7.30 ± 0.85 mg/g in TAC, 5.42 ± 0.51 mg/g with imperatorin 15 mg kg(-1)d(-1), ig) and the myocardial fibrosis. All of these improvements were blunted by L-NAME. Imperatorin treatment significantly activated phosphorylation of eNOS. Myocardial mRNA levels of natriuretic peptide precursor type B and protein inhibitor of NO synthase, which were increased in the TAC mice, were decreased in the imperatorin-treated ones. Imperatorin can attenuate cardiac hypertrophy both in vivo and in vitro, and halt the process leading from hypertrophy to heart failure by a NO-mediated pathway.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Cytoplasmic Dyneins; Furocoumarins; Gene Expression Regulation; Heart Failure; Male; Mice; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nitric Oxide; Nitric Oxide Synthase Type III; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases

To explore the effects of ethanolic extract of Radix Scrophulariae (EERS) on ventricular remodeling in rats.. Rats with coronary artery ligation (CAL) were randomly assigned to 5 groups: CAL model; CAL plus 40 mg/kg captopril; CAL plus 60 mg/kg, 120 mg/kg, 240 mg/kg EERS. Sham operation rats were randomly assigned to 2 groups, sham-operated control and sham-operated plus 120 mg/kg EERS. The rats were orally administered with the corresponding drugs or drinking water for 14 weeks. The left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with hematoxylin and eosin or picric acid/Sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of hydroxyproline (Hyp), matrix metalloproteinase 2 (MMP-2), angiotensin II (Ang II), aldosterone (ALD), endothelin 1 (ET-1), atrial natriuretic peptide (ANP), tumor necrosis factor α (TNF-α) and renin activity (RA) in myocardium or serum were determined. Real-time RT-PCR was used to detect the mRNA expressions of angiotensin converting enzyme (ACE), ET-1 and ANP.. EERS could significantly reduce the LVWI and HWI, decrease heart tissue concentrations of Hyp and collagen deposition, diminish cardiomyocyte cross-section area, reduce the tissue level of Ang II, ET-1, ANP and TNF-α. EERS could also down regulate the mRNA expression of ACE, ET-1 and ANP in myocardium.. EERS attenuates ventricular remodeling. The mechanisms may be related to restraining the excessive activation of RAAS, TNF-α and modulating some gene expressions associated with cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Chemical Analysis; Captopril; Cardiomegaly; Collagen; Coronary Vessels; Endothelin-1; Ethanol; Gene Expression Regulation; Hemodynamics; Hydroxyproline; Male; Matrix Metalloproteinase 2; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organ Size; Plant Extracts; Plant Roots; Random Allocation; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; RNA, Messenger; Scrophularia; Tumor Necrosis Factor-alpha; Ventricular Remodeling

The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats). Both biomarkers were measured by mean of two commercially available enzyme immunoassays and serum levels were correlated with heart weight and histopathological findings in the heart, with the aim of building an integrated assessment of the significance of these biomarkers. Results obtained demonstrated that NT-proANP and ANP can be accurately measured in the different rat strains, with NT-proANP concentrations higher than those of ANP, as expected because of its longer half-life. In addition, both correlated well with cardiac hypertrophy evaluated by means of heart weight and histopathological examination. NT-proANP and ANP represent reliable markers of cardiac hypertrophy in the rat.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Blood Pressure; Cardiomegaly; Half-Life; Heart Atria; Male; Organ Size; Radioimmunoassay; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Rats, Wistar

Because of the crucial role of the endocrine heart in maintaining homeostasis, considerable effort has been focused on the elucidation of the mechanistic underlying gene expression and secretion of the cardiac hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). However, much remains to be determined regarding specific molecular events involved in cardiocyte secretory function. In this work, we identified genes involved in the transcriptional response of the endocrine heart to volume overload (VO) and signaling pathways involved in its regulation. To this end, the cardiac atrial and ventricular transcriptomes were analyzed in the heart of rats subjected to experimentally induced aorto-caval shunt VO. Pathway analysis revealed unique gene expression profiles in the VO atria for G-protein signaling, notably a significant downregulation of Ras dexamethasone-induced protein 1 (RASD1). In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion. Concurrent knockdown of RASD1 and its effectors Gα(o1) or Gβ(1)γ(2) abrogated the endocrine response, demonstrating a previously unknown negative modulator role for RASD1. RASD1 thus emerges as a tonic inhibitor of ANF secretion and illustrates for the first time the concept of inhibitory protein regulators of ANF release. The novel molecular function identified herein for RASD1 is of considerable importance given its therapeutic implications for cardiovascular disease.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Cells, Cultured; Gene Expression Profiling; GTP-Binding Proteins; In Vitro Techniques; Male; Models, Animal; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; ras Proteins; Rats; Rats, Sprague-Dawley; RNA Interference; Signal Transduction

Mouse parathyroid hormone-related peptide (PTHrP) is a peptide hormone consisting of 139 amino acids. It is target of proteolysis resulting in circulation of N- and C-terminal peptides. C-terminal PTHrP peptides act in a PTH/PTHrP receptor-independent way with a minimal peptide sequence required to exert these effects covering amino acids 107-111 also known as osteostatin. Although effects of osteostatin on cardiac hypertrophy have been described in vitro, the in vivo relevance of these findings remained to be defined. The study was performed in two experimental series. In the first series, mice were randomly distributed into placebo or treatment group (each n=7) and osteostatin was administered via osmotic minipumps. In the second series, mice underwent a banding of the thoracic aorta to induce pressure overload and were again randomly distributed into placebo or treatment group (n=9 each). After 14 days, mice were anaesthetized and cardiac function, ECG, and cardiac hypertrophy were determined. Osteostatin increased the expression of ANF and reduced P-wave duration with little effects on cardiac performance in mice without pressure overload. In TAC banded mice, however, osteostatin significantly reduced TAC-induced loss of body weight, induced right ventricular hypertrophy, and reduced P-wave duration again. In osteostatin treated mice with pressure overload, the protein kinase C-dependent phosphorylation of connexin 43 was preserved. In summary, osteostatin attenuated pressure-overload-dependent loss of body weight without affecting left ventricular hypertrophy or left ventricular function but preserved atrial conduction. Osteostatin exerts moderate cardioprotective effects in mice under hemodynamic stress.

Topics: Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Connexin 43; Heart; Male; Mice; Mice, Inbred C57BL; Parathyroid Hormone-Related Protein; Peptide Fragments; Phosphorylation; RNA, Messenger; Ventricular Function, Left

Evidence has shown that endoplasmic reticulum stress (ERS) is associated with the pathogenesis of cardiac hypertrophy. The aim of this study was to investigate whether direct alleviation of ER stress by 4-phenylbutyric acid (PBA), a known chemical chaperone drug, could attenuate pressure-overload cardiac hypertrophy in mice. The effects of orally administered PBA (100mg/kg body weight daily for a week) were examined using mice undergoing transverse aortic constriction (TAC-mice), an animal model to produce pressure overload. TAC application for 1 week led to a 1.8-fold increase in the ratio of the heart weight over body weight (HW/BW) and up-regulation of the hypertrophy markers ANF and BNF accompanied by up-regulation of ERS markers (GRP78, p-PERK, and p-elF2α). The oral administration of PBA to the TAC-mice reduced hypertrophy (19%) and severely downregulated the fibrosis-related genes (transforming growth factor-β1, phospho-smad2, and pro-collagen isoforms). We conclude that ERS is induced as a consequence of remodeling during pathological hypertrophy and that PBA may help to relieve ERS and play a protective role against cardiac hypertrophy and possibly heart failure. We suggest PBA as a novel therapeutic agent for cardiac hypertrophy and fibrosis.

Topics: Administration, Oral; Animals; Aorta; Apoptosis; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Disease Models, Animal; DNA-Binding Proteins; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Hypertension; Mice; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phenylbutyrates; Pressure; Transcription Factors; Unfolded Protein Response

Cardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance with RNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit of RNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic response in cardiomyocytes. However, it is unclear whether ET-1-induced hypertrophy is affected by the CTD phosphatase, transcription factor IIF-interacting CTD phosphatase1 (FCP1).. We analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpression of FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes.. The level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression, whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [(3)H]-uridine incorporation showed that the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, the ET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, was suppressed by FCP1 and was not inhibited by dnFCP1.. ET-1-induced cardiac hypertrophy and CTD phosphorylation level are functionally regulated by FCP1. These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controlling phosphorylation level of the RNAPII CTD.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Endothelin-1; Gene Expression Regulation; Humans; Myocytes, Cardiac; Myosin Heavy Chains; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Sprague-Dawley; RNA; RNA Polymerase II; Transcription Factor TFIIH; Up-Regulation

Tissue-specific patterns of gene expression play an important role in the distinctive features of each organ. Small CTD phosphatases (SCPs) 1-3 are recruited by repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. SCPs are highly expressed in the heart and contain microRNAs (miR)-26b, 26a-2, and 26a-1 with the same seed sequence in their introns. Therefore, we tried to investigate the roles of miR-26b and its host gene in neonatal rat cardiomyocytes. Overexpression of miR-26b suppressed the mRNA expression levels of ANF, βMHC, and ACTA1 and reduced the cell surface area in cardiomyocytes. We confirmed that miR-26b targets the 3' untranslated region (3'UTR) of GATA4 and canonical transient receptor potential channel (TRPC) 3. Conversely, silencing of the endogenous miR-26b family enhanced the expression levels of TRPC3 and GATA4. On the other hand, overexpression of SCP1 induced the mRNA expression of ANF and βMHC and increased the cell surface area in cardiomyocytes. Next, we compared the effect of overexpression of SCP1 with its introns and SCP1 cDNA to observe the net function of SCP1 expression on cardiac hypertrophy. When the expression levels of SCP1 were the same, the overexpression of SCP1 cDNA had a greater effect at inducing cardiac hypertrophy than SCP1 cDNA with its intron. In conclusion, SCP1 itself has the potential to induce cardiac hypertrophy; however, the effect is suppressed by intronic miR-26b in cardiomyocytes. miR-26b has an antagonistic effect on its host gene SCP1.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; DNA-Binding Proteins; GATA4 Transcription Factor; Gene Expression Regulation; Genes, Reporter; Introns; Luciferases; Male; Mice; MicroRNAs; Myocytes, Cardiac; Nuclear Proteins; Organ Specificity; Rats; Regulatory Sequences, Nucleic Acid; RNA, Small Interfering; Transfection; TRPC Cation Channels

Acute arsenic (As(III)) exposure has been reported to cause cardiac toxicity, however this toxicity was never linked to the disturbance in cytochrome P450 (P450)-mediated arachidonic acid metabolism. Therefore, we investigated the effect of acute As(III) toxicity on the expression of P450 and soluble epoxide hydrolase (sEH) and their associated arachidonic acid metabolism in mice hearts. As(III) toxicity was induced by a single intraperitoneal injection of 12.5 mg/kg of As(III). Our results showed that As(III) treatment caused a significant induction of the cardiac hypertrophic markers in addition to Cyp1b1, Cyp2b, Cyp2c, Cyp4f, and sEH gene expression in mice hearts. Furthermore, As(III) increased sEH protein expression and activity in hearts with a consequent decrease in 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) formation. Whereas the formation of 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. As(III) also increased sEH mRNA and protein expression levels in addition to the hypertrophic markers which was reversed by knockdown of sEH in H9c2 cells. In conclusion, acute As(III) toxicity alters the expression of several P450s and sEH enzymes with a consequent decrease in the cardioprotective EETs which may represent a novel mechanism by which As(III) causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent As(III)-induced hypertrophy.

Topics: Animals; Arachidonic Acid; Arsenic; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cyclooxygenase 2; Cytochrome P-450 Enzyme System; Cytokines; Epoxide Hydrolases; Gene Expression Regulation; Gene Knockdown Techniques; Male; Mice; Mice, Inbred C57BL; Myocardium; Natriuretic Peptide, Brain; Rats; RNA, Messenger; RNA, Small Interfering; Solubility; Toxicity Tests, Acute

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. In recent years, evidences suggest that high-mobility group box 1 (HMGB1) protein, an inflammatory cytokine, participates in cardiac remodeling; however, the involvement of HMGB1 in the pathogenesis of cardiac hypertrophy remains unknown. The aim of this study was to investigate whether HMGB1 is sufficient to induce cardiomyocyte hypertrophy and to identify the possible mechanisms underlying the hypertrophic response. Cardiomyocytes isolated from 1-day-old Sprague-Dawley rats were treated with recombinant HMGB1, at concentrations ranging from 50 ng/mL to 200 ng/mL. After 24 hours, cardiomyocytes were processed for the evaluation of atrial natriuretic peptide (ANP) and calcineurin A expression. Western blot and real-time RT-PCR was used to detect protein and mRNA expression levels, respectively. The activity of calcineurin was also evaluated using a biochemical enzyme assay. HMGB1 induced cardiomyocyte hypertrophy, characterized by enhanced expression of ANP, and increased protein synthesis. Meanwhile, increased calcineurin activity and calcineurin A protein expression were observed in cardiomyocytes preconditioned with HMGB1. Furthermore, cyclosporin A pretreatment partially inhibited the HMGB1-induced cardiomyocyte hypertrophy. Our findings suggest that HMGB1 leads to cardiac hypertrophy, at least in part through activating calcineurin.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Western; Calcineurin; Cardiomegaly; Cells, Cultured; HMGB1 Protein; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Male; Nitric Oxide Synthase Type III; Signal Transduction

In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide- (specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)-stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling.. Mice with cardiac myocyte inducible PDE5 overexpression (P5(+)) were crossed to those lacking nitric oxide synthase 3 (N3(-)), and each model, the double cross, and controls were subjected to transaortic constriction. P5(+) mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3(-) mice were protected. However, P5(+)/N3(-) mice behaved similarly to P5(+) mice despite the lack of nitric oxide synthase 3-coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide-stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5(+) hearts exhibited higher oxidative stress, whereas P5(+)/N3(-) hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling.. These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide-derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Female; Heart Failure; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Nitric Oxide Synthase Type III; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Ventricular Remodeling

Blacks represent a high-risk population for salt-sensitive hypertension and heart disease, but the underlying mechanism remains unclear. Corin is a cardiac protease that regulates blood pressure by activating natriuretic peptides. A corin gene variant (T555I/Q568P) was identified in blacks with hypertension and cardiac hypertrophy. In this study, we tested the hypothesis that the corin variant contributes to the hypertensive and cardiac hypertrophic phenotype in vivo. Transgenic mice were generated to express wild-type (WT) or T555I/Q568P variant corin in the heart under the control of α-myosin heavy chain promoter. The mice were crossed into a corin knockout (KO) background to create KO/TgWT and KO/TgV mice that expressed WT or variant corin, respectively, in the heart. Functional studies showed that KO/TgV mice had significantly higher levels of proatrial natriuretic peptide in the heart compared with that in control KO/TgWT mice, indicating that the corin variant was defective in processing natriuretic peptides in vivo. By radiotelemetry, corin KO/TgV mice were found to have hypertension that was sensitive to dietary salt loading. The mice also developed cardiac hypertrophy at 12 to 14 months of age when fed a normal salt diet or at a younger age when fed a high-salt diet. The phenotype of salt-sensitive hypertension and cardiac hypertrophy in KO/TgV mice closely resembles the pathological findings in blacks who carry the corin variant. The results indicate that corin defects may represent an important mechanism in salt-sensitive hypertension and cardiac hypertrophy in blacks.

Topics: Animals; Atrial Natriuretic Factor; Black People; Blood Pressure; Blotting, Western; Cardiomegaly; Female; Humans; Hypertension; Male; Mice; Mice, Knockout; Mice, Transgenic; Myocardium; Polymorphism, Single Nucleotide; Serine Endopeptidases; Sodium Chloride, Dietary

Various experimental and clinical studies strongly support a cigarette smoke-heart disease association and suggest possible mechanisms, unfortunately, the involvement of genetic modulations remain unexplored. Thus, the main aim of the current study was to evaluate the effects of sub-chronic cigarette smoke exposure on the mRNA expression of cardiac hypertrophy genes, cytochrome P450 (CYP) enzymes, and the oxidative stress markers in heart rats. For this purpose, Wistar albino rats were exposed to increasing doses of passive cigarette smoke 2, 4, 8, and 24 cigarettes per day for 7 consecutive days. The mRNA expression of fifteen cardiac genes was determined using real-time polymerase chain reaction. Our results showed that the levels of hypertrophic genes; atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain were significantly induced, whereas the anti-hypertrophic gene α-myosin heavy chain was dramatically inhibited, in heart tissues of passive-smoke-exposed groups compared with normal-control groups. This was accompanied with a significant induction of CYP enzymes; CYP1A1, CYP2C11, CYP2E1, and CYP3A2, and the expression of oxidative stress genes, heme oxygenase 1, catalase, cyclooxygenase, and glutathione S-Transferase. The ability of cigarette smoke to induce cardiac hypertrophic genes, CYPs enzymes, and oxidative stress, collectively explore the molecular mechanism of cigarette smoke-induced cardiac diseases and brings further investigative attention to the public health issue of the injurious effects of chronic passive smoke exposure. In conclusion, sub-chronic environmental tobacco smoke exposure increases the incidence of cardiovascular diseases through modulation of cardiac genes.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Catalase; Cyclooxygenase 2; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Glutathione Transferase; Heme Oxygenase (Decyclizing); Isoenzymes; Male; Natriuretic Peptide, Brain; Oxidative Stress; Rats; Rats, Wistar; Tobacco Smoke Pollution; Ventricular Myosins

To investigate the negative regulation of microRNA-1 (miR-1) on L-type calcium channel beta2 subunit (Cavbeta 2) during cardiomyocyte hypertrophy and its mechanism.. Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (HJ2000). The targets of miR-1 were predicted by online database microCosm. The 3' untranslated region sequence of Cavbeta 2 was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression of atrial natriuretic peptide (ANP), beta-myosin heavy chain (beta-MHC), miR-1 and the Cavbeta 2 mRNA were detected by qRT-PCR. The protein expression of Cavbeta 2 was detected by Western blot. The level of miR-1 was up-regulated by miR-1 mimic transfection and the expression level of Cavbeta 2 was down-regulated by RNAi, then effects of which on cardiomyocyte hypertrophy were investigated.. (1) The expression of miR-1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the miR-1 level could suppress the increase of cell surface area, the expression of ANP and beta-MHC mRNA (P < 0.05). (2) Cavbeta 2 was the one of potential targets of miR-1 by prediction using online database microCosm. The luciferase activities of HEK293 cells with the plasmid containing miR-1 and wide type Cavbeta 3' UTR sequence was significantly decreased when compared with that of control group (P < 0.01). Up-regulation of the miR-1 level could suppress the protein expression of Cavbeta 2. (3) The expression of Cavbeta 2 was significantly increased in cardiomyocyte hypertrophy induced by ISO. Downregulation of Cavbeta by RNAi could markedly inhibit the increase of cell surface area, the expression of ANP and beta-MHC mRNA.. Cavbeta2 is one of potential targets of miR-1 by bioinformatics prediction. The experiment data confirms that Cavbeta2 is truly the target of miR-1. MiR-1 can negatively regulate the expression of Cavbeta 2, resulting in the decrease of intracellular Ca2+ content and the attenuation of cardiomyocyte hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Calcium Channels, L-Type; Cardiomegaly; Gene Expression Regulation; HEK293 Cells; Humans; MicroRNAs; Rats; Rats, Sprague-Dawley; Transfection; Ventricular Myosins

Cardiac hypertrophy in response to multiple stimuli has important physiological and pathological significances. GATA4 serves as a nuclear integrator of several signalling pathways during cardiac hypertrophy. Sp1 and Sp3 are also reported to be involved in this process. However, the mechanism by which GATA4 acts as a mediator, integrating these ubiquitously expressed transcriptional factors, is poorly understood. We found that the expression of GATA4 and Sp1 was up-regulated in the myocardium of a pressure overload hypertrophy rat model, as well in phenylephrine-induced (PE-induced) hypertrophic growth of neonatal cardiomyocytes. GST pull-down assays demonstrated that GATA4 could interact with Sp1 in vitro. Therefore, we proposed that GATA4 cooperates with Sp1 in regulating ANF expression, as its reactivation is closely linked with hypertrophy. Further studies demonstrated that GATA4 could activate the ANF promoter synergistically with Sp1 through direct interaction. In contrast, Sp3 exhibited antagonistic function, and overexpression of Sp3 repressed the transcriptional synergy between Sp1 and GATA4. We also found that Sp1 alone could activate the ANF promoter in cardiomyocytes, whereas Sp3 exerted negative effects on ANF expression. Bioinformatics analysis revealed novel Sp-binding sites on the ANF promoter. The recruitment of GATA4 and Sp1 on the ANF promoter was enhanced during phenylephrine-mediated hypertrophy, whereas the recruitment of Sp3 was reduced. The phosphorylation of GATA4 by ERK1/2 kinase could enhance the affinity between GATA4 and Sp1. Thus, our findings revealed the critical interaction of GATA4 and Sp1 in modulating ANF expression, indicating their involvement in cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Binding Sites; Cardiomegaly; Disease Models, Animal; GATA4 Transcription Factor; Gene Expression Regulation; HeLa Cells; Humans; Molecular Sequence Data; Phenylephrine; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Rats; Rats, Sprague-Dawley; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcription, Genetic

In vitro models of cardiac hypertrophy focus exclusively on applying "external" dynamic signals (electrical, mechanical, and chemical) to achieve a hypertrophic state. In contrast, here we set out to demonstrate the role of "self-organized" cellular architecture and activity in reprogramming cardiac cell/tissue function toward a hypertrophic phenotype. We report that in neonatal rat cardiomyocyte culture, subtle out-of-plane microtopographic cues alter cell attachment, increase biomechanical stresses, and induce not only structural remodeling, but also yield essential molecular and electrophysiological signatures of hypertrophy. Increased cell size and cell binucleation, molecular up-regulation of released atrial natriuretic peptide, altered expression of classic hypertrophy markers, ion channel remodeling, and corresponding changes in electrophysiological function indicate a state of hypertrophy on par with other in vitro and in vivo models. Clinically used antihypertrophic pharmacological treatments partially reversed hypertrophic behavior in this in vitro model. Partial least-squares regression analysis, combining gene expression and functional data, yielded clear separation of phenotypes (control: cells grown on flat surfaces; hypertrophic: cells grown on quasi-3-dimensional surfaces and treated). In summary, structural surface features can guide cardiac cell attachment, and the subsequent syncytial behavior can facilitate trophic signals, unexpectedly on par with externally applied mechanical, electrical, and chemical stimulation.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Adhesion; Cell Shape; Cells, Cultured; Electric Stimulation; Genetic Markers; Mechanotransduction, Cellular; Myocardial Contraction; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phenotype; Physical Stimulation; Rats; Signal Transduction; Stimulation, Chemical; Tissue Scaffolds

Myocardial hypertrophy is a common clinical finding leading to heart failure and sudden death. Mitofusin 2 (Mfn2), a hyperplasia suppressor protein, is downregulated in hypertrophic heart. This study examined the role of Mfn2 in myocardial hypertrophy and its potential signal pathway.. In in vitro studies, neonatal cardiac myocytes were isolated and cultured. Incubation of cultured cardiomycytes with angiotensin II (Ang II) inhibited gene expression of Mfn2; induced cell hypertrophy and protein synthesis; and activated protein kinase Akt. Pretreatment of cells with AdMfn2-a replication-deficient adenoviral vector encoding rat Mfn2 gene-upregulated Mfn2 expression and subsequently attenuated Ang II-induced cell hypertrophy; protein synthesis; and Akt activation. In in vivo studies, direct gene delivery of AdMfn2 into myocardium decreased the infusion of Ang II-induced atrial natriuretic factor (ANF, a hypertrophic marker) expression and cardiomyocyte cross-sectional area. Consistently, upregulation of Mfn2 in myocardium decreased the thicknesses of anterior and posterior walls of left ventricle (LV) and the ratio of LV mass/body weight in Ang II-treated rats. Of note, AdGFP (control for AdMfn2) did not affect the effects of Ang II in vitro or in vivo.. Upregulation of Mfn2 inhibits Ang II-induced myocardial hypertrophy. In this process, inhibition of Akt activation seems to play a significant role. These findings indicate Mfn2 is a critical protein in modulating myocyte hypertrophy.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression Regulation; GTP Phosphohydrolases; Membrane Proteins; Mitochondrial Proteins; Myocytes, Cardiac; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Up-Regulation

The present study was designed to develop an animal model of hypertension and cardiac hypertrophy associated with obesity in female rats. Furthermore, we studied the involvement of the natriuretic peptide system in the mechanisms of these conditions. Obesity was induced in Wistar rats by a high fat diet and ovariectomy. The rats were divided into four groups: ovariectomized or sham-operated with high-fat diet and ovariectomized or sham-operated with control diet. After 24 weeks of diet, rats were killed, and their tissues were removed. Cardiac atrial natriuretic peptide (ANP), clearance receptor (NPr-C) gene expression was determined by PCR. ANP concentrations were measured in plasma. Ovariectomized fat-fed rats (OF) showed increased body weight, visceral fat depot and blood pressure and decreased sodium excretion compared to other groups. Also, these rats showed higher heart-to-body weight and cell diameters of ventricular cardiomyocytes and lower cardiac ANP mRNA and plasma ANP than the control group. The adipocyte and renal NPr-C mRNA of OF rats were higher than the control group. These data showed that combined ovariectomy and high fat diet elicited obesity, hypertension and cardiac hypertrophy. These results suggest that the impairment of the natriuretic peptide system may be one of the mechanisms involved not only in development of hypertension but also in cardiac hypertrophy associated with obesity in ovariectomized rats.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Diet; Dietary Fats; Disease Models, Animal; Female; Gene Expression; Heart; Hypertension; Intra-Abdominal Fat; Obesity; Organ Size; Ovariectomy; Ovary; Rats; Rats, Wistar; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Sodium

Cardiac hypertrophy involves growth responses to a variety of stimuli triggered by increased workload. It is an independent risk factor for heart failure and sudden death. Mammalian target of rapamycin (mTOR) plays a key role in cellular growth responses by integrating growth factor and energy status signals. It is found in 2 structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC) 1 and mTORC2. The role of each of these branches of mTOR signaling in the adult heart is currently unknown.. We generated mice with deficient myocardial mTORC1 activity by targeted ablation of raptor, which encodes an essential component of mTORC1, during adulthood. At 3 weeks after the deletion, atrial and brain natriuretic peptides and β-myosin heavy chain were strongly induced, multiple genes involved in the regulation of energy metabolism were altered, but cardiac function was normal. Function deteriorated rapidly afterward, resulting in dilated cardiomyopathy and high mortality within 6 weeks. Aortic banding-induced pathological overload resulted in severe dilated cardiomyopathy already at 1 week without a prior phase of adaptive hypertrophy. The mechanism involved a lack of adaptive cardiomyocyte growth via blunted protein synthesis capacity, as supported by reduced phosphorylation of ribosomal S6 kinase 1 and 4E-binding protein 1. In addition, reduced mitochondrial content, a shift in metabolic substrate use, and increased apoptosis and autophagy were observed.. Our results demonstrate an essential function for mTORC1 in the heart under physiological and pathological conditions and are relevant for the understanding of disease states in which the insulin/insulin-like growth factor signaling axis is affected such as diabetes mellitus and heart failure or after cancer therapy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Atrial Natriuretic Factor; Autophagy; Cardiomegaly; Carrier Proteins; Cell Cycle Proteins; Energy Metabolism; Eukaryotic Initiation Factors; Gene Expression; Heart Failure; Heart Rate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myosin Heavy Chains; Natriuretic Peptide, Brain; Nonmuscle Myosin Type IIB; Phosphoproteins; Phosphorylation; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases, 90-kDa

Cardiac hypertrophy, a major determinant of heart failure, is associated with heat shock proteins (HSPs). HSP75 has been reported to protect against environmental stresses; however, its roles in cardiac hypertrophy remain unclear. Here, we generated cardiac-specific inducible HSP75 transgenic mice (TG) and cardiac hypertrophy was developed at 4 weeks after aortic banding in TG mice and wild-type littermates. The results revealed that overexpression of HSP75 prevented cardiac hypertrophy and fibrosis as assessed by heart weight/body weight ratio, heart weight/tibia length ratio, echocardiographic and hemodynamic parameters, cardiomyocyte width, left ventricular collagen volume, and gene expression of hypertrophic markers. Further studies showed that overexpression of HSP75 inhibited the activation of TAK/P38, JNK, and AKT signaling pathways. Thus, HSP75 likely reduces the hypertrophy and fibrosis induced by pressure overload through blocking TAK/P38, JNK, and AKT signaling pathways.

Topics: Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Cell Size; Collagen; Fibrosis; Heart Ventricles; HSP90 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Cells; Myocardium; Myosin Heavy Chains; Organ Size; Phosphorylation; Recombinant Proteins; Transcription, Genetic; Ventricular Pressure; Ventricular Remodeling

Pathological cardiac hypertrophy induced by adrenergic overactivation can subsequently develop to heart failure which remains as a leading cause of mortality worldwide. Tanshinone IIA is a lipid-soluble pharmacologically active compound extracted from the rhizome of the Chinese herb Salvia miltiorrhiza, a well-known traditional Chinese medicine used for the treatment of cardiovascular disorders. However, little is know about the effect of Tanshinone IIA on cardiac hypertrophy. The present study was aimed to investigate whether Tanshinone IIA prevents cardiac hypertrophy induced by isoproterenol (ISO) and to clarify its possible mechanisms. Cardiomyocytes hypertrophy was induced by ISO 10 μM for 48 h with or without Tanshinone IIA 10, 30, 100 μM pretreatment, and evaluated by determining the cell size and the expression of ANP, BNP, β-MHC, Calcineurin, and NFATc3 by real-time PCR and western blot. We found that Tanshinone IIA pretreatment attenuated the enlargement of cell surface area induced by ISO in cultured cardiomyocytes. The mRNA level of ANP, BNP and β-MHC was obviously elevated in ISO-treated cardiac cells, which was effectively inhibited by Tanshinone IIA. Moreover, we found that Tanshinone IIA pretreatment could prevent the augment of intracellular calcium transient in ISO-treated cardiomyocytes. The further study revealed that Calcineurin, NFATc3, ANP, BNP and β-MHC proteins were upregulated by ISO in ventricular myocytes, and Tanshinone IIA pretreatment significantly attenuate the increased expression of Calcineurin, NFATc3, ANP, BNP and β-MHC proteins. In summary, Tanshinone IIA attenuated cardiomyocyte hypertrophy induced by ISO through inhibiting Calcineurin/NFATc3 pathway, which provides new insights into the pharmacological role and therapeutic mechanism of Tanshinone IIA in heart diseases.

Topics: Abietanes; Animals; Atrial Natriuretic Factor; Calcineurin; Calcium Signaling; Cardiomegaly; Drugs, Chinese Herbal; Isoproterenol; Medicine, Chinese Traditional; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; NFATC Transcription Factors; Rats; RNA, Messenger; Up-Regulation

Activation of Ras signaling in cardiomyocytes has been linked to pathogenic myocardial hypertrophy progression and subsequent heart failure. Whether cardiomyopathy can regress once initiated needs to be established more fully. A 'tet-off' system was used to regulate expression of H-Ras-G12V in myocardium to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling in vivo. Ras activation at weaning for 2 wk caused hypertrophy, whereas activation for 4 to 8 wk led to cardiomyopathy and heart failure. Discontinuing H-Ras-G12V transgene expression after cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart:body weight ratios, demonstrating the reversibility of early pathogenic hypertrophy. Activation of Ras and downstream ERK 1/2 was associated with elevated expression of proliferating cell nuclear antigen and cyclins B1 and D1, indicating cell-cycle activation and reentry. Coordinate elevation of broad-spectrum cyclin-dependent kinase inhibitors (p21, p27, and p57) and Tyr15 phosphorylation of cdc2 signified the activation of cell-cycle checkpoints; absence of cell-cycle completion and cardiomyocyte replication were documented by using immunohistochemistry for mitosis and cytokinesis markers. After resolution of cardiomyopathy, cell-cycle activators and inhibitors examined returned to basal levels, a change that we interpreted as exit from the cell cycle. Cardiac cell-cycle regulation plays a role in recovery from pathogenic hypertrophy. The model we present provides a means to further explore the underlying mechanisms governing cell-cycle capacity in cardiomyocytes, as well as progression and regression of pathogenic cardiomyocyte hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Cycle; Cell Cycle Proteins; MAP Kinase Signaling System; Mice; Mice, Inbred Strains; Mice, Transgenic; Mitogen-Activated Protein Kinase 3; Myocardium; ras Proteins; Transgenes

We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy.. Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization.. We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes.. The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-β-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression.. These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Caveolae; Caveolin 3; Cyclic GMP; Heart Failure; Immunoenzyme Techniques; In Vitro Techniques; Mice; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; Natriuretic Peptide, Brain; NFATC Transcription Factors; RNA, Messenger

Repeated sauna therapy (ST) increases endothelial nitric oxide synthase (eNOS) activity and improves cardiac function in heart failure as well as peripheral blood flow in ischemic limbs. The present study investigates whether ST can increase coronary vascularity and thus attenuate cardiac remodeling after myocardial infarction (MI). We induced MI by ligating the left coronary artery of Wistar rats. The rats were placed in a far-infrared dry sauna at 41°C for 15 min and then at 34°C for 20 min once daily for 4 wk. Cardiac hemodynamic, histopathological, and gene analyses were performed. Despite the similar sizes of MI between the ST and non-ST groups (51.4 ± 0.3 vs. 51.1 ± 0.2%), ST reduced left ventricular (LV) end-diastolic (9.7 ± 0.4 vs. 10.7 ± 0.5 mm, P < 0.01) and end-systolic (8.6 ± 0.5 vs. 9.6 ± 0.6 mm, P < 0.01) dimensions and attenuated MI-induced increases in LV end-diastolic pressure. Cross-sectional areas of cardiomyocytes were smaller in ST rats and associated with a significant reduction in myocardial atrial natriuretic peptide mRNA levels. Vascular density was reduced in the noninfarcted myocardium of non-ST rats, and the density of cells positive for CD31 and for α-smooth muscle actin was decreased. These decreases were attenuated in ST rats compared with non-ST rats and associated with increases in myocardial eNOS and vascular endothelial growth factor mRNA levels. In conclusion, ST attenuates cardiac remodeling after MI, at least in part, through improving coronary vascularity in the noninfarcted myocardium. Repeated ST might serve as a novel noninvasive therapy for patients with MI.

Topics: Actins; Analysis of Variance; Animals; Atrial Natriuretic Factor; Cardiomegaly; Chi-Square Distribution; Coronary Vessels; Disease Models, Animal; Gene Expression Regulation; Hemodynamics; Male; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; RNA, Messenger; Steam Bath; Time Factors; Ultrasonography; Vascular Endothelial Growth Factor A; Ventricular Function, Left; Ventricular Pressure; Ventricular Remodeling

Reactive oxygen species (ROS) induce matrix metalloproteinase (MMP) activity that mediates hypertrophy and cardiac remodeling. Adiponectin (APN), an adipokine, modulates cardiac hypertrophy, but it is unknown if APN inhibits ROS-induced cardiomyocyte remodeling. We tested the hypothesis that APN ameliorates ROS-induced cardiomyocyte remodeling and investigated the mechanisms involved. Cultured adult rat ventricular myocytes (ARVM) were pretreated with recombinant APN (30 μg/ml, 18 h) followed by exposure to physiologic concentrations of H(2)O(2) (1-200 μM). ARVM hypertrophy was measured by [(3)H]leucine incorporation and atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) gene expression by RT-PCR. MMP activity was assessed by in-gel zymography. ROS was induced with angiotensin (ANG)-II (3.2 mg·kg(-1)·day(-1) for 14 days) in wild-type (WT) and APN-deficient (APN-KO) mice. Myocardial MMPs, tissue inhibitors of MMPs (TIMPs), p-AMPK, and p-ERK protein expression were determined. APN significantly decreased H(2)O(2)-induced cardiomyocyte hypertrophy by decreasing total protein, protein synthesis, ANF, and BNP expression. H(2)O(2)-induced MMP-9 and MMP-2 activities were also significantly diminished by APN. APN significantly increased p-AMPK in both nonstimulated and H(2)O(2)-treated ARVM. H(2)O(2)-induced p-ERK activity and NF-κB activity were both abrogated by APN pretreatment. ANG II significantly decreased myocardial p-AMPK and increased p-ERK expression in vivo in APN-KO vs. WT mice. ANG II infusion enhanced cardiac fibrosis and MMP-2-to-TIMP-2 and MMP-9-to-TIMP-1 ratios in APN-KO vs. WT mice. Thus APN inhibits ROS-induced cardiomyocyte remodeling by activating AMPK and inhibiting ERK signaling and NF-κB activity. Its effects on ROS and ultimately on MMP expression define the protective role of APN against ROS-induced cardiac remodeling.

Topics: Adiponectin; AMP-Activated Protein Kinases; Analysis of Variance; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Genes, Reporter; Hydrogen Peroxide; Hypertrophy, Left Ventricular; Male; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; NADPH Oxidases; Natriuretic Peptide, Brain; NF-kappa B; Oxidants; Oxidative Stress; Phosphorylation; Rats; Reactive Oxygen Species; Recombinant Proteins; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Ventricular Remodeling

The overexpression of the transcription factor, E2F1, induces hypertrophy and apoptosis with cell cycle re-entry in cardiomyocytes in vitro and in vivo, suggesting that targeting E2F1 may have therapeutic potential. Accordingly, we tested the hypothesis that blocking the E2F1-mediated signal transduction pathway prevents cardiac hypertrophy by treating E2F1 knockout mice (E2F1-/-) with either isoproterenol (ISO) or Angiotensin II (ANG). Echocardi-ography was used to measure left ventricular mass index and myocardial performance index, a measure of combined systolic and diastolic left ventricular function. In control mice (E2F1+/+) both ISO and ANG treatments induced cardiac hypertrophy, and impaired ventricular function in ANG treated mice. In contrast to previously published work, E2F1-/- mice also demonstrated a similar pattern of cardiac hypertrophy and function after either treatment. Atrial natriuretic peptide, a molecular marker of hypertrophy and necropsy-determined body weight-normalized left ventricle mass were similarly increased in ISO and ANG treated E2F1+/+ and E2F-/- mice, supporting the echocardiographic data. These data indicate that E2F1 is not necessary for the development of cardiac hypertrophy although studies using an overexpression approach suggest a causal role of E2F1. The reason for this discrepancy is unclear, although it is possible that other E2F-family members (e.g., E2F2) may play a compensatory role. In conclusion, our data demonstrate that cardiac hypertrophy can be induced in an E2F1-independent fashion and suggest that in contrast to previous reports, targeting E2F1 may not be a good therapeutic approach.

Topics: Angiotensin II; Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Hypertrophic; Cardiotonic Agents; Cell Cycle; E2F1 Transcription Factor; Gene Expression Regulation; Humans; Isoproterenol; Male; Mice; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; RNA, Messenger; Signal Transduction; Vasoconstrictor Agents

To investigate the role of miRNA-199a on cardiac hypertrophy.. (1) Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC, n = 6) and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the change of microRNAs (miRNAs). (2) Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats. The myocytes were divided into two groups: adenovirus miRNA-199a (Ad-miRNA-199a) or adenovirus vector (Ad-vector). They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000. qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain (αMHC, myh6), β-myosin heavy chain (βMHC, myh7) and atrial natriuretic peptide (ANP, Nppa). Software Axio Vision was used to detect the change of cardiomyocytes surface areas. (3) Neonatal rat ventricular myocytes were divided into two groups: antisense oligonucleotide-miRNA-199a (As-miRNA-199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups: A: control (ctl), B: phenylephrine (PE), C: PE + As-ctl, D: PE + As-miRNA-199a. qRT-PCR was used to detect the change of myh6, myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas.. (1) qRT-PCR results showed that miRNA-1, miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA-199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA-199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7 were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups.. miRNA-199a may play a regulatory role in cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Male; MicroRNAs; Myosin Heavy Chains; Rats; Rats, Sprague-Dawley; RNA, Messenger

Hypertension is the main risk factor for left ventricular hypertrophy and development of diastolic heart failure. There is no yet treatment, which can effectively reduce mortality in patients suffering from heart failure with preserved systolic function. We tested whether the calcium sensitizer levosimendan and the AT1-receptor antagonist valsartan could protect from salt-induced hypertension, cardiovascular mortality and heart failure in Dahl/Rapp salt-sensitive rats fed for 7 weeks with a high salt diet (8% NaCl). Levosimendan (1 mg/kg/day via drinking water) and valsartan (30 mg/kg in the food) monotherapies and their combination prevented mortality in Dahl/Rapp rats. The drug combination evoked an additive effect on blood pressure, cardiac hypertrophy, cardiomyocyte cross-sectional area, target organ damage and myocardial ANP mRNA expression. There was a close correlation between systolic blood pressure and cardiac hypertrophy, cardiac and renal damage. As compared to Dahl/Rapp controls kept on low-salt diet (NaCl 0.3%). The high salt rats exhibited impaired diastolic relaxation as assessed by isovolumic relaxation time. Levosimendan alone and in combination with valsartan, improved diastolic relaxation without significantly improving systolic function. Our findings are evidence for an additive effect between levosimendan and valsartan on blood pressure and a blood pressure-dependent protection against the development of salt-induced target organ damage. The present study also demonstrates that levosimendan, alone or in combination with valsartan, can correct diastolic dysfunction induced by salt-dependent hypertension.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cardiovascular Diseases; Diet, Sodium-Restricted; Echocardiography; Heart; Heart Failure; Heart Rate; Hydrazones; Hypertension; Kidney; Male; Myocytes, Cardiac; Pyridazines; Rats; Rats, Inbred Dahl; RNA, Messenger; Simendan; Sodium Chloride, Dietary; Sodium, Dietary; Tetrazoles; Valine; Valsartan; Vasodilator Agents; Ventricular Remodeling

C57BL/6-Kit(W-sh/W-sh) mice are generally regarded as a mast cell-deficient model, as they lack the necessary kit receptor for mast cell development. Further characterization of this strain, however, indicates that C57BL/6-Kit(W-sh/W-sh) mice also have a disruption in the Corin gene. Corin is a transmembrane serine protease critical for processing atrial natriuretic peptide (ANP) from pro-ANP through proteolytic cleavage. Pro-ANP is produced, stored and released by cardiac myocytes in response to atrial stretch and the stress generated by increased afterload such as increased ventricular pressure from aortic stenosis or myocardial infarction. ANP inhibits the effects of the renin-angiotensin system to preserve homeostasis under conditions of increased hemodynamic load, and changes in the level of its activating enzyme Corin have been observed during the progression to heart failure. Here, we investigate the effect of increased hemodynamic load on Corin-deficient C57BL/6-Kit(W-sh/W-sh) mice. Ten-week old male mice were subjected to transverse aortic constriction for 8 weeks and were monitored for changes in cardiac structure and function by echocardiography. Hearts were collected 8 weeks after surgery for molecular and histological analyses. Corin-deficient C57BL/6-Kit(W-sh/W-sh) mice developed rapidly progressive and substantial left ventricular dilation, hypertrophy, and markedly impaired cardiac function during the 8 weeks after surgery, compared to wildtype mice. Concomitant with this we observed increased levels of ANP transcript, but a lack of prepro-ANP or pro-ANP protein in heart tissue extracted from Corin-deficient mice. Surprisingly, fibrosis was not increased in Corin-deficient mice when compared to wildtype mice. These data indicate that Corin's involvement in ANP processing is a key element in the heart's response to increased hemodynamic load. Further, C57BL/6-Kit(W-sh/W-sh) strain is an effective model for investigating the involvement of Corin and, conversely, a less than optimal model for investigating mast cell, and immunological, functions in certain cardiovascular pathologies.

Topics: Animals; Aortic Diseases; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Echocardiography, Doppler; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Reverse Transcriptase Polymerase Chain Reaction; Serine Endopeptidases; Ventricular Dysfunction, Left

Activation of nuclear factor kappaB (NF-kappaB) has been found necessary for cardiac hypertrophic growth in vivo and in vitro experiments. Adiponectin, an adipocyte-derived polypeptide, suppresses cardiac hypertrophy in response to pressure overload. Here we investigated the potential effect of adiponectin on NF-kappaB activation in hypertrophic neonatal rat ventricular myocytes (NRVMs) and related signal transduction pathway. We treated NRVMs with globular adiponectin (gAd) before angiotensin II (AngII) stimulation. Pretreating cells with gAd reduced the increased incorporation of [(3)H]-leucine and the mRNA levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) stimulated by AngII, indicating gAd inhibited AngII-induced cardiac hypertrophic signaling. Moreover, gAd pretreatment suppressed inhibitory protein kappaB (I-kappaB) phosphorylation and decreased p65 nuclear translocation, DNA-binding and transcription activity of NF-kappaB. Meanwhile, gAd promoted AMP-activated protein kinase (AMPK) phosphorylation, which is a downstream signaling mediator of adiponectin. Pharmacological activator of AMPK could inhibit AngII-induced NF-kappaB translocation, and inhibitor of AMPK or a dominant-negative AMPK adenovirus suppressed gAd-mediated inhibition of I-kappaB phosphorylation and NF-kappaB activation. When AMPK was inhibited, the suppressive effect of gAd on ANP mRNA expression was reduced. Our data indicate that gAd inhibits cardiac hypertrophic signaling through AMPK mediated suppression of NF-kappaB activation.

Topics: Adiponectin; AMP-Activated Protein Kinases; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Enzyme Activation; Heart Ventricles; Humans; I-kappa B Proteins; Myocytes, Cardiac; NF-kappa B; Phosphorylation; Rats

The mixed-lineage kinases (MLKs) act upstream of mitogen-activated protein kinases, but their role in cardiac biology and pathology is largely unknown.. We investigated the effect of a MLK1-3 inhibitor CEP-11004 on G protein-coupled receptor agonist-induced stress response in neonatal rat cardiac myocytes in culture.. CEP-11004 administration dose-dependently attenuated phenylephrine and endothelin-1 (ET-1)-induced c-Jun N-terminal kinase activation. MLK inhibition also reduced ET-1- and phenylephrine-induced phosphorylation of p38 mitogen-activated protein kinase. In contrast, phenylephrine-induced extracellular signal-regulated kinase phosphorylation was further up-regulated by CEP-11004. ET-1 increased activator protein-1 binding activity 3.5-fold and GATA-binding protein 4 (GATA-4) binding activity 1.8-fold, both of which were attenuated with CEP-11004 administration by 59% and 63% respectively. Phenylephrine induced activator protein-1 binding activity by 2.6-fold, which was decreased by 81% with CEP-11004 administration. Phenylephrine also induced a 3.7-fold increase in the transcriptional activity of B-type natriuretic peptide (BNP), which was attenuated by 41% with CEP-11004 administration. In agreement, MLK inhibition also reduced hypertrophic agonist-induced secretion of immunoreactive atrial natriuretic peptide and BNP.. These results showed that inhibition of the MLK1-3 signalling pathway was sufficient for suppressing the activity of key nuclear effectors (GATA-4 and activator protein-1 transcription factors) in cardiac hypertrophy, and attenuated the agonist-induced atrial natriuretic peptide secretion and activation of BNP gene transcription.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Carbazoles; Cardiomegaly; Cell Nucleus; Endothelin-1; Genes, jun; Heart; Hypertrophy; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Transcription Factor AP-1; Transcription Factors

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.

Topics: Adrenergic beta-Agonists; Angiotensin II; Animals; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Cell Line; Cyclic GMP-Dependent Protein Kinases; Humans; Isoproterenol; Kidney; Membrane Potentials; Mice; Mice, Knockout; Myocytes, Cardiac; Patch-Clamp Techniques; Receptors, Atrial Natriuretic Factor; RGS Proteins; TRPC Cation Channels; TRPC6 Cation Channel; Vasoconstrictor Agents

Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3',5'-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Cardiomegaly; Catalytic Domain; Cell Line; Cyclic GMP; Guanylate Cyclase; Heart Failure; Humans; Kidney; Natriuretic Peptide, Brain; Oligopeptides; Peptides; Phosphopeptides; Phosphorylation; Rats; Receptors, Atrial Natriuretic Factor; Second Messenger Systems

The β(2)-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.

Topics: Actins; Adrenergic beta-2 Receptor Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Cell Size; Cells, Cultured; Clenbuterol; Fibroblasts; Gene Expression Regulation; Insulin-Like Growth Factor I; Myocytes, Cardiac; Natriuretic Peptide, Brain; Paracrine Communication; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection

It has been shown that angiotensin II (Ang II) is involved in cardiac remodeling mediated by NADPH oxidase-dependent reactive oxygen species (ROS). Accordingly, NADPH oxidase-dependent ROS may play a role in cardiac hypertrophy induced by pressure overload. In the present study, we sought to determine whether inhibition of NADPH oxidase prevents cardiac hypertrophy. After abdominal aorta banding to induce cardiac hypertrophy, rats were treated for 8 weeks with apocynin (Apo) or captopril (Cap). Measures of cardiac hypertrophy were evaluated. Treatment with Cap or Apo reduced the left ventricle/body weight ratio (LV/BW), LV transnuclear myocyte diameter, and atrial natriuretic factor (ANF) mRNA expression relative to those of untreated rats subjected to aorta banding. The activity of NADPH oxidase and the ROS levels were decreased in treated animals. Cap, but not Apo, decreased Ang II levels and inhibited expression of p22phox and p67phox in LVs. In conclusion, local expression of Ang II appears to contribute to pressure overload-induced cardiac hypertrophy by upregulating NADPH oxidase expression and promoting ROS synthesis. Inhibition of NADPH oxidase and elimination of ROS may prevent or repair damage due to cardiac hypertrophy.

Topics: Acetophenones; Angiotensin II; Animals; Antioxidants; Atrial Natriuretic Factor; Base Sequence; Captopril; Cardiomegaly; DNA Primers; Enzyme Inhibitors; Male; NADPH Oxidases; Phosphoproteins; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger

Heparin cofactor II (HCII), a serine protease inhibitor, inhibits tissue thrombin action after binding with dermatan sulfate proteoglycans in the extracellular matrix of the vascular system. We previously reported that heterozygous HCII-deficient (HCII(+/-)) humans and mice demonstrate acceleration of vascular remodeling, including atherosclerosis. However, the action of HCII on cardiac remodeling never has been determined. HCII(+/+) and HCII(+/-) mice at age 25 weeks were infused with angiotensin II (Ang II; 2.0 mg/kg/d) for 2 weeks by an osmotic mini-pump. Echocardiography revealed acceleration of cardiac concentric remodeling in HCII(+/-) mice and larger left atrial volume in HCII(+/-) mice than in HCII(+/+) mice. Histopathologic studies showed more prominent interstitial fibrosis in both the left atrium and left ventricle in HCII(+/-) mice than in HCII(+/+) mice. Daily urinary excretion of 8-hydroxy-2'-deoxyguanosine, a parameter of oxidative stress, and dihydroethidium-positive spots, indicating superoxide production in the myocardium, were markedly increased in Ang II-treated HCII(+/-) mice compared to those in HCII(+/+) mice. Cardiac gene expression levels of atrial natriuretic peptides and brain natriuretic peptides, members of the natriuretic peptide family, Nox 4, Rac-1, and p67(phox) as components of NAD(P)H oxidase, and transforming growth factor-beta1 and procollagen III were more augmented in HCII(+/-) mice than in HCII(+/+) mice. However, administration of human HCII protein attenuated all of those abnormalities in Ang II-treated HCII(+/-) mice. Moreover, human HCII protein supplementation almost abolished cardiac fibrosis in Ang II-treated HCII(+/+) mice. The results indicate that HCII has a protective role against Ang II-induced cardiac remodeling through suppression of the NAD(P)H oxidase-transforming growth factor-beta1 pathway.

Topics: Analysis of Variance; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Fibrosis; Heart Atria; Heparin Cofactor II; Mice; Mice, Transgenic; Myocardium; Natriuretic Peptide, Brain; Oxidative Stress; Transforming Growth Factor beta1; Ventricular Remodeling

An increasing amount of evidence demonstrates the beneficial role of oxytocin (OT) in the cardiovascular system. Similar actions are attributed to genistein, an isoflavonic phytoestrogen. The treatment with genistein activates the OT system in the aorta of ovariectomized (OVX) Sprague-Dawley (SD) rats. The objective of this study was to determine the effects of low doses of genistein on the OT-induced effects in rat hypertension. The hypothesis tested was that treatment of OVX spontaneously hypertensive rats (SHRs) with genistein improves heart structure and heart work through a mechanism involving the specific OT receptor (OTR). OVX SHRs or SD rats were treated with genistein (in microg/g body wt sc, 10 days) in the presence or absence of an OT antagonist (OTA) [d(CH(2))(5), Tyr(Me)(2), Orn(8)]-vasotocin or a nonspecific estrogen receptor antagonist (ICI-182780). Vehicle-treated OVX rats served as controls. RT-PCR and Western blot analysis demonstrated that left ventricular (LV) OTR, downregulated by ovariectomy, increased in response to genistein. In SHRs or SD rats, this effect was blocked by OTA or ICI-182780 administration. The OTR was mainly localized in microvessels expressing the CD31 marker and colocalized with endothelial nitric oxide synthase. In SHRs, the genistein-stimulated OTR increases were associated with improved fractional shortening, decreased blood pressure (12 mmHg), decreased heart weight-to-body weight ratio, decreased fibrosis, and lowered brain natriuretic peptide in the LV. The prominent finding of the study is the detrimental effect of OTA treatment on the LV of SHRs. OTA treatment of OVX SHRs resulted in a dramatic worsening of ejection fractions and an augmented fibrosis. In conclusion, these results demonstrate that cardiac OTRs are involved in the regulation of cardiac function of OVX SHRs. The decreases of OTRs may contribute to cardiac pathology following menopause.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Female; Fibrosis; Fulvestrant; Genistein; Hypertension; Myocardial Contraction; Myocardium; Natriuretic Peptide, Brain; Ovariectomy; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Receptors, Estrogen; Receptors, Oxytocin; RNA, Messenger; Vasotocin; Ventricular Function, Left; Ventricular Pressure; Ventricular Remodeling

Recent evidence has shown that prolonged exposure to exogenous tissue factor (TF) can alter the cellular functions of cardiomyocytes resulting in cardiac dysfunction. The effect of TF may arise from local inflammation within or in the vicinity of the heart. The aim of this study was to investigate the effect of TF on cardiomyocyte proliferation and growth. H9c2 rat cardiomyocytes were exposed to a range of concentrations of recombinant TF (rTF) (1.3-52 ng/ml) for up to 10 days and the outcome on cell proliferation and induction of apoptosis measured. At lower concentrations examined (1.3 ng/ml), rTF had a proliferative influence on the H9c2 cells. In contrast, elevated concentrations of rTF (52 ng/ml) induced cellular apoptosis as indicated by increased caspase-3 activity and nuclear localisation of p53. Moreover, incubation with intermediate concentrations of rTF (13 ng/ml) resulted in an initial increase in proliferation but subsequently, led to cellular apoptosis by day 7 of the incubation. In order to determine if these effects induced hypertrophic cell growth, expression of mechano-growth factor (MGF) was analysed. Incubation of cells with rTF resulted in enhanced expression of MGF particularly at the intermediate concentrations of rTF (13 ng/ml) as well as mean cellular transverse diameter. In addition, there was a rapid increase in the expression of atrial natriuretic factor (ANF) in the cells, on incubation with rTF but diminished rapidly when exposed to higher concentrations of rTF. These data indicate that exposure to increasing concentrations of rTF can accelerate the rate of cardiomyocyte turnover which may ultimately lead to depletion of viable cells within the heart. Moreover, at lower concentrations of rTF, the induction of cell proliferation together with hypertrophic markers indicates that rTF may contribute to the induction and progression of cardiac hypertrophy.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; Insulin-Like Growth Factor I; Myocytes, Cardiac; Rats; Recombinant Proteins; Thromboplastin

Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.. The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.. Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.. Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Topics: Adrenomedullin; Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Gene Transfer Techniques; Genetic Vectors; Humans; Myocytes, Cardiac; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Pressure; Rats; Superoxide Dismutase

Angiotensin-(1-7) displays antihypertensive and antiproliferative properties although its effect on cardiac remodeling and hypertrophy in hypertension has not been fully elucidated. The present study was designed to examine the effect of chronic angiotensin-(1-7) treatment on myocardial remodeling, cardiac hypertrophy and underlying mechanisms in spontaneous hypertension. Adult male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were treated with or without angiotensin-(1-7) or the angiotensin-(1-7) antagonist A-779 for 24 weeks. Mean arterial pressure, left ventricular geometry, expression of the hypertrophic markers ANP and β-MHC, collagen contents (type I and III), collagenase (MMP-1), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of MMPs-1 (TIMP-1) were evaluated in WKY and SHR rats with or without treatment. Our data revealed that chronic angiotensin-(1-7) treatment significantly suppressed hypertension, left ventricular hypertrophy, expression of ANP and β-MHC as well as myocardial fibrosis in SHR rats, the effects of which were nullified by the angiotensin-(1-7) receptor antagonist A-779. In addition, angiotensin-(1-7) treatment significantly counteracted hypertension-induced changes in the mRNA expression of MMP-2 and TIMP-1 and collagenase activity, the effects of which were blunted by A-779. In vitro study revealed that angiotensin-(1-7) directly increased the activity of MMP-2 and MMP-9 while decreasing the content of TIMP-1 and TIMP-2. Taken together, our results revealed a protective effect of angiotensin-(1-7) against cardiac hypertrophy and collagen deposition, which may be related to concerted changes in MMPs and TIMPs levels. These data indicated the therapeutic potential of angiotensin-(1-7) in spontaneous hypertension-induced cardiac remodeling.

Topics: Angiotensin I; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Collagen; Collagenases; Fibrosis; Hypertension; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Myocardium; Myosin Heavy Chains; Peptide Fragments; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tissue Inhibitor of Metalloproteinases

To observe the effect of Qi-Benefiting, Blood-Activating Recipe on the different pathologic stage of myocardial hypertrophy induced by ISO in rats.. Myocardial hypertrophy rats were induced by isoproterenol, and treated with Qi-Benefiting, Blood-Activating Recipe for 5,10 and 15 weeks, hemodynamic parameters, LVMI, HMI were determined, ANP and BNP were analysed.. Qi-Benefiting, Blood-Activating Recipe could increase cardiac output and improve hemodynamic parameters all after 5, 10 and 15 weeks' treatment (P<0.05). It could decrease the contents of ANP and BNP after 5, 10 and 15 weeks' treatment (P<0.05). Qi-Benefiting, Blood-Activating Recipe could significantly decrease the levels of HWI and LVMI after 5, 10 and 15 weeks' treatment (P<0.05).. Qi-Benefiting, Blood-Activating Recipe can significant improve hemodynamic status, increase cardiac output and decrease the level of neurohormonal factors.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Output; Cardiomegaly; Drug Combinations; Drugs, Chinese Herbal; Isoproterenol; Male; Myocardium; Natriuretic Peptide, Brain; Plants, Medicinal; Qi; Random Allocation; Rats; Rats, Sprague-Dawley; Ventricular Function, Left

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Myosins; Cardiomegaly; Cell Differentiation; Cell Enlargement; Cell Line; Cell Size; DNA Damage; Doxorubicin; Drug Synergism; Gene Expression; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Myoblasts, Cardiac; Myocytes, Cardiac; Myosin Heavy Chains; Myosin Light Chains; Phosphorylation; Proteins; Rats; Tretinoin

Angiotensin II (AngII) type 1-receptor blockers (ARBs) have been effectively used not only in the treatment of hypertension but also in cardiac protection. However, whether and why there are differences in these effects still remain unclear. Here we compared the effects of five commonly used ARBs (Candesartan, Olmesartan, Losartan, Telmisartan and Valsartan) on pressure overload-induced cardiac hypertrophy in mice model. Pressure overload was produced by constriction of the transverse aorta (TAC) for 2 weeks, which induced a significant elevation of blood pressure; ARBs or saline was administered through a stomach tube; Cardiac hypertrophy was evaluated by transthoracic echocardiography, cardiac histology and specific gene expression analyses. Although all the five ARBs, which did not repress the elevation of left ventricular pressure after TAC, attenuated the development of cardiac hypertrophy in the wild-type mice, the degrees of regression by Candesartan, Olmesartan and Losartan tended to be larger than those by Telmisartan and Valsartan. Furthermore, in angiotensinogen-knockout mice lacking endogenous AngII, TAC-induced cardiac hypertrophy was regressed by Candesartan, Olmesartan and Losartan but not by Telmisartan and Valsartan administration. Our data suggest that Candesartan, Olmesartan and Losartan can effectively inhibit pressure overload-induced cardiac hypertrophy even in the absence of AngII, whereas Telmisartan and Valsartan could exert the inhibitory effects only in the presence of AngII.

Topics: Analysis of Variance; Angiotensin II Type 1 Receptor Blockers; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Benzimidazoles; Benzoates; Biphenyl Compounds; Blood Pressure; Cardiomegaly; Echocardiography; Gene Expression; Hemodynamics; Imidazoles; Losartan; Male; Mice; Myocardium; Reverse Transcriptase Polymerase Chain Reaction; Telmisartan; Tetrazoles; Treatment Outcome; Valine; Valsartan

The lysosomal cysteine peptidase cathepsin L (CTSL) is an important lysosomal proteinase involved in a variety of cellular functions including intracellular protein turnover, epidermal homeostasis, and hair development. Deficiency of CTSL in mice results in a progressive dilated cardiomyopathy. In the present study, we tested the hypothesis that cardiac overexpression of human CTSL in the murine heart would protect against cardiac hypertrophy in vivo. The effects of constitutive human CTSL expression on cardiac hypertrophy were investigated using in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding (AB) in CTSL transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive overexpression of human CTSL in the murine heart attenuated the hypertrophic response, markedly reduced apoptosis, and fibrosis. Cardiac function was also preserved in hearts with increased CTSL levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the Akt/GSK3beta signaling cascade. Our in vitro studies further confirmed that CTSL expression in cardiomyocytes blunts cardiac hypertrophy through blocking of Akt/GSK3beta signaling. The study indicates that CTSL improves cardiac function and inhibits cardiac hypertrophy, inflammation, and fibrosis through blocking Akt/GSK3beta signaling.

Topics: Angiotensin II; Animals; Animals, Newborn; Apoptosis; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Caspases; Cathepsin L; Cathepsins; Cells, Cultured; Cysteine Endopeptidases; Echocardiography; Female; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

This study was carried out in order to investigate the effects of epigallocatechin gallate (EGCG) on myocardial fibrosis and cell proliferation in cardiac hypertrophy. Cardiac hypertrophy was established in rats by abdominal aortic constriction, and EGCG at doses of 25, 50 and 100 mg/kg was administered intragastrically for 6 weeks. The results showed that in the rats with cardiac hypertrophy, EGCG at 25 - 100 mg/kg dose-dependently reduced heart weight indices, decreased atrial natriuretic polypeptide and endothelin levels in plasma, but increased nitrite (the oxidative product of NO) levels in the serum and in the myocardium. EGCG also reduced the hydroxyproline concentration and decreased the proliferating cell nuclear antigen expression in the hypertrophic myocardium. EGCG remarkably inhibited pressure overload-induced c-myc increase in Western blot analysis. In cultured newborn rat cardiac fibroblasts, treatment with EGCG at 12.5 - 200 mg/L for 6 - 48 h decreased cell proliferation induced by serum. EGCG at 12.5 - 100 mg/L dose-dependently inhibited cell proliferation and DNA synthesis of fibroblasts induced by angiotensin II (Ang II) at 1 mumol/L. EGCG also significantly increased nitrite levels in culture medium, and up-regulated inducible nitric oxide synthase protein expression if compared with the Ang II group. The inhibitory effect of EGCG on cell proliferation induced by Ang II was partly blocked by pretreatment with N(omega)-nitro- L-arginine methyl ester hydrochloride. These results suggest that EGCG inhibits the proliferation of cardiac fibroblasts both in vivo and in vitro, thereby preventing myocardial fibrosis in cardiac hypertrophy. EGCG might exert its cardiac protective action through induction of NO production.

Topics: Angiotensin II; Animals; Antioxidants; Atrial Natriuretic Factor; Camellia sinensis; Cardiomegaly; Catechin; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endothelins; Enzyme Inhibitors; Fibroblasts; Fibrosis; Hydroxyproline; Male; Myocardium; Nitric Oxide Synthase Type II; Nitrites; Organ Size; Plant Extracts; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Transcription Factors

Estrogen has been shown to protect the heart and attenuate myocardial hypertrophy and left ventricular remodelling through as yet to be defined mechanisms. In the present study we examined concentration-dependent effects of estrogen on hypertrophy of adult rat cardiomyocytes, potential underlying mechanisms related to intracellular pH (pHi) and possible sex-dependent responses. Cardiomyocytes were isolated from adult male and female Sprague-Dawley rats and used immediately for pHi determinations or cultured and subsequently treated for 24 h with 17beta-estradiol to assess hypertrophic responses. Fluorometric measurements with the pHi-sensitive dye BCECF demonstrated that at 1 pM 17beta-estradiol increased pHi (+0.05 pH units in females and +0.12 pH units in males, P<0.05) by a rapid non-genomic mechanism that was blocked by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 microM). Treatment with 1 pM 17beta-estradiol for 24 h increased cell size (females: 20%, P<0.05; males: 29%, P<0.05) and ANP expression (females: 414%, P<0.05; males: 497%, P<0.05) in a NHE-1-, and ERK1/2 MAPK-dependent manner. At 1 nM, 17beta-estradiol decreased pHi (females: -0.24 pH units, P<0.05; males: -0.07 pH units, P<0.05) which was also prevented by AVE, although at this concentration the hormone had no direct hypertrophic effect but instead prevented hypertrophy induced by phenylephrine. Our results show that low levels of estrogen produce cardiomyocyte hypertrophy through ERK/NHE-1 activation and intracellular alkalinization whereas an antihypertrophic effect is seen at high concentrations. These effects may further our understanding of the role of estrogen in heart disease particularly associated with hypertrophy.

Topics: Adrenergic alpha-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Estradiol; Estrogens; Female; Fluoresceins; Fluorescent Dyes; Heart Ventricles; Hydrogen-Ion Concentration; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Phenylephrine; Rats; Rats, Sprague-Dawley; Sex Characteristics; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Time Factors

Acute coronary artery occlusion triggers the release of atrial natriuretic peptide (ANP) from the heart. ANP affects vasodilation, natriuresis, and inflammation, but the integrated biological effects of ANP on myocardial infarction are unknown. To elucidate these effects, the left anterior coronary artery was ligated in anesthetized, ANP-deficient (ANP(-/-)) and congenic wild-type (ANP(+/+)) mice. The survival of ANP(-/-) mice was markedly better (56%) at 30 days postinfarction than the survival of ANP(+/+) mice (20%, P < 0.01). Surviving mice were comparable initially, but ANP(-/-) mice developed more cardiac hypertrophy (P < 0.001) and had lower contractility indexes 30 days after infarction (P < 0.05). An analysis 24 h after coronary occlusion showed that ANP(-/-) mice had smaller infarcts than ANP(+/+) mice (62.6 +/- 12.1 vs. 100.8 +/- 3.8%, P < 0.001) adjusted for comparable areas at risk for ischemia. The administration of ANP to ANP(-/-) mice via osmotic minipumps significantly enlarged infarct size to levels comparable with those observed in ANP(+/+) mice (P < 0.05). There was no difference in neutrophil migration into the noninfarcted myocardium of ANP(-/-) mice undergoing actual versus sham-operated coronary occlusion. By comparison, after coronary occlusion, the neutrophil infiltration into the myocardium was enhanced in ANP(+/+) (P < 0.0005) and ANP(-/-) mice administered ANP (P < 0.0005). The expression of P-selectin, a molecule that mediates neutrophil adhesion, was significantly greater after coronary occlusion in the vasculature of ANP(+/+) or ANP(-/-) mice treated with ANP than in ANP(-/-) mice (P < 0.002). Taken together, these results indicate that ANP increases P-selectin, neutrophil infiltration, infarct size, and mortality following experimental coronary occlusion.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Coronary Occlusion; Disease Models, Animal; Hemodynamics; Inflammation; Infusion Pumps, Implantable; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Contraction; Myocardial Infarction; Myocardium; Neutrophil Infiltration; P-Selectin; Time Factors

Atorvastatin blunts the response of cardiomyocytes to catecholamines by reducing isoprenylation of G gamma subunits. We examined whether atorvastatin exerts similar effects in vivo and protects the rat heart from harmful effects of catecholamines.. Rats were treated with atorvastatin (1 or 10 mg/kg x day) or H(2)O for 14 days per gavage. All three animal groups were subjected to restraint stress on day 10 and to infusions of isoprenaline (ISO; 1 mg/kg x day) or NaCl via minipumps for the last 4 days. Heart rate was measured by telemetry, left ventricular atrial natriuretic peptide (ANP) transcript levels by RT-PCR, and left atrial contractile function in organ baths. Heart rate was similar in all six study groups. In animals pre-treated with water, infusion of ISO induced an increase in heart-to-body weight ratio (HW/BW) by approximately 20%, an increase in ANP mRNA by approximately 350%, and a reduction in the inotropic effect of isoprenaline in left atrium by approximately 50%. In animals pre-treated with high-dose atorvastatin, the effects of ISO on HW/BW, ANP, and left atrial force were approximately 40, 50, and 40% smaller, respectively. Low dose atorvastatin had similar, albeit smaller effects. Atorvastatin treatment of NaCl-infused rats had only marginal effects. In cardiac homogenates from atorvastatin-treated rats (both NaCl- and ISO-infused), G gamma and G alpha(s) were partially translocated from the membrane to the cytosol.. In the rat heart, treatment with atorvastatin results in translocation of cardiac membrane G gamma and G alpha(s) to the cytosol. This mechanism might contribute to protecting the heart from harm induced by chronic isoprenaline infusion without affecting heart rate.

Topics: Adrenergic beta-Agonists; Animals; Atorvastatin; Atrial Natriuretic Factor; Cardiomegaly; Cell Membrane; Circadian Rhythm; Cytosol; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Protein gamma Subunits; Heart; Heart Rate; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Infusion Pumps, Implantable; Isoproterenol; Male; Myocardial Contraction; Myocardium; Protein Transport; Pyrroles; Rats; Rats, Wistar; RNA, Messenger

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) have pivotal roles in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We here tested whether a novel CaM antagonist, DY-9760e inhibits ET-1-induced hypertrophy through inhibition of CaMKII and ERK activities. We first confirmed that Ca(2+) oscillation induced by ET-1 treatment elicits transient activation of CaMKII and ERK in cultured cardiomyocytes. DY-9760e treatment with 3 microM totally and partially inhibited the ET-1-induced CaMKII and ERK activation, respectively. The ET-1-induced ERK activation was also partially blocked by a CaMKII inhibitor, KN93. To confirm involvement of CaMKII activity in the ERK activation by ET-1 and A23187, cultured cardiomyocytes were transfected with a constitutively active CaMKII. The transfection with the active CaMKII elicited ERK activation in cultured cardiomyocytes and cotransfection with dominant negative CaMKII eliminated its ERK activation. Consistent with inhibitory actions of DY-9760e on the ET-1-induced CaMKII and ERK activation, induction of hypertrophy-related genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was significantly inhibited by DY-9760e treatment. Combination treatment with DY-9760e and U0126, a MEK inhibitor, totally blocked the ET-1-induced ANP and BNP expression. DY-9760e treatment (3 microM) significantly inhibited the ET-1-induced hypertrophy and combination treatment with DY-9760e and U0126 totally blocked the ET-1-induced hypertrophy in cultured cardiomyocytes. These results suggest that DY-9760e elicits antihypertrophic action on ET-1-induced cardiac hypertrophy through inhibition of CaMKII and ERK activation and that CaMKII activity in part mediates ET-1-induced ERK activation.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzylamines; Butadienes; Calcimycin; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cell Proliferation; Cell Size; Cells, Cultured; DNA Replication; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Indazoles; Ionophores; Myocytes, Cardiac; Natriuretic Peptide, Brain; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Wistar; RNA, Messenger; Sulfonamides; Time Factors; Transfection

The mitochondrially expressed manganese-dependent superoxide dismutase (MnSOD, SOD2) is an essential antioxidative enzyme that is necessary for normal heart function. In this study, we investigated the heart function of mice that were exposed to increased oxidative stress for time periods of up to 6 months due to decreased MnSOD activity caused by heterozygous deletion of the MnSOD gene.. We generated a mouse strain in which the gene encoding MnSOD was exchanged against a cassette containing the SOD cDNA under the control of the tetracycline response element. After breeding with mice carrying the tetracycline receptor, compound mice express MnSOD depending on the presence of tetracycline. Without tetracycline receptor the MnSOD gene is fully inactivated, and animals show an MnSOD-deficient phenotype. Using echocardiographic recordings, we found an impairment of left ventricular functions: MnSOD+/- mice displayed a decrease in fraction shortening and ejection fraction and an increase in left ventricular internal diameter in systole. Furthermore, MnSOD+/- mice developed heart hypertrophy with accompanying fibrosis and necrosis revealed by immunhistochemical analysis. Although we did not find an increase in apoptosis in MnSOD+/- hearts under normal conditions, we observed an increase of the number of apoptotic cells and vascular senescence after treatment with doxorubicin.. Our study demonstrates that lifelong reduction of MnSOD activity has a negative effect on normal heart function. This animal model presents a valuable tool to investigate the mechanism of heart pathology reported in patients bearing different polymorphic variants of the MnSOD gene and to develop new therapeutic strategies through manipulation of the antioxidative defence system.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Antioxidants; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Doxorubicin; Doxycycline; Fibrosis; Heart Function Tests; Heterozygote; Homozygote; Mice; Mice, Transgenic; Mutation; Myocardium; Necrosis; Oxidative Stress; Superoxide Dismutase

Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin-II (Ang-II)-induced hypertrophy in cultured neonatal rat cardiomyocytes.. Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang-II-induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor L-NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor-beta1 (TGF-beta1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit.. The results indicate that EPO significantly attenuates Ang-II-induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO-treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or L-NAME partially abolished the anti-hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down-regulated the protein expression of TGF-beta1.. We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K-Akt-eNOS-NO pathway and the down-regulation of TGF-beta1.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Base Sequence; Blotting, Western; Cardiomegaly; Cells, Cultured; DNA Primers; Erythropoietin; Heart; In Vitro Techniques; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; Rats; RNA, Messenger

Elevated plasma glucocorticoid level is an independent predictor of increased mortality risk in chronic heart failure, but local biosynthesis and pathophysiological roles of glucocorticoids in the heart remain unclear.. Dahl salt-sensitive rats on high-salt diet and mice with transthoracic aortic banding (TAC) operation (TAC mice), both of which finally represent heart failure, were assessed at compensatory hypertrophic stage. As a model of cardiac-specific activation of steroidogenesis, alpha-myosin heavy chain-steroidogenic acute regulatory protein transgenic mice were used.. In hypertrophied hearts of Dahl salt-sensitive rats and TAC mice, the gene expressions of steroidogenic acute regulatory protein and CYP11A, rate limiting factors of steroid biosynthesis, were significantly upregulated and cardiac corticosterone level was increased compared with age-matched control. Although transgenic mice represented no morphological changes at basal condition, TAC induced greater increases in a ratio of left ventricular weight to body weight (4.8 +/- 0.2 vs.4.3 +/- 0.1 mg/g, P < 0.05) and left ventricular corticosterone level (104.5 +/- 13.3 vs. 69.8 +/- 3.8 pg/mg, P < 0.05) in the transgenic mice than in littermates. In neonatal cardiomyocytes, corticosterone increased atrial natriuretic peptide expression, protein synthesis and cell surface area, and provided the additive hypertrophic effects on phenylephrine-induced hypertrophied myocytes. These effects were prevented by glucocorticoid receptor blockade but not by mineralocorticoid receptor blockade.. In hypertrophied hearts, cardiac steroidogenesis was activated with an increase in cardiac glucocorticoid level. Glucocorticoid had potential of augmenting cardiac hypertrophy via glucocorticoid receptor even under the activation of alpha-adrenoceptor-mediated hypertrophic signaling. Cardiac steroidogenesis system and local glucocorticoid may play important roles in the development of hypertrophy and the progression to heart failure.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Corticosterone; Disease Progression; Gene Expression; Glucocorticoids; Heart Failure; Male; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Phenylephrine; Rats; Rats, Inbred Dahl; Steroids

We investigated cardiac hypertrophy elicited by rosiglitazone treatment at the level of protein synthesis/degradation, mTOR, MAPK and AMPK signalling pathways, cardiac function and aspects of carbohydrate/lipid metabolism. Hearts of rats treated or not with rosiglitazone (15 mg/kg day) for 21 days were evaluated for gene expression, protein synthesis, proteasome and calpain activities, signalling pathways, and function by echocardiography. Rosiglitazone induced eccentric heart hypertrophy associated with increased expression of ANP, BNP, collagen I and III and fibronectin, reduced heart rate and increased stroke volume. Rosiglitazone robustly increased heart glycogen content ( approximately 400%), an effect associated with increases in glycogenin and UDPG-PPL mRNA levels and glucose uptake, and a reduction in glycogen phosphorylase expression and activity. Cardiac triglyceride content, lipoprotein lipase activity and mRNA levels of enzymes involved in fatty acid oxidation were also reduced by the agonist. Rosiglitazone-induced cardiac hypertrophy was associated with an increase in myofibrillar protein content and turnover (increased synthesis and an enhancement of calpain-mediated myofibrillar degradation). In contrast, 26S beta5 chymotryptic proteasome activity and mRNA levels of 20S beta2 and beta5 and 19S RPN 2 proteasome subunits along with the ubiquitin ligases atrogin and CHIP were all reduced by rosiglitazone. These morphological and biochemical changes were associated with marked activation of the key growth-promoting mTOR signalling pathway, whose pharmacological inhibition with rapamycin completely blocked cardiac hypertrophy induced by rosiglitazone. The study demonstrates that both arms of protein balance are involved in rosiglitazone-induced cardiac hypertrophy, and establishes the mTOR pathway as a novel important mediator therein.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Western; Body Weight; Cardiomegaly; Eating; Echocardiography; Glucosyltransferases; Glycogen; Glycogen Phosphorylase; Glycogen Synthase; Glycoproteins; Hemodynamics; Hypoglycemic Agents; Lipoprotein Lipase; Male; Myofibrils; Natriuretic Peptide, Brain; Proteasome Endopeptidase Complex; Protein Kinases; Rats; Rats, Sprague-Dawley; Rosiglitazone; Thiazolidinediones; TOR Serine-Threonine Kinases; UTP-Glucose-1-Phosphate Uridylyltransferase

Cardiac energy metabolism is a determinant of the response to hypertrophic stimuli. To investigate how it responds to physiological or pathological stimuli, we compared the energetic status in models of hypertrophy induced by physiological stimuli (pregnancy or treadmill running) and by pathological stimulus (spontaneously hypertensive rats, SHR) in 15 week-old female rats, leading to a 10% cardiac hypertrophy. Late stage of compensated hypertrophy was also studied in 25 week-old SHR (35% of hypertrophy). Markers of cardiac remodelling did not follow a unique pattern of expression: in trained rats, only ANF was increased; in gravid rats, calcineurin activation and BNP expression were reduced while beta-MHC expression was enhanced; all markers were clearly up-regulated in 25 week-old SHR. Respiration of permeabilized fibers revealed a 17% increase in oxidative capacity in trained rats only. Mitochondrial enzyme activities, expression of the master regulator PGC-1alpha and mitochondrial transcription factor A, and content of mitochondrial DNA were not consistently changed, suggesting that compensated hypertrophy does not involve alterations of mitochondrial biogenesis. Mitochondrial fatty acid utilization tended to increase in trained rats and decreased by 14% in 15 week-old SHR. Expression of markers of lipid oxidation, PPARalpha and its down-stream targets MCAD and CPTI, was up-regulated after training and tended to decrease in gravid and 15 week-old SHR rats. Taken together these results show that there is no univocal pattern of cardiac adaptation in response to physiological or pathological hypertrophic stimuli, suggesting that other factors could play a role in determining adaptation of energy metabolism to increased workload.

Topics: Acyl-CoA Dehydrogenase; Animals; Atrial Natriuretic Factor; Cardiomegaly; Carnitine O-Palmitoyltransferase; Disease Models, Animal; Energy Metabolism; Female; Glucose Transporter Type 4; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Myosin Heavy Chains; Natriuretic Peptide, Brain; Organ Size; Oxygen Consumption; Polymerase Chain Reaction; PPAR alpha; Pregnancy; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Rats

The related cytoplasmic non-receptor tyrosine kinases Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) have been implicated in phenylephrine-induced G-protein-coupled receptor-mediated signaling mechanisms leading to cardiomyocyte hypertrophy. We report that, in phenylephrine-stimulated neonatal rat ventricular myocytes (NRVM), Pyk2 augments expression of the hypertrophic marker atrial natriuretic factor (ANF) but reduces cytoskeletal organization and cell spreading. In contrast, FAK attenuates ANF production but does not alter cytoskeletal organization and cell spreading. Pyk2 and FAK exhibit differential localization in both unstimulated and phenylephrine-stimulated myocytes. Pyk2 catalytic activity is required for Pyk2 to augment ANF secretion but is not necessary to reduce cell spreading. Pyk2 autophosphorylation is required but not sufficient for Pyk2 to augment ANF secretion. Expression of the Pyk2 FERM domain as an autonomous fragment inhibits phenylephrine-mediated ANF secretion and reduces cell spreading. In addition, expression of the Pyk2 FERM domain inhibits the ability of Pyk2 to augment ANF secretion; this is correlated with reduced Pyk2 autophosphorylation. These data indicate that Pyk2 and FAK have different roles and occupy different positions in signaling pathways leading to the development of cardiomyocyte hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Myosins; Cardiomegaly; Cardiotonic Agents; Cell Movement; Cells, Cultured; Cytoskeleton; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; Myocytes, Cardiac; Myosin Light Chains; Phenylephrine; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Transfection

Myotrophin-induced activation of NF-kappaB has been shown to be associated with cardiac hypertrophy (CH) that progresses to heart failure (HF). In the present study, we examined the cause-and-effect relationship between myotrophin and NF-kappaB activation using small hairpin RNA (shRNA) against myotrophin both in vitro (using neonatal rat myocytes) and in vivo [using myotrophin transgenic (Myo-Tg) mice, which overexpress myotrophin in the heart, develop CH, and gradually progress to HF]. Among several lentiviral vectors expressing myotrophin shRNAs, L-sh-109 showed the best silencing effect at both the mRNA (155.3 +/- 5.9 vs. 32.5 +/- 5.5, P < 0.001) and protein levels associated with a significant reduction of atrial natriuretic factor (ANF) and NF-kappaB. In vivo, when L-sh-109 was delivered directly into the hearts of 10-wk-old Myo-Tg mice, we observed a significant regression of cardiac mass (8.0 vs. 5.7 mg/g, P < 0.001) and myotrophin gene expression (54.5% over untreated Myo-Tg mice, P < 0.001) associated with a reduction in ANF and NF-kappaB signaling components. Our data suggest that using RNA interference to silence the myotrophin gene prevents NF-kappaB activation, associated with an attenuation of CH. This strategy could be an excellent therapeutic means for the treatment of CH and HF.

Topics: Animals; Atrial Natriuretic Factor; Avian Proteins; Cardiomegaly; Cells, Cultured; Cytokines; Disease Progression; Gene Expression; Genetic Therapy; Heart Failure; Intercellular Signaling Peptides and Proteins; Lentivirus; Mice; Mice, Transgenic; Myocytes, Cardiac; Myosin Heavy Chains; NF-kappa B; RNA, Messenger; RNA, Small Interfering

Submandibular salivary glands are the major source of epidermal growth factor (EGF) in mice. Acute secretion of EGF from these glands protects the heart against catecholamine-induced injury. Little is known about chronic adrenergic stimulation of salivary glands and the contribution of accumulated EGF to the adaptive hypertrophic response of the heart to such chronic adrenergic stimulation. Here we show that the EGF content of submandibular glands did not recover to normal values 24 h after a single phenylephrine injection or an aggressive encounter. Repeated (twice a day for 2 days) adrenergic stimulation resulted in an almost 90% decrease in EGF content in the submandibular glands. In these conditions, new adrenergic stimulation did not result in an increase in plasma EGF concentration, or in the activation of liver ErbB1 (the EGF receptor). Chronic isoproterenol or phenylephrine administration (7 days) induced atrial natriuretic factor expression in the heart and an increase in both ventricular weight and protein. The surgical removal of submandibular glands (sialoadenectomy) did not affect these adaptive responses of the heart. We conclude that EGF from submandibular glands does not contribute to heart hypertrophy, one of the adaptive responses induced by chronic adrenergic stimulation.

Topics: Adrenergic alpha-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Epidermal Growth Factor; Heart; Isoproterenol; Male; Mice; Myocardium; Organ Size; Phenylephrine; Submandibular Gland

Recently, we reported that histone deacetylase (HDAC) inhibitors block cardiac hypertrophy and that activation of HDAC2, one of the class I HDACs, is required for hypertrophy. In the present study, we tried to find the downstream target of HDAC inhibitor by utilizing cardiomyocytes and H9c2 cells. Both trichostatin A (TSA, class I and II HDAC inhibitor) and SK7041 (SK, class I HDAC blocker) attenuated the expression level and promoter activity of Nppa (natriuretic polypeptide precursor type A) and Myh7 (myosin heavy polypeptide 7), which are fetal genes associated with hypertrophy. Promoter-mapping revealed that the Nppa promoter region from -130 to approximately -105, which contains binding sites for Krüppel-like factor 4 (KLF4), is responsible for the HDAC inhibitor-mediated inhibition. SK-induced repression of Nppa promoter activity was attenuated when the KLF4-binding element was deleted or disrupted. Klf4 was upregulated by HDAC inhibitors, whereas it was down-regulated by phenylephrine in cardiomyocytes or by partial aortic constriction in mice. Klf4 successfully recruited the proximal Nppa promoter region flanking the KLF4-binding element in cardiomyocytes, and the recruitment was reduced by treatment with phenylephrine, which was recovered by SK. Overexpression of Klf4 blocked the agonist-induced increase in cardiomyocyte size, [(3)H]-leucine incorporation, and Nppa promoter activation. However, promoter activity was not prominently inhibited when the KLF4-binding element was disrupted or when a small inhibitory RNA to KLF4 was transfected into cells. Hypertrophic phenotypes were enhanced in Klf4-knockdown cells. These results suggest that KLF4, a novel anti-hypertrophic transcriptional regulator, mediates the HDAC inhibitor-induced prevention of cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Down-Regulation; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Myosin Heavy Chains; Phenotype; Protein Binding; Rats; Response Elements; Stress, Physiological

The heart adapts to an increased workload through the activation of a hypertrophic response within the cardiac ventricles. This response is characterized by both an increase in the size of the individual cardiomyocytes and an induction of a panel of genes normally expressed in the embryonic and neonatal ventricle, such as atrial natriuretic peptide (ANP). ANP and brain natriuretic peptide (BNP) exert their biological actions through activation of the natriuretic peptide receptor-1 (Npr1). The current study examined mice lacking Npr1 (Npr1(-/-)) activity and investigated the effects of the absence of Npr1 signaling during cardiac development on embryo viability, cardiac structure and gene and protein expression. Npr1(-/-)embryos were collected at embryonic day (ED) 12.5, 15.5 and neonatal day 1 (ND 1). Npr1(-/-)embryos occurred at the expected Mendelian frequency at ED 12.5, but knockout numbers were significantly decreased at ED 15.5 and ND 1. There was no indication of cardiac structural abnormalities in surviving embryos. However, Npr1(-/-)embryos exhibited cardiac enlargement (without fibrosis) from ED 15.5 as well as significantly increased ANP mRNA and protein expression compared to wild-type (WT) mice, but no concomitant increase in expression of the hypertrophy-related transcription factors, Mef2A, Mef2C, GATA-4, GATA-6 or serum response factor (SRF). However, there was a significant decrease in Connexin-43 (Cx43) gene and protein expression at mid-gestation in Npr1(-/-)embryos. Our findings suggest that the mechanism by which natriuretic peptide signaling influences cardiac development in Npr1(-/-) mice is distinct from that seen during the development of pathological cardiac hypertrophy and fibrosis. The decreased viability of Npr1(-/-)embryos may result from a combination of cardiomegaly and dysregulated Cx43 protein affecting cardiac contractility.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Blotting, Western; Cardiomegaly; Connexin 43; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Heart; Immunoenzyme Techniques; Male; Mice; Mice, Knockout; Natriuretic Peptide, Brain; Receptors, Atrial Natriuretic Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transcription Factors

beta-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of beta-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which beta-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of beta-catenin onto the Anf promoter. Thus, we document a direct positive role of beta-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for beta-catenin/TCF/LEF1.

Topics: Animals; Atrial Natriuretic Factor; beta Catenin; Cardiomegaly; Gene Expression Regulation; Gene Knockdown Techniques; Lymphoid Enhancer-Binding Factor 1; Myocytes, Cardiac; Phenylephrine; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; TCF Transcription Factors; Transcription, Genetic; Up-Regulation

PNC-2 is a peptide corresponding to an effector domain (residues 96-110) of ras-p21 that strongly and specifically blocks mitogenic signal transduction by oncogenic but not activated, normally-expressed wild-type ras-p21 protein. Since myocardial hypertrophy can be induced both by oncogenic and overexpressed wild-type ras-p21, we investigated whether PNC-2 can block norepinephrine (NE)-induced, ras-dependent myocardial hypertrophy in cardiac myocytes. Since PNC-2 blocks oncogenic ras-p21-induced activation of JNK and ERK, we further determined whether this peptide blocks activation of these kinases in NE-treated myocytes. Using cultured neonatal rat ventricular myocytes (NRVM), we found that NE alone significantly increased NRVM surface area, (3)H-leucine uptake, protein/DNA ratio, and atrial nartiuretic factor (ANF) mRNA levels in these cells. However, pretreatment of the NRVM with PNC-2 linked on its carboxyl terminal end to a transmembrane-penetrating leader sequence (PNC-2-leader) resulted in strong inhibition of NE-mediated cell growth and (3)H-leucine uptake and in significantly lower protein/DNA ratios. Induction of ANF mRNA levels was likewise inhibited by PNC-2-leader. In contrast, no inhibition of any of these NE-induced events was observed with a negative control peptide, X13-leader. Western blot analysis showed that JNK and ERK1/2 activity, but not p38 activity, was increased in NRVM within 5 min of exposure to NE (2 microM). Pretreatment with PNC-2-leader decreased ERK1/2 and JNK activity to basal levels. We conclude that a synthetic peptide designed to block oncogenic ras can also counter the effects of NE-induced hypertrophy associated with overexpression of ras p21 by blocking JNK/ jun and ERK activation. PNC-2 may provide a prototype for novel therapy in cardiac conditions associated with activation of NE.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; DNA; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; JNK Mitogen-Activated Protein Kinases; Molecular Sequence Data; Myocytes, Cardiac; Norepinephrine; Oncogene Protein p21(ras); p38 Mitogen-Activated Protein Kinases; Peptides; Phosphorylation; Protein Biosynthesis; Protein Sorting Signals; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Signal Transduction

Mitogen-activated protein kinase kinase 7 (MKK7) induces cardiac hypertrophy by activating the c-Juns NH2-terminal kinases (JNK). It has been reported that growth arrest and DNA-damage-inducible beta (GADD45Beta) binds to MKK7 directly and blocks its catalytic activity, mediates the inhibition of JNK signaling. However, the potential role of GADD45Beta on cardiac hypertrophy has not been investigated. In this study, we found co-infection of cardiomyocytes with adenoviral vectors expressing MKK7 and GADD45B could counteract the characteristic hypertropic responses, including an increase in cell size and elevated atrial natriuretic factor (ANP) expression which induced by overexpression of MKK7. Furthermore, siRNA-mediated knockdown of GADD45B could also cause cardiomyocytes hypertrophy. GeneChip data showed that GADD45B mRNA decreased significantly in patients with hypertrophy cardiomyopathy (HCM) compared with healthy subjects. Association study indicated that haplotype (rs2024144-rs3783501) of GADD45B affected the thickness of inter-ventricular septum in patients with HCM. Dual-luciferase assay showed that C-A haplotype displayed significantly increased transcription activity compared to T-G haplotype.

Topics: Alleles; Animals; Antigens, Differentiation; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Enhancer Elements, Genetic; Genes, Reporter; Haplotypes; Humans; MAP Kinase Kinase 7; Myocytes, Cardiac; Polymorphism, Genetic; Rats; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Ventricular Septum

Cardiac hypertrophy, an adaptive process to an increased hemodynamic overload, includes not only an increase in cell size but also qualitative changes in constituent proteins. Although swelling-activated chloride channels (I(Cl,swell)) chronically activate in hypertrophied atrial myocytes, the role of I(Cl,swell) in regulation of atrial natriuretic peptide (ANP) release is poorly understood. We investigated the effects of I(Cl,swell) on ANP release and contractility and its modification in hypertrophied rat atria. To stimulate I(Cl,swell), hypoosmotic HEPES buffered solution (0.8T, 0.7T and 0.6T) was perfused into isolated perfused beating atria. The hypoosmotic HEPES buffered solution increased ANP release as compared to isoosmotic buffered solution (1T) in an osmolarity-reduction dependent manner. Atrial contractility and extracellular fluid translocation did not change. Exposure to hypoosmotic buffer (0.8T) containing low chloride (8mM), tamoxifen or diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) significantly attenuated hypoosmolarity-induced ANP release. The pretreatment with genistein, okdaic acid, U73122, GF109203x, and staurosporine attenuated hypoosmolarity-induced ANP release whereas orthovanadate augmented it significantly. In hypertrophied atria from renal hypertensive rats, hypoosmolarity-induced ANP release was markedly attenuated and DIDS-induced decrease in ANP release and negative inotropy were augmented as compared to sham-operated rat atria. Therefore, we suggest that I(Cl,swell) may partly participate hypoosmolarity-induced ANP release through protein tyrosine kinase and phospholipase C-protein kinase C pathway. The modification of responses of ANP release to hypoosmolarity and DIDS in hypertrophied atria may relate to changes in I(Cl,swell) activity by persistent high blood pressure.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Atrial Natriuretic Factor; Cardiomegaly; Chloride Channels; Estrenes; Extracellular Fluid; Genistein; Heart Atria; Hypertension, Renal; Indoles; Maleimides; Okadaic Acid; Osmotic Pressure; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Staurosporine; Tamoxifen; Vanadates

An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.

Topics: Angiotensin II; Angiotensinogen; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Separation; Cells, Cultured; Coculture Techniques; Diuretics, Osmotic; Endothelin-1; Fibrosis; Leucine; Mannitol; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Peptidyl-Dipeptidase A; Rats; Rats, Wistar; Receptors, Angiotensin; Renin; Thymidine; Transforming Growth Factor beta1; Tritium

Corin is a cardiac serine protease that acts as the pro-atrial natriuretic peptide (ANP) convertase. Recently, 2 single-nucleotide polymorphisms (SNPs) (T555I and Q568P) in the human corin gene have been identified in genetic epidemiological studies. The minor I555/P568 allele, which is more common in African Americans, is associated with hypertension and cardiac hypertrophy. In this study, we examined the effect of T555I and Q568P amino acid substitutions on corin function. We found that corin frizzled-like domain 2, where T555I/Q568P substitutions occur, was required for efficient pro-ANP processing in functional assays. Mutant corin lacking this domain had 30+/-5% (P<0.01) activity compared to that of wild type. Similarly, corin variant T555I/Q568P had a reduced (38+/-7%, P<0.01) pro-ANP processing activity compared to that of wild type. The variant also exhibited a low activity (44+/-15%, P<0.05) in processing pro-brain natriuretic peptide (BNP). We next examined the biochemical basis for the loss of activity in T555I/Q568P variant and found that the zymogen activation of the corin variant was impaired significantly, as indicated by the absence of the activated protease domain fragment. This finding was confirmed in human embryonic kidney (HEK)293 cells and murine HL-1 cardiomyocytes. Thus, our results show that the corin gene SNPs associated with hypertension and cardiac hypertrophy impair corin zymogen activation and natriuretic peptide processing activity. Our data suggest that corin deficiency may be an important mechanism in hypertensive and heart diseases.

Topics: Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Enzyme Precursors; Genetic Variation; Humans; Hypertension; Kidney; Membrane Proteins; Mutagenesis; Natriuretic Peptide, Brain; Natriuretic Peptides; Polymorphism, Single Nucleotide; Protein Structure, Tertiary; Serine Endopeptidases; Substrate Specificity; Transfection

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-kappaB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33-->alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and beta-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.

Topics: Active Transport, Cell Nucleus; Amino Acid Substitution; Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cell Nucleus; Cell Proliferation; Cells, Cultured; Female; Intercellular Signaling Peptides and Proteins; Mutation, Missense; Myocytes, Cardiac; Protein Biosynthesis; Protein Structure, Secondary; Rats; Rats, Inbred WKY; Structure-Activity Relationship; Transcription Factor RelA; Ventricular Myosins

The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of protein synthesis, growth and proliferation in mammalian cells, is implicated in the development of cardiac hypertrophy. Ras homolog enriched in brain (Rheb) positively regulates mTORC1. We have studied whether Rheb is sufficient to activate mTOR signaling and promote protein synthesis and cardiac hypertrophy in adult rat ventricular cardiomyocytes (ARVC). Rheb was overexpressed via an adenoviral vector in isolated ARVC. Overexpression of Rheb in ARVC activated mTORC1 signaling, several components of the translational machinery and stimulated protein synthesis. Our direct visualization approach to determine ARVC size revealed that overexpression of Rheb also induced cell growth and indeed did so to similar extent to the hypertrophic agent, phenylephrine (PE). Despite potent activation of mTORC1 signaling, overexpression of Rheb did not induce expression of the cardiac hypertrophic marker mRNAs for brain natriuretic peptide and atrial natriuretic factor, while PE treatment did markedly increase their expression. All the effects of Rheb were blocked by rapamycin, confirming their dependence on mTORC1 signaling. Our findings reveal that Rheb itself can activate both protein synthesis and cell growth in ARVC and demonstrate the key role played by mTORC1 in the growth of cardiomyocytes.

Topics: Adenoviridae; Animals; Anti-Bacterial Agents; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cardiotonic Agents; Cell Proliferation; Cell Size; Cells, Cultured; Genetic Vectors; Heart Ventricles; Male; Monomeric GTP-Binding Proteins; Myocytes, Cardiac; Natriuretic Peptide, Brain; Neuropeptides; Phenylephrine; Protein Biosynthesis; Protein Kinases; Ras Homolog Enriched in Brain Protein; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Polyamines (putrescine, spermidine and spermine) are essential for cell growth and differentiation. Nitric oxide exhibits antihypertrophic functions and inhibits cardiac remodelling. However, the metabolism of polyamines and the potential interactions with nitric oxide in cardiac hypertrophy remain unclear. We randomly divided Wistar rats into four treatment groups: controls, isoproterenol (ISO), ISO and L-arginine, and L-arginine. Isoproterenol (5 mg/kg/day, subcutaneously) and/or L-arginine (800 mg/kg/day, intraperitoneally) was administered once daily for 7 days. The expression of atrial natriuretic peptide mRNA was determined by reverse transcription-polymerase chain reaction, and fibrogenesis of heart was assessed by Van Gieson staining. Polyamines were measured with high-performance liquid chromatography, and plasma nitric oxide content and lactate dehydrogenase (LDH) activity were determined with a spectrophotometer. The expression levels of ornithine decarboxylase, spermidine/spermine N1-acetyltransferase (SSAT), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were analysed by Western blot. Heart-to-body weight ratio, left ventricle-to-body weight ratio, atrial natriuretic peptide mRNA expression, collagen fibres and LDH activity were elevated, both ornithine decarboxylase and SSAT proteins were up-regulated, and total polyamines were increased in the group treated with ISO. Additionally, the expression of iNOS was up-regulated, eNOS was down-regulated, and nitric oxide levels were low. Notably, cotreatment with L-arginine reversed most of these changes except for SSAT expression,which was further up-regulated. We propose that increased polyamines and decreased nitric oxide are involved in cardiac hypertrophy induced by ISO and suggest that L-arginine pre-treatment can attenuate cardiac hypertrophy through the regulation of key enzymes of the polyamine and nitric oxide pathways.

Topics: Adrenergic beta-Agonists; Animals; Arginine; Atrial Natriuretic Factor; Biogenic Polyamines; Blood Pressure; Blotting, Western; Cardiomegaly; Chromatography, High Pressure Liquid; Electrocardiography; Heart; Heart Rate; Isoproterenol; Male; Myocardium; Nitric Oxide; Organ Size; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction

To investigate the role of mitogen activated protein kinase kinase 1 (MAPKK, MEK1) and regulated kinase1/2 (ERK1/2) on cardiac hypertrophy induced by rat parathyroid hormone1-34 (rPTH1-34).. Neonatal rat cardiomyocytes was treated with or without 10(-7) mol/L rPTH1-34 in the absence or presence 2 x 10(-5) mol/L PD98059, a MEK1 inhibitor. Cellular diameter was measured by Motic Images Advanced 3.0 software and the synthetic rate of protein in cardiac myocytes was detected by 3H-leucine incorporation, mRNA expression of atrial natriuretic peptide (ANP) was measured by RT-PCR and protein expression of ERK1/2 and p-ERK1/2 was measured by Western blot.. rPTH1-34 (10(-7) mol/L) significantly increase cellular diameter (+13.6 microm), 3H-leucine incorporation (+898 cpm/well), ANP mRNA expression (+73.9%), and p-ERK1/2 protein expression (+15%) compared to control cells (all P < 0.05) and these effects could be significantly attenuated by PD98059: cellular diameter (-7.1 microm), 3H-leucin e incorporation (-644 cpm/well), ANP mRNA expression (-52.2%), and protein expression of p-ERK1/2 (-18%) (all P < 0.05 vs. PTH group). PD98059 did not affect control cells without PTH treatment (all P > 0.05).. PD98059 attenuates PTH induced cardiac hypertrophy in vitro via inhibiting the expression of ERK1/2 and p-ERK1/2.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Flavonoids; Gene Expression Regulation; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Myocytes, Cardiac; Parathyroid Hormone; Rats; Rats, Wistar

Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality. We have reported that chronic intermittent hypoxia (CIH), a direct consequence during OSA, leads to left ventricular (LV) remodeling and dysfunction in rats. The present study is to determine LV myocardial cellular injury that is possibly associated with LV global dysfunction. Fifty-six rats were exposed either to CIH (nadir O(2) 4-5%) or sham (handled normoxic controls, HC), 8 h/day for 6 wk. At the end of the exposure, we studied LV global function by cardiac catheterization, and LV myocardial cellular injury by in vitro analyses. Compared with HC, CIH animals demonstrated elevations in mean arterial pressure and LV end-diastolic pressure, but reductions in cardiac output (CIH 141.3 +/- 33.1 vs. HC 184.4 +/- 21.2 ml x min(-1) x kg(-1), P < 0.01), maximal rate of LV pressure rise in systole (+dP/dt), and maximal rate of LV pressure fall in diastole (-dP/dt). CIH led to significant cell injury in the left myocardium, including elevated LV myocyte size, measured by cell surface area (CIH 3,564 +/- 354 vs. HC 2,628 +/- 242 microm(2), P < 0.05) and cell length (CIH 148 +/- 23 vs. HC 115 +/- 16 microm, P < 0.05), elevated terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-stained positive cell number (CIH 98 +/- 45 vs. HC 15 +/- 13, P < 0.01), elevated caspase-3 activity (906 +/- 249 vs. 2,275 +/- 1,169 pmol x min(-1) x mg(-1), P < 0.05), and elevated expression of several remodeling gene markers, including c-fos, atrial natriuretic peptide, beta-myosin heavy chain, and myosin light chain-2. However, there was no difference between groups in sarcomere contractility of isolated LV myocytes, or in LV collagen deposition on trichrome-stained slices. In conclusion, CIH-mediated LV global dysfunction is associated with myocyte hypertrophy and apoptosis at the cellular level.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiac Myosins; Cardiac Output; Cardiomegaly; Caspase 3; Cell Size; Chronic Disease; Collagen; Disease Models, Animal; Hypertrophy; Hypoxia; Male; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Myosin Light Chains; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Research Design; RNA, Messenger; Ventricular Dysfunction, Left; Ventricular Pressure; Ventricular Remodeling

The role of the angiotensin II type 2 (AT2) receptor in cardiac hypertrophy remains controversial. We studied the effects of AT2 receptors on chronic pressure overload-induced cardiac hypertrophy in transgenic mice selectively overexpressing AT2 receptors in ventricular myocytes. Left ventricular (LV) hypertrophy was induced by ascending aorta banding (AS). Transgenic mice overexpressing AT2 (AT2TG-AS) and nontransgenic mice (NTG-AS) were studied after 70 days of aortic banding. Nonbanded NTG mice were used as controls. LV function was determined by catheterization via LV puncture and cardiac magnetic resonance imaging. LV myocyte diameter and interstitial collagen were determined by confocal microscopy. Atrial natriuretic polypeptide (ANP) and brain natriuretic peptide (BNP) were analyzed by Northern blot. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2, inducible nitric oxide synthase (iNOS), endothelial NOS, ERK1/2, p70S6K, Src-homology 2 domain-containing protein tyrosine phosphatase-1, and protein serine/threonine phosphatase 2A were analyzed by Western blot. LV myocyte diameter and collagen were significantly reduced in AT2TG-AS compared with NTG-AS mice. LV anterior and posterior wall thickness were not different between AT2TG-AS and NTG-AS mice. LV systolic and diastolic dimensions were significantly higher in AT2TG-AS than in NTG-AS mice. LV systolic pressure and end-diastolic pressure were lower in AT2TG-AS than in NTG-AS mice. ANP, BNP, and SERCA2 were not different between AT2TG-AS and NTG-AS mice. Phospholamban (PLB) and the PLB-to-SERCA2 ratio were significantly higher in AT2TG-AS than in NTG-AS mice. iNOS was higher in AT2TG-AS than in NTG-AS mice but not significantly different. Our results indicate that AT2 receptor overexpression modified the pathological hypertrophic response to aortic banding in transgenic mice.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Blotting, Northern; Blotting, Western; Cardiomegaly; Collagen; Hypertension; Male; Mice; Mice, Transgenic; Microscopy, Confocal; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Receptor, Angiotensin, Type 2; RNA, Messenger; Survival Analysis; Ventricular Function, Left

In this study, we used an experimental model of cardiac hypertrophy to explore the role of the kallikrein-kinin system (KKS) in cardiac protection in transgenic rats harboring the human tissue kallikrein gene, TGR(hKLK1). Tissue kallikrein cleaves low-molecular-weight kininogen to produce kinin peptides, which bind to kinin receptors and trigger a wide spectrum of biological effects. The transgene, under the control of the zinc-inducible metallothionein promoter, was expressed in most tissues including the heart, kidney, lung and brain. These animals were subjected to treatment with thyroid hormone in order to promote cardiac hypertrophy. Induction of cardiac hypertrophy revealed a marked protective effect caused by the expression of the kallikrein transgene, evidenced by the significantly reduced cardiac weight gain and the lower enhancement in the cardiac expression of atrial natriuretic peptide and collagen III, markers for hypertrophy and fibrosis, respectively. In conclusion, our data show that expression of tissue kallikrein exerts antihypertrophic and antifibrotic actions in the heart.

Topics: Animals; Animals, Genetically Modified; Atrial Natriuretic Factor; Cardiomegaly; Collagen Type III; Fibrosis; Male; Myocardium; Rats; Rats, Sprague-Dawley; Thyroxine; Tissue Kallikreins

Cardiac hypertrophy by activation of the beta-adrenergic receptor (beta AR) is mediated more efficiently by the beta1-AR than by the beta2-AR. We investigated the signalling mechanism by which the beta1-AR mediates cardiac hypertrophy.. Experiments were performed in cultured neonatal rat cardiomyocytes. Hypertrophy was determined by the protein/DNA content and atrial natriuretic factor transcription. Phosphorylation of Akt and Src was assessed by immunoblotting. Isoproterenol (ISO, 10 microM), a non-selective beta-AR agonist, caused selective downregulation of the beta1-AR (control beta1 vs. beta2: 35 vs. 65%, Bmax 78 +/- 4 fmol/mg; 4 h, 10 vs. 90%, 61 +/- 5 fmol/mg). Concanavalin A (Con A, 0.5 microg/mL), an inhibitor of endocytosis, prevented downregulation of beta1-ARs by ISO treatment (4 h, 35 vs. 65%, 73 +/- 8 fmol/mg), suggesting that beta1-ARs selectively undergo endocytosis. Interference with beta1-AR endocytosis by Con A, carboxyl terminal peptide of beta-AR kinase-1, dominant negative (DN) beta-arrestin-1, or DN dynamin inhibited beta-adrenergic hypertrophy, suggesting that the endocytosis machinery plays a key role in mediating beta-adrenergic hypertrophy. Activation of Akt by the beta1-AR was blocked by inhibition of the endocytosis machinery, suggesting that endocytosis mediates activation of Akt. Akt plays a critical role in beta-adrenergic hypertrophy, since DN Akt blocked ISO-induced hypertrophy. beta-Adrenergic activation of Akt is mediated by Src, which associates with the endocytosis machinery and is necessary and sufficient to mediate beta-adrenergic hypertrophy.. Activation of the endocytosis machinery is required for activation of Akt, which, in turn, critically mediates beta1-AR-induced cardiac hypertrophy.

Topics: Adrenergic beta-1 Receptor Agonists; Adrenergic beta-Agonists; Animals; Animals, Newborn; Arrestins; Atrial Natriuretic Factor; beta-Arrestin 1; beta-Arrestins; Cardiomegaly; Cell Size; Cells, Cultured; Concanavalin A; Dynamins; Endocytosis; Enzyme Activation; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Isoproterenol; Myocytes, Cardiac; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Receptors, Adrenergic, beta-1; Signal Transduction; src-Family Kinases; Time Factors; Transcription, Genetic

A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors and increases its DNA binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, p300-targeted acetylated sites of GATA4 during myocardial cell hypertrophy have not been identified. By mutational analysis, we showed that 4 lysine residues located between amino acids 311 and 322 are required for synergistic activation of atrial natriuretic factor and endothelin-1 promoters by GATA4 and p300. A tetra-mutant GATA4, in which these 4 lysine residues were simultaneously mutated, retained the ability to localize in nuclei and to interact with cofactors including FOG-2, GATA6, and p300 but lacked p300-induced acetylation, DNA binding, and transcriptional activities. Furthermore, coexpression of the tetra-mutant GATA4 with wild-type GATA4 impaired the p300-induced acetylation, DNA binding, and transcriptional activities of the wild type. When we expressed the tetra-mutant GATA4 in neonatal rat cardiac myocytes using a lentivirus vector, this mutant suppressed phenylephrine-induced increases in cell size, protein synthesis, and expression of hypertrophy-responsive genes. However, its expression did not affect the basal state. Thus, we have identified the most critical lysine residues acting as p300-mediated acetylation targets in GATA4 during hypertrophic responses in cardiac myocytes. The results also demonstrate that GATA4 with simultaneous mutation of these sites specifically suppresses hypertrophic responses as a dominant-negative form, providing further evidence for the acetylation of GATA4 as one of critical nuclear events in myocardial cell hypertrophy.

Topics: Acetylation; Active Transport, Cell Nucleus; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cell Nucleus; Cells, Cultured; E1A-Associated p300 Protein; Endothelin-1; GATA4 Transcription Factor; GATA6 Transcription Factor; Mutation; Myocytes, Cardiac; Phenylephrine; Protein Processing, Post-Translational; Rats; Transcription, Genetic

Mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor, have pressure-independent cardiac hypertrophy. However, the mechanism underlying GC-A-mediated inhibition of cardiac hypertrophy remains to be elucidated. In the present report, we examined the role of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for G(q) and G(i), in the antihypertrophic effects of GC-A.. In cultured cardiac myocytes, treatment of atrial natriuretic peptide stimulated the binding of guanosine 3',5'-cyclic monophosphate-dependent protein kinase (PKG) I-alpha to RGS4, PKG-dependent phosphorylation of RGS4, and association of RGS4 and Galpha(q). In contrast, blockade of GC-A by an antagonist, HS-142-1, attenuated the phosphorylation of RGS4 and association of RGS4 and Galpha(q). Moreover, overexpressing a dominant negative form of RGS4 diminished the inhibitory effects of atrial natriuretic peptide on endothelin-1-stimulated inositol 1,4,5-triphosphate production, [(3)H]leucine incorporation, and atrial natriuretic peptide gene expression. Furthermore, expression and phosphorylation of RGS4 were significantly reduced in the hearts of GC-A knockout (GC-A-KO) mice compared with wild-type mice. For further investigation, we constructed cardiomyocyte-specific RGS4 transgenic mice and crossbred them with GC-A-KO mice. The cardiac RGS4 overexpression in GC-A-KO mice significantly reduced the ratio of heart to body weight (P<0.001), cardiomyocyte size (P<0.01), and ventricular calcineurin activity (P<0.05) to 80%, 76%, and 67% of nontransgenic GC-A-KO mice, respectively. It also significantly suppressed the augmented cardiac expression of hypertrophy-related genes in GC-A-KO mice.. These results provide evidence that GC-A activates cardiac RGS4, which attenuates Galpha(q) and its downstream hypertrophic signaling, and that RGS4 plays important roles in GC-A-mediated inhibition of cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Female; Male; Mice; Mice, Knockout; Mice, Transgenic; Myocardium; Myocytes, Cardiac; RGS Proteins; Signal Transduction

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+-H+ exchanger (NHE1) and Cl- -HCO3- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 +/- 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 microm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 +/- 0.3 microm and 2.7 +/- 0.3 microm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 +/- 0.4 Hz to 0.77 +/- 0.04 Hz following 24 h treatment; these Ca2+ -handling abnormalities were completely prevented by ETZ (0.28 +/- 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Topics: Age Factors; Angiotensin II; Animals; Atrial Natriuretic Factor; Calcium; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cardiomegaly; Cardiotonic Agents; Cation Transport Proteins; Cells, Cultured; Chloride-Bicarbonate Antiporters; Ethoxzolamide; Gene Expression; Humans; Kidney; Membrane Potentials; Membrane Transport Proteins; Mice; Myocytes, Cardiac; Phenylephrine; Rats; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfate Transporters; Transfection; Vasoconstrictor Agents

Glucose metabolism in the heart requires oxidation of cytosolic NADH from glycolysis. This study examines shuttling reducing equivalents from the cytosol to the mitochondria via the activity and expression of the oxoglutarate-malate carrier (OMC) in rat hearts subjected to 2 wk (Hyp2, n = 6) and 10 wk (Hyp10, n = 8) of pressure overload hypertrophy vs. that of sham-operated rats (Sham2, n = 6; and Sham10, n = 7). Moderate aortic banding produced increased atrial natriuretic factor (ANF) mRNA expression at 2 and 10 wk, but only at 10 wk did hearts develop compensatory hypertrophy (33% increase, P < 0.05). Isolated hearts were perfused with the short-chain fatty acid [2,4-(13)C(2)]butyrate (2 mM) and glucose (5 mM) to enable dynamic-mode (13)C NMR of intermediate exchange across OMC. OMC flux increased before the development of hypertrophy: Hyp2 = 9.6 +/- 2.1 vs. Sham2 = 3.7 +/- 1.2 muM.min(-1).g dry wt(-1), providing an increased contribution of cytosolic NADH to energy synthesis in the mitochondria. With compensatory hypertrophy, OMC flux returned to normal: Hyp10 = 3.9 +/- 1.7 vs. Sham10 = 3.8 +/- 1.2 muM.g(-1).min(-1). Despite changes in activity, no differences in OMC expression occurred between Hyp and Sham groups. Elevated OMC flux represented augmented cytosolic NADH shuttling, coupled to increased nonoxidative glycolysis, in response to hypertrophic stimulus. However, development of compensatory hypertrophy moderated the pressure-induced elevation in OMC flux, which returned to control levels. The findings indicate that the challenge of pressure overload increases cytosolic redox state and its contribution to mitochondrial oxidation but that hypertrophy, before decompensation, alleviates this stress response.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blood Pressure; Butyrates; Cardiomegaly; Carrier Proteins; Cytosol; Disease Models, Animal; Glycolysis; Heart Rate; Hypertension; Ligation; Magnetic Resonance Spectroscopy; Male; Mitochondria, Heart; Mitochondrial Proteins; NAD; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide decrease blood pressure and cardiac hypertrophy by activating natriuretic peptide receptor A (NPR-A), a transmembrane guanylyl cyclase also known as guanylyl cyclase A. Inactivation of NPR-A is a potential mechanism for the renal hyporesponsiveness observed in congestive heart failure (CHF) but direct data supporting this hypothesis are lacking. We examined whether NPR-A activity was reduced in CHF, and if so, by what mechanism. In two separate trials, CHF was induced in mice by 8-wk transverse aortic constriction. Sham controls underwent surgery without constriction. The constricted animals developed severe heart failure as indicated by increased heart weight, increased left ventricular end diastolic and systolic diameters, and decreased left ventricular ejection fractions. Kidney membranes were assayed for guanylyl cyclase activity or used to purify NPR-A by sequential immunoprecipitation/SDS-PAGE. Maximal ANP-dependent guanylyl cyclase activities were reduced by 44 or 43% in kidney membranes from CHF animals in two independent trials. Basal cyclase activities were also reduced by 31% in the second trial. The amount of phosphorylated NPR-A was reduced by 25 or 24% in kidney membranes from CHF animals as well. SYPRO Ruby staining suggested that NPR-A protein levels were similar between treatments in the first trial. However, more accurate estimates of NPR-A protein levels by immunoprecipitation/Western analysis in the second trial indicated that NPR-A protein was reduced by 30%. We conclude that reduced NPR-A protein levels, not receptor dephosphorylation, explain the renal hyporesponsiveness to natriuretic peptides in CHF.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Constriction, Pathologic; Echocardiography; Guanylate Cyclase; Heart Failure; Immunoprecipitation; Kidney; Male; Mice; Mice, Inbred C57BL; Organometallic Compounds; Osmolar Concentration; Phosphorylation; Receptors, Atrial Natriuretic Factor; Staining and Labeling

[Arg8]-vasopressin (AVP) is an essential hormone for maintaining osmotic homeostasis and is known to be a potent vasoconstrictor that regulates the cardiovascular system. In the present study, cardiomyocytes were isolated from neonatal mice and used to investigate the effects of AVP on cardiac hypertrophy. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that vasopressin V1A receptor mRNA, but not V1B or V2 receptor mRNA, was expressed in primary cultured neonatal mouse cardiomyocytes. By exposing the cultured neonatal cardiomyocytes to AVP for 24 h, cell surface areas were significantly increased, suggesting that AVP could induce cardiomyocyte growth. We then investigated the expression level of the atrial natriuretic peptide (ANP), which is a marker of cardiac hypertrophy. Stimulation with AVP increased the expression of cardiomyocyte ANP mRNA in a dose- and time-dependent manner. Immunocytochemical studies showed that stimulation with AVP significantly increased the expression of the ANP protein as well. Furthermore, AVP administration activated extracellular signal-regulated kinase (ERK)1/2 in cardiomyocytes. The effects of AVP on these parameters were significantly inhibited by a selective vasopressin V1A receptor antagonist, OPC-21268, and were not observed in cardiomyocytes from mice lacking the vasopressin V1A receptor. In vivo cardiac hypertrophy in response to pressure overload was attenuated in vasopressin V1A receptor-deficient (V1AR-KO) mice. Taken together, our data suggest that AVP promotes cardiomyocyte hypertrophy via the vasopressin V1A receptor, which is in part regulated by the pathway of ERK1/2 signaling.

Topics: Animals; Animals, Newborn; Arginine Vasopressin; Atrial Natriuretic Factor; Cardiomegaly; Cell Enlargement; Cells, Cultured; Disease Models, Animal; DNA; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Immunohistochemistry; Male; Mice; Mice, Knockout; Myocytes, Cardiac; Phosphorylation; Piperidines; Protein Biosynthesis; Quinolones; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription, Genetic; Vasoconstrictor Agents

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.

Topics: Amidines; Animals; Aortic Diseases; Atrial Natriuretic Factor; Benzylamines; Cardiomegaly; Chronic Disease; Enzyme Inhibitors; Fibrosis; Heart Failure; Hypertension; Matrix Metalloproteinase 2; Mice; Mice, Knockout; Myocardium; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Protein Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Ribosomal Protein S6; Systole; TOR Serine-Threonine Kinases; Vasoconstriction

Cardiac hypertrophy is associated with a dramatic change in the gene expression profile of cardiac myocytes. Many genes important during development of the fetal heart but repressed in the adult tissue are reexpressed, resulting in gross physiological changes that lead to arrhythmias, cardiac failure, and sudden death. One transcription factor thought to be important in repressing the expression of fetal genes in the adult heart is the transcriptional repressor REST (repressor element 1-silencing transcription factor). Although REST has been shown to repress several fetal cardiac genes and inhibition of REST function is sufficient to induce cardiac hypertrophy, the molecular mechanisms employed in this repression are not known. Here we show that continued REST expression prevents increases in the levels of the BNP (Nppb) and ANP (Nppa) genes, encoding brain and atrial natriuretic peptides, in adult rat ventricular myocytes in response to endothelin-1 and that inhibition of REST results in increased expression of these genes in H9c2 cells. Increased expression of Nppb and Nppa correlates with increased histone H4 acetylation and histone H3 lysine 4 methylation of promoter-proximal regions of these genes. Furthermore, using deletions of individual REST repression domains, we show that the combined activities of two domains of REST are required to efficiently repress transcription of the Nppb gene; however, a single repression domain is sufficient to repress the Nppa gene. These data provide some of the first insights into the molecular mechanism that may be important for the changes in gene expression profile seen in cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Chromatin; Co-Repressor Proteins; DNA-Binding Proteins; Endothelin-1; Gene Expression Regulation; Guanylate Cyclase; Heart; Heart Ventricles; Histones; Humans; Macromolecular Substances; Male; Myocytes, Cardiac; Nerve Tissue Proteins; Rats; Rats, Wistar; Receptors, Atrial Natriuretic Factor; Repressor Proteins; Transcription, Genetic

Histone deacetylase (HDAC) determines the acetylation status of histones and, thereby, controls the regulation of gene expression. HDAC inhibitors have been shown to inhibit cardiomyocyte growth in vitro and in vivo. We assessed whether HDAC inhibitors exert a beneficial effect on the remodeling heart in infarcted rats. At 24 h after ligation of the left anterior descending artery, male Wistar rats were randomized to vehicle, HDAC inhibitors [valproic acid (VPA) and tributyrin], an agonist of HDAC (theophylline), VPA + theophylline, or tributyrin + theophylline for 4 wk. Significant ventricular hypertrophy was detected as increased myocyte size at the border zone isolated by enzymatic dissociation after infarction. Cardiomyocyte hypertrophy and collagen formation at the remote region and border zone were significantly attenuated by VPA and tributyrin with a similar potency compared with that induced by the vehicle. Left ventricular shortening fraction was significantly higher in the VPA- and tributyrin-treated groups than in the vehicle-treated group. Increased synthesis of atrial natriuretic peptide mRNA after infarction was confirmed by RT-PCR, consistent with the results of immunohistochemistry and Western blot for acetyl histone H4. The beneficial effects of VPA and tributyrin were abolished by theophylline, implicating HDAC as the relevant target. Inhibition of HDAC by VPA or tributyrin can attenuate ventricular remodeling after infarction. This might provide a worthwhile therapeutic target.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Cell Size; Collagen; Coronary Vessels; Disease Models, Animal; Echocardiography; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Fibrosis; Histone Acetyltransferases; Histones; Immunohistochemistry; Ligation; Male; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Polymerase Chain Reaction; Rats; Rats, Wistar; Research Design; RNA, Messenger; Theophylline; Triglycerides; Valproic Acid; Ventricular Function, Left; Ventricular Remodeling

Myofibrillogenesis regulator-1 (MR-1) augments cardiomyocytes hypertrophy induced by angiotensin II (Ang II) in vitro. However, its roles in cardiac hypertrophy in vivo remain unknown. Here, we investigate whether MR-1 can promote cardiac hypertrophy induced by Ang II in vivo and elucidate the molecular mechanisms of MR-1 on cardiac hypertrophy. We used a model of Ang II-induced cardiac hypertrophy by infusion of Ang II in female mice. In wild-type mice subjected to the Ang II infusion, cardiac hypertrophy developed after 2 weeks. In mice overexpressing human MR-1 (transgenic), however, cardiac hypertrophy was significantly greater than in wild-type mice as estimated by heart weight:body weight ratio, cardiomyocyte area, and echocardiographic measurements, as well as cardiac atrial natriuretic peptide and B-type natriuretic peptide mRNA and protein levels. Our further results showed that cardiac inflammation and fibrosis observed in wild-type Ang II mice were augmented in transgenic Ang II mice. Importantly, increased nuclear factor kappaB activation was significantly increased higher in transgenic mice compared with wild-type mice after 2 weeks of Ang II infusion. In vitro experiments also revealed that overexpression of MR-1 enhanced Ang II-induced nuclear factor kappaB activation, whereas downregulation of MR-1 blocked it in cardiac myocytes. In conclusion, our results suggest that MR-1 plays an aggravative role in the development of cardiac hypertrophy via activation of the nuclear factor kappaB signaling pathway.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Endomyocardial Fibrosis; Female; Gene Expression Regulation; Humans; Mice; Mice, Transgenic; Muscle Proteins; Myocardium; Natriuretic Peptide, Brain; NF-kappa B; RNA, Messenger; Signal Transduction; Vasoconstrictor Agents; Ventricular Remodeling

Our aim was to determine if the expression pattern of CLP-1 in developing heart is consistent with its role in controlling RNA transcript elongation by transcriptional elongation factor b (P-TEFb) and if the inhibitory control exerted over P-TEFb by CLP-1 is released under hypertrophic conditions.. We performed immunoblot and immunofluorescence analysis of CLP-1 and the P-TEFb components cdk9 and cyclin T in fetal mouse heart and 2 day post-natal mouse cardiomyocytes to determine if they are co-localized. We induced hypertrophy in rat cardiomyocytes either by mechanical stretch or treatment with hypertrophic agents such as endothelin-1 and phenylephrine to determine if CLP-1 is released from P-TEFb in response to hypertrophic stimuli. The involvement of the Jak/STAT signal transduction pathway in this process was studied by blocking this pathway with the Jak2 kinase inhibitor, AG490, and assessing the association of CLP-1 with P-TEFb complexes.. We found that CLP-1 is expressed along with P-TEFb components in developing heart during the period in which knockout mice lacking the CLP-1 gene develop cardiac hypertrophy and die. Under conditions of hypertrophy induced by mechanical stretch or agonist treatment, CLP-1 dissociates from the P-TEFb complex, a finding consistent with the de-repression of P-TEFb kinase activity seen in hypertrophic cardiomyocytes. Blockage of Jak/STAT signaling by AG490 prevented release of CLP-1 from P-TEFb despite the ongoing presence of hypertrophic stimulation by mechanical stretch.. CLP-1 expression in developing heart and isolated post-natal cardiomyocytes colocalizes with P-TEFb expression and therefore has the potential to regulate RNA transcript elongation by controlling P-TEFb cdk9 kinase activity in heart. We further conclude that the dissociation of CLP-1 from P-TEFb is responsive to hypertrophic stimuli transduced by cellular signal transduction pathways. This process may be part of the genomic stress response resulting in increased RNA transcript synthesis in hypertrophic cardiomyocytes.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Cyclin T; Cyclin-Dependent Kinase 9; Cyclins; Fluorescent Antibody Technique; Gene Expression Regulation, Developmental; Immunohistochemistry; Janus Kinase 2; Mice; Mice, Knockout; Myocytes, Cardiac; Positive Transcriptional Elongation Factor B; Rats; RNA-Binding Proteins; Transcription Factors

Cardiovascular disease is the leading cause of morbidity and mortality in both men and women, but the incidence for women rises sharply after menopause. This has been mainly attributed in the reduction of the female sex hormone estrogen during menopause, suggesting that estrogen may have cardioprotective effects, although how estrogen exerts its cardioprotective effects is not fully understood. Moreover, the beneficial effect of estrogen on end-organ damage such as cardiac hypertrophy (CH) remains unclear. The aim of the present study was to examine the interaction between estrogen and the natriuretic peptide system (NPS) and their possible roles during the development of CH by using the proANP heterozygous atrial natriuretic peptide (ANP +/-) mouse as a model of salt-sensitive CH. Male, female ANP +/- mice, and also ovariectomized (Ovx) female ANP +/- mice treated with oil or estrogen were fed either a normal or high salt (HS) diet. After a 5-week treatment period, marked CH was noted in the male and oil-injected Ovx female ANP +/- mice treated with HS. The cardiac NPS, i.e. ANP, B-type natriuretic peptide, and natriuretic peptide receptor-A, was activated in these ANP +/- mice. Interestingly, the female and estrogen-injected Ovx female ANP +/- mice did not exhibit CH, and the cardiac NPS remained unchanged. Collectively, we provide direct evidence that estrogen has the ability to resist the induction of salt-induced CH in ANP +/- mice. Furthermore, the development of hypertrophy may be activating the cardiac NPS in an attempt to blunt these structural changes.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Estrogens; Female; Guanylate Cyclase; Heterozygote; Male; Mice; Mice, Mutant Strains; Models, Animal; Myocardium; Natriuretic Peptide, Brain; Neprilysin; Receptors, Atrial Natriuretic Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride, Dietary

Inactivation of peroxisome proliferator-activated receptor (PPARs) isoforms alpha, beta/delta, and gamma mediate distinct facets of hypertrophic transformation of adult cardiac myocytes. PPARs are ligand-activated transcription factors that modulate the transcriptional regulation of fatty acid metabolism and the hypertrophic response in neonatal cardiac myocytes. The purpose of this study was to determine the role of PPAR isoforms in the morphologic and metabolic phenotype transformation of adult cardiac myocytes in culture, which, in medium containing 20% fetal calf serum, undergo hypertrophy-like cell growth associated with downregulation of regulatory proteins of fatty acid metabolism. Expression and DNA-binding activity of PPARalpha, PPARbeta/delta, and PPARgamma rapidly decreased after cell isolation and remained persistently reduced during the 14-day culture period. Cells progressively increased in size and developed both re-expression of atrial natriuretic factor and downregulation of regulatory proteins of fatty acid metabolism. Supplementation of the medium with fatty acid (oleate 0.25 mM/palmitate 0.25 mM) prevented inactivation of PPARs and downregulation of metabolic genes. Furthermore, cell size and markers of hypertrophy were markedly reduced. Selective activation of either PPARalpha or PPARbeta/delta completely restored expression of regulatory genes of fatty acid metabolism but did not influence cardiac myocyte size and markers of hypertrophy. Conversely, activation of PPARgamma prevented cardiomyocyte hypertrophy but had no effect on fatty acid metabolism. The results indicate that PPAR activity markedly influences hypertrophic transformation of adult rat cardiac myocytes. Inactivation of PPARalpha and PPARbeta/delta accounts for downregulation of the fatty acid oxidation pathway, whereas inactivation of PPARgamma enables development of hypertrophy.

Topics: Acyl-CoA Dehydrogenase; Animals; Atrial Natriuretic Factor; Cardiomegaly; Carnitine O-Palmitoyltransferase; CD36 Antigens; Cells, Cultured; Male; Myocytes, Cardiac; Oleic Acid; Palmitates; Phenoxyacetates; PPAR alpha; PPAR delta; PPAR gamma; PPAR-beta; Pyrimidines; Rats; RNA, Messenger; Thiazolidinediones

Primary systemic carnitine deficiency is an autosomal recessive disorder caused by a decreased renal reabsorption of carnitine because of mutations of the carnitine transporter OCTN2 gene, and hypertrophic cardiomyopathy is a common clinical feature of homozygotes. Although heterozygotes for OCTN2 mutations are generally healthy with normal cardiac performance, heterozygotes may be at risk for cardiomyopathy in the presence of additional risk factors, such as hypertension. To test this hypothesis, we investigated the effects of surgically induced pressure overload on the hearts of heterozygous mutants of a murine model of OCTN2 mutation, juvenile visceral steatosis mouse (jvs/+). Eleven-week-old jvs/+ mice and age-matched wild-type mice were used. At baseline, there were no differences in physical characteristics between wild-type and jvs/+ mice. However, plasma and myocardial total carnitine levels in jvs/+ mice were lower than in wild-type mice. Both wild-type and jvs/+ mice were subjected to ascending aortic constriction with or without 1% l-carnitine supplementation for 4 weeks. At 4 weeks after ascending aortic constriction, jvs/+ mice showed an exaggeration of cardiac hypertrophy and pulmonary congestion, further increased gene expression of atrial natriuretic peptide in the left ventricles, further deterioration of left ventricular fractional shortening, reduced myocardial phosphocreatine:adenosine triphosphate ratio, and increased mortality compared with wild-type mice; l-carnitine supplementation prevented these changes in jvs/+ mice subjected to ascending aortic constriction. In conclusion, cardiomyopathy and heart failure with energy depletion may be induced by pressure overload in heterozygotes for OCTN2 mutations and could be prevented by l-carnitine supplementation.

Topics: Adenosine Triphosphate; Animals; Aorta; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cardiomyopathies; Carnitine; Constriction; Echocardiography; Genetic Predisposition to Disease; Heterozygote; Hypertension; Lung; Male; Mice; Mice, Mutant Strains; Mutation; Myocardium; Organ Size; Organic Cation Transport Proteins; Phosphocreatine; RNA, Messenger; Solute Carrier Family 22 Member 5

To investigate cardiomyocyte hypertrophy and hormonal profile in cardiac hypertrophy resulting from sequentially applied overloads.. We studied Sprague-Dawley rats with renovascular hypertension (RV), where pressure overload predominates, or deoxycorticosterone acetate (DOCA)-salt (DS), where volume overload predominates, at 2 and 4 weeks of treatment, and the combination of both models in inverse sequence: RV 2 weeks/DS 2 weeks (RV2/DS2) and DS 2 weeks/RV 2 weeks (DS2/RV2), and their sham controls (Sh).. Blood pressure and cardiomyocyte diameter increased to a similar extent in RV and DS at 2 and 4 weeks and in combined models. Cardiomyocyte length increased remarkably in the DS4 group. Circulating atrial natriuretic peptide (ANP) was elevated in all hypertensive groups after 2 and 4 weeks. The RV2/DS2 group showed similar plasma ANP levels to RV4, but DS2/RV2 exhibited a three-fold increase in ANP levels (P<0.001 versus Sh4, DS2 and DS4). Atrial ANP mRNA remained unchanged in all groups. DS treatment alone or in combination with RV increased left ventricular ANP mRNA, meanwhile only RV treatment increased left ventricular B-type natriuretic peptide (BNP) mRNA. Ventricular ANP expression levels, but not circulating ANP, correlated with both cardiomyocyte diameter (r=0.859, P<0.01) and length (r=0.848, P<0.01). Renal expression of natriuretic peptide receptor C (NPR-C) was unchanged in RV4 but decreased to a similar extent in the DS4 group and both combined treatments.. Morphometric patterns seem to be more related to the paracrine function of the heart than to the secretion of ANP and the endocrine function. Plasma ANP in the DS2/RV2 group could indicate a different evolution of the remodelling process. ANP expression seems to be a more sensitive marker for volume than for pressure overload.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Body Weight; Cardiomegaly; DNA Primers; Male; Myocardium; Organ Size; Polymerase Chain Reaction; Pressure; Radioimmunoassay; Rats; Rats, Sprague-Dawley

Better control of cardiovascular function in patients on peritoneal dialysis (PD) is critical because PD patients have a tendency to overhydration, which has been proved to be associated with cardiovascular and patient outcome. In the general population, lipid metabolism is also considered to be an important indicator of future cardiovascular events. Icodextrin has been used to improve ultrafiltration volume without increasing dextrose load. We therefore expected that parameters of lipid metabolism and cardiovascular function could both be improved, or at least maintained, after icodextrin use in PD patients. We therefore analyzed those parameters in 14 prevalent PD patients who required a switch from dextrose to icodextrin solution for the long dwell at 1 year before the switch, at the time of the switch, and at 1 and 2 years after the switch. In the study patients, cardiovascular remodeling evaluated by ultrasonographic left ventricular mass index was diminished, but the intima media area of the cervical artery was elevated after icodextrin use. Intima media thickness did not change over time. Biochemical indices such as brain natriuretic peptide, atrial natriuretic peptide, lipoprotein A, total cholesterol, and triglycerides were all lower after icodextrin use. These results indicate that icodextrin has the potential to improve lipid metabolism, volemic status, and cardiac hypertrophy in prevalent PD patients. However, atherosclerotic vascular change is refractory to improvement.

Topics: Arteries; Atrial Natriuretic Factor; Cardiomegaly; Dialysis Solutions; Glucans; Glucose; Hemodialysis Solutions; Humans; Icodextrin; Lipid Metabolism; Middle Aged; Natriuretic Peptide, Brain; Neck; Peritoneal Dialysis; Tunica Intima; Tunica Media; Ultrasonography; Ventricular Remodeling; Water-Electrolyte Balance

Although studies have shown that endothelial nitric oxide synthase (eNOS) homozygous knockout mice (eNOS-/-) develop left ventricular (LV) hypertrophy, well compensated at least to 24 wks, uncertainty still exists as to the cardiac functional and molecular mechanistic consequences of eNOS deficiency at later time-points. To bridge the gap in existent data, we examined whole hearts from eNOS-/- and age-matched wild-type (WT) control mice ranging in age from 18 to 52 wks for macroscopic and microscopic histopathology, LV mRNA and protein expression using RNA Dot blots and Western blots, respectively, and LV function using isolated perfused work-performing heart preparations. Heart weight to body weight (HW/BW in mg/g) ratio increased significantly as eNOS-/- mice aged (82.2%, P < 0.001). Multi-focal replacement fibrosis and myocyte degeneration/death were first apparent in eNOS-/- mouse hearts at 40 wks. Progressive increases in LV atrial natriuretic factor (ANF) and alpha-skeletal actin mRNA levels both correlated significantly with increasing HW/BW ratio in aged eNOS-/- mice (r = 0.722 and r = 0.648, respectively; P < 0.001). At 52 wks eNOS-/- mouse hearts exhibited basal LV hypercontractility yet blunted beta adrenergic receptor (betaAR) responsiveness that coincided with a significant reduction in the LV ratio of phospholamban to sarcoplasmic reticulum Ca2+-ATPase-2a protein levels and was preceded by a significant upregulation in LV steady-state mRNA and protein levels of the 28 kDa membrane-bound form of tumor necrosis factor-alpha. We conclude that absence of eNOS in eNOS-/- mice results in a progressive concentric hypertrophic cardiac phenotype that is functionally compensated with decreased betaAR responsiveness, and is associated with a potential cytokine-mediated alteration of calcium handling protein expression.

Topics: Actins; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Gene Expression Regulation, Enzymologic; Gene Silencing; Heart Rate; Heart Ventricles; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Myocytes, Cardiac; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Organ Size; Perfusion; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tumor Necrosis Factor-alpha; Ventricular Dysfunction, Left

We have previously shown that deficiency for the z-disc protein calsarcin-1 (CS1) sensitizes the heart to calcineurin signaling and to stimuli of pathological hypertrophy. In the present study we asked whether overexpression of CS1 might exhibit antihypertrophic effects, and therefore we tested this hypothesis both in vitro and in vivo.. Adenoviral gene transfer of CS1 into neonatal cardiomyocytes inhibited hypertrophy as a result of Gq-agonist stimulation, including angiotensin-II (Ang-II), endothelin-1, and phenylephrine. Consistently, Adenoviral gene transfer of CS1 also led to the reduction of increased levels of atrial natriuretic factor (mRNA) and the calcineurin-sensitive gene MCIP1.4, suggesting that CS1 inhibits calcineurin-dependent signaling. Furthermore, we generated CS1-overexpressing transgenic mice (CS1Tg). Unchallenged CS1Tg mice did not exhibit a pathological phenotype as assessed by echocardiography and analysis of cardiac gene expression. Likewise, when subjected to long-term infusion of Ang-II, both CS1Tg and wild-type mice developed a similar degree of arterial hypertension. Yet, in contrast to wild-type mice, Ang-II-treated CS1Tg animals did not display cardiac hypertrophy. Despite the absence of hypertrophy, both fractional shortening and dP/dt(max) were preserved in CS1Tg Ang-II-treated mice as assessed by echocardiography and cardiac catheterization, respectively. Moreover, induction of the hypertrophic gene program (atrial natriuretic factor, brain natriuretic peptide) was markedly blunted, and expression of the calcineurin-dependent gene MCIP1.4 was significantly reduced in CS1Tg mice, again consistent with an inhibitory role of CS1 on calcineurin.. The sarcomeric protein CS1 prevents Ang-II-induced cardiomyocyte hypertrophy at least in part via inhibition of calcineurin signaling. Thus, overexpression of CS1 might represent a novel approach to attenuate pathological cardiac hypertrophy.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcineurin; Calcium-Binding Proteins; Cardiomegaly; Carrier Proteins; Genetic Therapy; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Muscle Proteins; RNA, Messenger; Signal Transduction

The aim of the present study was to determine whether the antidiabetic agent pioglitazone has a direct inhibiting effect on myocardial hypertrophy induced by high glucose and insulin in primary cultured neonatal rat cardiomyocytes. Culture preparations of ventricular muscle cells newborn rats were utilized. At 72 h of culture age, the cardiomyocytes were incubated for another 48 h with 25.5 mmol/L glucose plus 0.1 micromol/L insulin (group 2), 25.5 mmol/L glucose and 0.1 micromol/L insulin plus 10 micromol/L pioglitazone (group 3), 10 micromol norepinephrine (group 4), respectively. Cells cultured continuously in medium served as control (group 1). Cellular surface area, protein content, atrial natriuretic factor (ANF) mRNA, and cardiotrophin-1 (CT-1) mRNA were assessed after treatment with different agents. All those parameters increased significantly after treatment with high glucose and insulin as compared with control (P < 0.01). These effects were inhibited markedly by pioglitazone. The cellular surface area and ANF mRNA in group 3 were decreased as compared with group 2 (P < 0.01). The protein content and CT-1 mRNA in group 3 were also decreased as compared with group 2 (P < 0.05). We concluded that a the cellular level myocardial hypertrophy induced by high glucose and insulin was inhibited directly by pioglitazone in primary cultured cardiac myocytes. CT-1 may be involved in myocardial hypertrophy induced by high glucose andinsulin and inhibiting effects of pioglitazone on myocardial hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Cytokines; Glucose; Hypoglycemic Agents; Insulin; Muscle Proteins; Myocytes, Cardiac; Pioglitazone; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones

The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Biomarkers; Blotting, Western; Cardiomegaly; Cell Size; Cells, Cultured; Eicosapentaenoic Acid; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phosphorylation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxP-targeted mice (Gata4fl/fl) were crossed with mice containing a beta-myosin heavy chain (beta-MHC) or alpha-MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Cell Survival; GATA4 Transcription Factor; Gene Deletion; Gene Expression Regulation; Mice; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains

Cardiotrophin-1 (CT-1) is a cytokine involved in the growth and survival of cardiac cells via activation of the Janus activated kinase/signal transducer activator of transcription (JAK/STAT). Statins, 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have effects that extend beyond cholesterol reduction and inhibit vascular smooth muscle cell (VSMC) proliferation and cardiac hypertrophy. However, whether stains also can inhibitin vitromyocardial hypertrophy or not still remains elusive. The purpose of this study was to explore the effects of simvastatin on the hypertrophy of cultured rat cardiomyocytes induced by CT-1 and to investigate whether this effect was mediated via JAK-STAT signaling pathway.. Primary cardiomyocytes from 2-day-old (P2) rats were cultured, stimulated with CT-1, and treated with various concentration of simvastatin. Incorporation of [(3)H] leucine, reverse transcription-polymerase chain reaction and western blotting techniques were used to investigate cardiacmyocyte size, ANP mRNA and JAK-STAT protein expression. Simvastatin was proved, in a dose-independent manner, to decrease cardiacmyocytes size as well as protein synthesis, and inhibit ANP mRNA synthesis and JAK-STAT protein expression induced by CT-1 in cardiacmyocytes.. These results suggest that simvastatin can ameliorate cardiacmyocytes hypertrophyin vitrovia JAK-STAT signaling pathways. The present study provides a novel understanding and alternative therapeutic strategy for cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Cytokines; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Myocytes, Cardiac; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Simvastatin; STAT Transcription Factors

The activation of the calcineurin-nuclear factor of activated T cells cascade during the development of pressure-overload cardiac hypertrophy has been previously reported in a number of studies. In addition, numerous pharmacological studies involving calcineurin inhibitors such as FK506 and cyclosporine A have now demonstrated that these agents can prevent such hypertrophic responses in the heart. However, little is known regarding the roles of the calcineurin downstream effector--nuclear factor of activated T cells. Our present study has further examined the roles of nuclear factor of activated T cells in pressure-overload cardiac hypertrophy by employing a recently developed cell-permeable nuclear factor of activated T cells inhibitor peptide. Rat hearts were subjected to pressure overload attributable by 4 weeks of aortic banding, and then treated with this cell-permeable nuclear factor of activated T cells inhibitor peptide and a control peptide. Treatment with the inhibitor was found to significantly decrease the heart weight/body weight ratio, the size of cardiac myocytes, and the serum brain natriuretic peptide and atrial natriuretic peptide levels. These results suggest that nuclear factor of activated T cells functions in a key role in the development of cardiac hypertrophy during pressure overload. Inhibition of nuclear factor of activated T cells by a specific inhibitor peptide is a suitable method for characterization of the molecular mechanisms underlying cardiac hypertrophy as well as in the search for new promising therapies for disease.

Topics: Animals; Aorta, Abdominal; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cell Membrane Permeability; Male; Natriuretic Peptide, Brain; NFATC Transcription Factors; Oligopeptides; Rats; Rats, Wistar; Signal Transduction; Ventricular Pressure

Although in mice, the dynamics of gene expression during heart development is well characterized, information on humans is scarce due to the limited availability of material. Here, we analyzed the transcriptional distribution of Mlc-2a, Mlc-1v, Mlc-2v, and atrial natriuretic factor (ANF) in human embryonic hearts between 7 and 18 weeks of gestation and in healthy and hypertrophic adult hearts by in situ hybridization and compared expression with that in mice. Strikingly, Mlc-2a, Mlc-1v, and ANF, which are essentially chamber-restricted in mice by mid-gestation, showed a broader distribution in humans. On the other hand, Mlc-2v may prove to be an adequate ventricular marker in humans in contrast to mouse. This study emphasizes the importance of careful comparative human-animal analyses during embryonic development and adulthood, as avoiding erroneous extrapolations may be critical to develop new and successful myocardial replacement therapies.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Electrophysiology; Gene Expression Regulation, Developmental; Heart; Humans; Mice; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Myosin Light Chains; Protein Isoforms

Postnatal cardiac myocytes respond to diverse signals by hypertrophic growth and activation of a fetal gene program. In an effort to discover regulators of cardiac hypertrophy, we performed a eukaryotic expression screen for activators of the atrial natriuretic factor (ANF) gene, a cardiac-specific marker of hypertrophic signaling. We discovered that a family of transcriptional coactivators, called CAMTAs, promotes cardiomyocyte hypertrophy and activates the ANF gene, at least in part, by associating with the cardiac homeodomain protein Nkx2-5. The transcriptional activity of CAMTAs is governed by association with class II histone deacetylases (HDACs), which negatively regulate cardiac growth. Mice homozygous for a mutation in a CAMTA gene are defective in cardiac growth in response to pressure overload and neurohumoral signaling, whereas mice lacking HDAC5, a class II HDAC, are sensitized to the prohypertrophic actions of CAMTA. These findings reveal a transcriptional regulatory mechanism that modulates cardiac growth and gene expression by linking hypertrophic signals to the cardiac genome.

Topics: Animals; Atrial Natriuretic Factor; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Cardiomegaly; Chlorocebus aethiops; COS Cells; Gene Expression Regulation, Developmental; Genetic Testing; Heart; Histone Deacetylases; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Mice; Mice, Knockout; Mutation; Myocardium; Myocytes, Cardiac; Protein Kinase C; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Signal Transduction; Trans-Activators; Transcription Factors; Transcriptional Activation

Mice deficient for the AP-1 transcription factor JunD, the only Jun protein constitutively expressed and clearly detectable in the mammalian heart, develop enhanced cardiac hypertrophy in response to chronic pressure overload. Catecholamines inducing alpha-adrenergic receptor-mediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. In the present study we analyzed the mechanistic role of JunD in cardiomyocyte hypertrophy in vitro in response to alpha-adrenergic agonist phenylephrine (PE).. Cardiomyocytes were isolated from 1- to 3-day-old rats and transfected with adenoviruses expressing LacZ or wild-type JunD, or with expression vectors encoding LacZ, wild-type JunD, mutated JunD forming only JunD homodimers (JunDeb1), mutated JunD lacking the JNK site (JunD-Delta 162), or c-Jun. After stimulation with PE (10(-5) mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [3H]-leucine incorporation) and mRNA expression of JunD, c-Jun, c-Fos, and atrial natriuretic peptide (ANP) were analyzed. Transcriptional activation was determined by luciferase activity in cardiomyocytes transfected with AP-1 or ANP luciferase reporter plasmids. Gel shift assays with an AP-1 consensus oligonucleotide were performed to analyze AP-1 DNA binding activities.. PE augmented mRNA levels of c-Jun and c-Fos, but decreased JunD transcript levels. Adenoviral over-expression of wild-type JunD blunted PE-induced hypertrophic growth and expression of ANP mRNA. Over-expression of JunD in cardiomyocytes caused enhanced AP-1 protein-DNA binding, without increasing the transcriptional response from AP-1 or ANP luciferase reporter plasmids at baseline or upon PE stimulation. Moreover, over-expression of JunDeb1 attenuated transcription from AP-1 or ANP luciferase reporter plasmids and blunted c-Jun-mediated acceleration of AP-1 transcriptional activity at baseline and in response to PE.. Our observations establish a novel role for JunD as a negative regulator of cardiomyocyte hypertrophy in response to hypertrophic stimuli by inhibiting AP-1 transcriptional activity.

Topics: Adrenergic alpha-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; DNA; Electrophoretic Mobility Shift Assay; Myocytes, Cardiac; Phenylephrine; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha; RNA, Messenger; Stimulation, Chemical; Transcription Factor AP-1; Transcription, Genetic; Transfection

Preferential and specific down-regulation of genes involved in fatty acid (FA) uptake and metabolism is considered a hallmark of severe hypertrophic remodeling and progression to cardiac failure. Therefore, we investigated the time course of changes in cardiac metabolic gene expression (1) in mice subjected to regional myocardial infarction (MI) for 4 days, 1 month, or 3 months and (2) in mice overexpressing calcineurin (Cn) which initially develop concentric hypertrophy progressing after the age of 4 weeks to dilated cardiomyopathy and failure. In both models, hypertrophy was characterized by increased expression of beta-myosin heavy chain protein and atrial natriuretic factor mRNA, indicative of marked structural remodeling. Fractional shortening progressively decreased from 31% to 15.1% and 3.7% 1 and 3 months after MI, respectively. One month post-MI, the expression of several metabolic genes, i.e., acyl-CoA synthetase (-50%), muscle-type carnitine palmitoyl transferase 1 (-37%) and citrate synthase (-28%), was significantly reduced in the surviving myocardium. Despite overt signs of cardiac failure 3 months post-MI, the expression of these genes had returned to normal levels. In hearts of both 4- and 6-week-old Cn mice, genes involved in both FA and glucose metabolism and mitochondrial citrate synthase were down-regulated, reflecting an overall decline in metabolic gene expression, rather than a specific and preferential down-regulation of genes involved in FA uptake and metabolism. These findings challenge the concept that specific and sustained down-regulation of genes involved in FA uptake and metabolism represents a hallmark of the development of cardiac hypertrophy and progression to failure.

Topics: Animals; Atrial Natriuretic Factor; Body Weight; Calcineurin; Cardiomegaly; Collagen Type I; Disease Progression; Down-Regulation; Echocardiography; Fatty Acids; Gene Expression; Heart; Heart Failure; Lipid Metabolism; Male; Mice; Myocardial Infarction; Myosin Heavy Chains; Organ Size; Oxidation-Reduction; Phenotype; RNA, Messenger

We examined the role of Jak2 kinase phosphorylation in the development of pressure overload hypertrophy in mice subjected to transverse aortic constriction (TAC) and treated with tyrphostin AG490, a pharmacological inhibitor of Jak2.. Control mice (sham), subjected to TAC for 15 days (TAC) or to TAC and treated with 48 microg/kg/day i.p. of tyrphostin AG490 (TAC+AG490) were evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy.. Mice subjected to TAC alone developed concentric hypertrophy that accompanied activation of the components of the Jak/STAT signaling pathway manifested by an increase in phosphorylation of Jak2 and STAT3. We also observed increased phosphorylation of MAPK p44/p42, p38 MAPK and JNK in the TAC group, as well as, an increase in expression of MKP-1 phosphatase which negatively regulates MAPK kinases. Treatment of aortic constricted mice with tyrphostin AG490 failed to develop hypertrophy and showed a marked reduction in phosphorylation of Jak2 and STAT3. There was, however, in TAC and AG490 treated mice, a notable increase in the phosphorylation state of the MAPK p44/42, whereas MKP-1 phosphatase was downregulated.. These findings suggest that Jak2 kinase plays an important role in left ventricular remodeling during pressure overload hypertrophy. Pharmacological inhibition of Jak2 kinase during pressure overload blocks the development of concentric hypertrophy.

Topics: Actins; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Disease Models, Animal; Echocardiography; Heart Ventricles; Janus Kinase 2; Ligation; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Phosphorylation; Protein Kinase Inhibitors; RNA, Messenger; STAT3 Transcription Factor; Tyrphostins; Ventricular Function, Left; Ventricular Myosins; Ventricular Remodeling

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Endothelin-1; Myocytes, Cardiac; Rats; Rats, Wistar; RNA, Messenger; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers

The role of the angiotensin II type 2 receptor (AT2) during alterations in cardiac size remains largely unclear. Through employment of an AT2 antagonist, the present study explored a possible involvement of the AT2 receptor during salt-induced cardiac hypertrophy in the proatrial natriuretic peptide gene-disrupted mouse (ANP-/-). ANP-/- mice received either saline solution or the AT2 antagonist, PD123319, and were then placed on a high salt diet (8.0% NaCl) for 3 weeks. Cardiac and pulmonary size, expression of the renin-angiotensin system (RAS), and the behaviour of various hypertrophy marker genes were assessed. PD123319 caused enhanced expression of the systemic RAS, yet the cardiac RAS was largely unaffected. Although AT2 blockade did not alter whole cardiac mass, right ventricle mass, as well as pulmonary mass-to-body mass ratios were significantly decreased. Collagen type I was decreased in the latter tissues, likely contributing to the regression in mass. Several players essential in the maintenance of myocardial extracellular matrix homeostasis including B-type natriuretic peptide, matrix metalloproteinase-2, tumour necrosis factor, and transforming growth factor were also significantly altered by PD123319. These data suggest that AT2 blockade is involved in significant changes in myocardial extracellular matrix components translating into decreases in tissue mass in the salt-sensitive ANP-/- animal.

Topics: Angiotensin II Type 2 Receptor Blockers; Animals; Atrial Natriuretic Factor; Cardiomegaly; Collagen Type I; Heart; Imidazoles; Kidney; Lung; Matrix Metalloproteinase 2; Mice; Mice, Transgenic; Mutation, Missense; Natriuretic Peptide, Brain; Organ Size; Peptidyl-Dipeptidase A; Pyridines; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Atrial Natriuretic Factor; Renin-Angiotensin System; RNA, Messenger; Sodium Chloride, Dietary; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Vasoconstrictor Agents

1. Atrial natriuretic peptide (ANP)-null mice (Nppa(-/-)) exhibit cardiac hypertrophy at baseline and adverse cardiac remodelling in response to transverse aortic constriction (TAC)-induced pressure overload stress. Previous studies have suggested that natriuretic peptides could potentially oppose mineralocorticoid signalling at several levels, including suppression of adrenal aldosterone production, inhibition of mineralocorticoid receptor (MR) activation or suppression of MR-mediated production of pro-inflammatory factors. Thus, we hypothesized that the MR blocker eplerenone would prevent the exaggerated left ventricular (LV) remodelling/fibrosis and dysfunction after TAC in Nppa(-/-). 2. In the present study, Nppa(-/-) and wild-type Nppa(+/+) mice fed eplerenone- or vehicle (oatmeal)-supplemented chow since weaning were subjected to TAC or sham operation. The daily dose of eplerenone administered was approximately 200 mg/kg. At 1 week after TAC, LV size and function were evaluated by echocardiogram and LV cross-sections were stained with picrosirius red for collagen volume measurement. Total RNA was extracted from the LV for real-time polymerase chain reaction analysis of osteopontin. 3. Eplerenone had no effect on baseline hypertrophy observed in sham-operated Nppa(-/-) compared with Nppa(+/+) mice. Eplerenone attenuated the TAC-induced increase in LV weight in both genotypes and completely prevented LV dilation, systolic dysfunction and interstitial collagen deposition seen in Nppa(-/-) mice after TAC. However, serum aldosterone levels were lower in Nppa(-/-) compared with Nppa(+/+) wild types. No interaction between eplerenone and genotype in osteopontin mRNA levels was observed. 4. Eplerenone prevents adverse cardiac remodelling related to pressure overload in ANP-deficient mice, mainly due to an antifibrotic effect. The mechanism whereby ANP deficiency leads to excess hypertrophy, fibrosis and early failure following TAC is increased profibrotic signals resulting from excess or unopposed MR activation, rather than increased levels of aldosterone.

Topics: Aldosterone; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Eplerenone; Heart; Hypertrophy; Male; Mice; Mice, Knockout; Myocardium; Spironolactone; Ventricular Remodeling

We recently reported that a transcriptional repressor, neuron-restrictive silencer factor (NRSF), represses expression of fetal cardiac genes, including atrial and brain natriuretic peptide (ANP and BNP), by recruiting class I histone deacetylase (HDAC) and that attenuation of NRSF-mediated repression contributes to the reactivation of fetal gene expression during cardiac hypertrophy. The molecular mechanism by which the activity of the NRSF-HDAC complex is inhibited in cardiac hypertrophy remains unresolved, however. In the present study, we show that class II HDACs (HDAC4 and 5), which are Ca/calmodulin-dependent kinase (CaMK)-responsive repressors of hypertrophic signaling, associate with NRSF and participate in NRSF-mediated repression. Blockade of the CaMK-class II HDAC signaling pathway using a CaMK-resistant HDAC5 mutant, a CaMK inhibitor (KN62) or a dominant-negative CaMK mutant inhibited ET-1-inducible ANP and BNP promoter activity, but that inhibitory effect was abolished by mutation of the neuron-restrictive silencer element (NRSE) within the ANP and BNP promoter. In addition, adenovirus-mediated expression of a dominant-negative NRSF mutant abolished the inhibitory effect of KN62 on ET-1-inducible endogenous ANP gene expression in ventricular myocytes. Finally, the interaction between NRSF and class II HDACs was decreased in both in vitro and in vivo models of cardiac hypertrophy. These findings show that ET-1-induced CaMK signaling disrupts class II HDAC-NRSF repressor complexes, thereby enabling activation of ANP and BNP gene transcription in ventricular myocytes, and shed light on a novel mechanism by which the fetal cardiac gene program is reactivated.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Disease Models, Animal; DNA Probes; Endothelin-1; Histone Deacetylases; Humans; In Vitro Techniques; Mice; Mutation; Myocytes, Cardiac; Rats; Repressor Proteins; Signal Transduction; Transcription Factors

Ca(2+) signaling through CaMKII is critical in regulating myocyte function with regard to excitation-contraction-relaxation cycles and excitation-transcription coupling. To investigate the role of nuclear CaMKII in cardiac function, transgenic mice were designed and generated to target the expression of a CaMKII inhibitory peptide, AIP (KKALRRQEAVDAL), to the nucleus. The transgenic construct consists of the murine alpha-myosin heavy chain promoter followed by the expression unit containing nucleotides encoding a four repeat concatemer of AIP (AIP(4)) and a nuclear localization signal (NLS). Western blot and immunohistochemical analyses demonstrate that AIP(4) is expressed only in the nucleus of cardiac myocytes of the transgenic mice (NLS-AIP(4)). The function of cytoplasmic CaMKII is not affected by the expression of AIP(4) in the nucleus. Inhibition of nuclear CaMKII activity resulted in reduced translocation of HDAC5 from nucleus to cytoplasm in NLS-AIP(4) mouse hearts. Loss of nuclear CaMKII activity causes NLS-AIP(4) mice to have smaller hearts than their nontransgenic littermates. Transcription factors including CREB and NFkappaB are not regulated by cardiac nuclear CaMKII. With physiological stresses such as pregnancy or aging (8 months), NLS-AIP(4) mice develop hypertrophy symptoms including enlarged atria, systemic edema, sedentariness, and morbidity. RT-PCR analyses revealed that the hypertrophic marker genes, such as ANF and beta-myosin heavy chain, were upregulated in pregnancy stressed mice. Our results suggest that absence of adequate Ca2+signaling through nuclear CaMKII regulated pathways leads to development of cardiac disease.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Calcium Signaling; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cell Nucleus; Genetic Markers; Heart; Mice; Mice, Transgenic; Molecular Sequence Data; Myocardium; Myosin Heavy Chains; Nuclear Localization Signals; Organ Size; Peptides; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Ventricular Myosins

Pioglitazone, one of the synthetic peroxisome proliferator-activated receptor (PPARgamma) agonists, has been found to inhibit inflammatory response. However, it is not known yet whether the preventive effect of pioglitazone on cardiac hypertrophy is related to its antiinflammatory function. The objective of this study was to investigate the role of pioglitazone in attenuation of cardiac hypertrophy and its relation to the inhibitory effect on the inflammatory cytokine expression in cultured neonatal rat cardiomyocytes. The mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), interleukin (IL)-1beta, IL-6, and PPARgamma was measured by using RT-PCR. Cardiomyocyte hypertrophy was induced by stimulating angiotensin II (Ang II) and evaluated both by measuring surface area of cardiac myocyte and 3H-leucine incorporation. The expressions of IL-1beta, IL-6, ANP, and BNP were significantly enhanced, whereas that of PPARgamma was significantly reduced in Ang II-induced hypertrophic cardiomyocytes. Pioglitazone decreased cardiac myocyte surface area and inhibited 3H-leucine incorporation into cardiomyocytes. Furthermore, pioglitazone upregulated the suppressed expression of PPARgamma and attenuated the increased IL-1beta and IL-6 expression. The effect of pioglitazone might be associated with PPARgamma activation and the consequent antiinflammatory function in prevention and treatment of cardiac hypertrophy.

Topics: Angiotensin II; Animals; Anti-Inflammatory Agents; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Interleukin-1beta; Interleukin-6; Leucine; Myocytes, Cardiac; Natriuretic Peptide, Brain; Pioglitazone; PPAR gamma; Rats; Rats, Wistar; RNA, Messenger; Thiazolidinediones

To investigate the effects of pioglitazone on cardiac hypertrophy in vitro and in vivo.. Angiotensin II was used to establish hypertrophy of cardiac myocytes and pioglitazone was applied to these myocytes in various dosages in vitro. ANP and BNP mRNA expression was evaluated by RT-PCR, and the rate of protein synthesis in CM by 3H-leucine incorporation in cardiac myocytes. Left ventricular hypertrophy was induced by incomplete ligation of abdominal aorta of rats and pioglitazone (20 mg x kg(-1). day(-1)) was administrated one week prior to the operation until 4 weeks after the operation. Cytokines mRNA expression in left ventricle was measured by RT-PCR, left ventricular wall thickness and myocyte diameter were determined by pathological method.. Pioglitazone inhibited ANP and BNP mRNA expression and 3H-leucine incorporation in neonatal rat cardiac myocytes induced by angiotensin II in a dose-dependent manner in vitro. Furthermore, pioglitazone reduced the mRNA expression of proinflammatory cytokines, including interleukin-1 beta and cardiotrophin-1, and inhibited the pressure overload-induced increase in the ratio of heart weight to body weight, left ventricular wall thickness and myocyte diameter of rats in vivo.. Pioglitazone inhibits cardiac hypertrophy of rats in vitro and in vivo, and may play a role in prevention and treatment of cardiovascular diseases characterized by cardiac hypertrophy in future.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Cytokines; Disease Models, Animal; Interleukin-1beta; Male; Myocytes, Cardiac; Natriuretic Peptide, Brain; Pioglitazone; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thiazolidinediones

Our recent studies suggest that 1,25-dihydroxyvitamin D3 functions as an endocrine suppressor of renin biosynthesis. Genetic disruption of the vitamin D receptor (VDR) results in overstimulation of the renin-angiotensin system (RAS), leading to high blood pressure and cardiac hypertrophy. Consistent with the higher heart-to-body weight ratio, the size of left ventricular cardiomyocytes in VDR knockout (KO) mice was markedly increased compared with wild-type (WT) mice. As expected, levels of atrial natriuretic peptide (ANP) mRNA and circulating ANP were also increased in VDRKO mice. Treatment of VDRKO mice with captopril reduced cardiac hypertrophy and normalized ANP expression. To investigate the role of the cardiac RAS in the development of cardiac hypertrophy, the expression of renin, angiotensinogen, and AT-1a receptor in the heart was examined by real-time RT-PCR and immunostaining. In VDRKO mice, the cardiac renin mRNA level was significantly increased, and this increase was further amplified by captopril treatment. Consistently, intense immunostaining was detected in the left ventricle of captopril-treated WT and VDRKO mice by use of an anti-renin antibody. Levels of cardiac angiotensinogen and AT-1a receptor mRNAs were unchanged in the mutant mice. These data suggest that the cardiac hypertrophy seen in VDRKO mice is a consequence of activation of both the systemic and cardiac RAS and support the notion that 1,25-dihydroxyvitamin D(3) regulates cardiac functions, at least in part, through the RAS.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Captopril; Cardiomegaly; Mice; Mice, Knockout; Myocytes, Cardiac; Receptors, Calcitriol; Renin-Angiotensin System

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes (CMs) and progressive fibrosis are known to occur simultaneously. To investigate the nature of interactions between ventricular CMs and cardiac fibroblasts (CFs) in these conditions, we have established a "dedifferentiated model" of adult murine CMs in coculture with CFs. In such a model, which is recognized to study cardiac cell hypertrophy in vitro, dedifferentiated CMs in culture and in coculture were characterized by immunopositive staining to ANP (atrial natriuretic peptide) and beta-myosin heavy chain (beta-MHC). The results confirm that ANP secretion by CMs was significantly increased during the cultures. The increase size of cultured CMs was significantly higher in CM/CF cocultures than in CM cultures which was also observed when CMs were cultured with fibroblast conditioned medium (FCM). In addition, fibroblast proliferation studies showed that CMs favored fibroblast adhesion and/or growth at the beginning of the coculture and fibroblast proliferation throughout the time course of the coculture. Furthermore, a significant level of interleukin-6 (IL-6) production was detected by ELISA in CM/CF cocultures. A similar higher increase was observed when CMs were cultured in the presence of FCM. These results demonstrate that CFs enhance myocyte hypertrophy and that CMs regulate fibroblast adhesion and/or proliferation, suggesting a paracrine interaction between CMs and CFs which could involve IL-6.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cell Proliferation; Cells, Cultured; Coculture Techniques; Cytokines; Fibroblasts; Hypertrophy; Mice; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Ventricular Myosins

Cardiac hypertrophy is a complex and nonhomogenous response to various stimuli. In this study, we used high-density oligonucleotide microarray to examine gene expression profiles during physiological hypertrophy, pathological hypertrophy, and heart failure in Dahl salt-sensitive rats. There were changes in 404/3,160 and 874/3,160 genes between physiological and pathological hypertrophy and the transition from hypertrophy to heart failure, respectively. There were increases in stress response genes (e.g., heat shock proteins) and inflammation-related genes (e.g., pancreatitis-associated protein and arachidonate 12-lipoxygenase) in pathological processes but not in physiological hypertrophy. Furthermore, atrial natriuretic factor and brain natriuretic protein showed distinctive changes that are very specific to different conditions. In addition, we used a resampling-based gene score-calculating method to define significantly altered gene clusters, based on Gene Ontology classification. It revealed significant alterations in genes involved in the apoptosis pathway during pathological hypertrophy, suggesting that the apoptosis pathway may play a role during the transition to heart failure. In addition, there were significant changes in glucose/insulin signaling, protein biosynthesis, and epidermal growth factor signaling during physiological hypertrophy but not during pathological hypertrophy.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Echocardiography; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation; Heart Failure; Hypertrophy; Inflammation; Insulin; Natriuretic Peptide, Brain; Oligonucleotide Array Sequence Analysis; Pancreatitis-Associated Proteins; Physical Conditioning, Animal; Rats; Rats, Inbred Dahl; RNA; Signal Transduction

Atrial dilatation is an important risk factor for atrial fibrillation (AF). In the present study, we monitored the electrophysiological changes during progressive atrial dilatation in chronically instrumented goats.. In 8 goats, 2 screw-in leads with piezoelectric crystals were implanted transvenously in the right atrium. After 2 weeks, atrial diameter and effective refractory period were measured. AF paroxysms were induced by burst pacing to determine the baseline AF cycle length and stability of AF. After His-bundle ablation, the above measurements were repeated once a week. After 4 weeks of complete AV block, the free wall of the right atrium was mapped and the atrium was fixed in formalin for histological analysis. After His-bundle ablation, the ventricular rate decreased from 113.8+/-4.8 to 44.6+/-2.5 bpm. Right atrial diameter increased gradually by 13.5+/-3.9% during 4 weeks of AV block (P<0.01). The duration of induced AF paroxysms increased from 4.6 seconds to 6.4 minutes (P<0.05). Atrial effective refractory period and AF cycle length remained constant. Spontaneous paroxysms of AF were not observed. Atrial mapping during rapid pacing revealed that slow conduction (<30 cm/s) was present in 3.7+/-1.0% of the mapped area (control, 0.9+/-0.5%, P<0.05). Histological analysis showed hypertrophy without atrial fibrosis. Connexin40 and connexin43 expression was unchanged.. Chronic AV block in the goat leads to progressive atrial dilatation, prolongation of induced AF paroxysms, and local conduction delays. The increase in AF stability was not a result of a shortening of atrial refractoriness or atrial fibrosis.

Topics: Aldosterone; Angiotensin II; Animals; Atrial Fibrillation; Atrial Natriuretic Factor; Cardiac Pacing, Artificial; Cardiomegaly; Cell Size; Chronic Disease; Connexin 43; Connexins; Female; Gap Junction alpha-5 Protein; Goats; Heart Block; Heart Conduction System; Hemodynamics; Hypertrophy; Myocytes, Cardiac; Neural Conduction; Norepinephrine; Refractory Period, Electrophysiological

Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure. In cardiomyocytes, the hormone is synthesized as a precursor, proatrial natriuretic peptide (pro-ANP), which is proteolytically converted to active ANP. Corin is a cardiac transmembrane serine protease that has been shown to process pro-ANP in vitro, but its physiological importance had not been established. Here, we show that corin-deficient (Cor-/-) mice develop normally during embryogenesis and survive to postnatal life. Cor-/- mice have elevated levels of pro-ANP but no detectable levels of ANP as compared with WT littermates. Infusion of an active recombinant soluble corin transiently restores pro-ANP conversion, resulting in the release of circulating biologically active ANP. Using radiotelemetry to assess blood pressure, we find that Cor-/- mice have spontaneous hypertension as compared with WT mice, and it is enhanced after dietary salt loading. Pregnant Cor-/- mice demonstrate late-gestation proteinuria and enhanced high blood pressure during pregnancy. In addition, Cor-/- mice exhibit cardiac hypertrophy resulting in a mild decline in cardiac function later in life. Thus, our data establish corin as the physiological pro-ANP convertase and indicate that corin deficiency may contribute to hypertensive heart disease.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Embryonic Development; Female; Hypertension; Male; Mice; Pregnancy; Pregnancy Complications; Protein Precursors; Proteinuria; Serine Endopeptidases; Sodium Chloride

Cyclic guanosine monophosphate (cGMP), which is implicated in cardiac cell growth and function, is synthesized by cytoplasmic soluble guanylyl cyclase (GC) stimulated via nitric oxide (NO) and by particulate membrane-bound GC activated via natriuretic peptides. We investigated possible cGMP elevation in the left ventricle (LV) of rats developing physiologic LV hypertrophy during gestation. Furthermore, expression of estrogen receptors (ER) and oxytocin receptors (OTR) was evaluated because their activation stimulates NO and atrial natriuretic peptide (ANP) release from the heart. Compared with nonpregnant controls, Sprague-Dawley rats on day 7 of gestation had similar heart weights, but, on days 14 and 21, ventricular mass increased by 12% and 28% respectively (P< 0.05). LV cGMP concentration was elevated at day 14 of gestation (3.25 +/- 0.12 vs 4.65 +/- 0.17 pmol/g wet weight, P< 0.01) but decreased at day 21 (2.45 +/- 0.09 pmol/g, P< 0.05) to increase again on postpartum day 1 (6.01 +/- 0.15 pmol/g) and day 4 (9.21 +/- 1.79 pmol/g). Changes in endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), OTR and ERalpha, but not ERbeta, proteins paralleled the pregnancy-related cGMP changes in the LV. In contrast, ANP mRNA of the LV remained at control level throughout gestation but increased postpartum, whereas brain natriuretic peptide (BNP) expression declined at term and increased postpartum. The particulate GC natriuretic peptide receptors (GC-A and GC-B) transcripts were already lower at day 14 of gestation. Natriuretic peptide clearance receptor (NPR-C) transcript was not altered on days 7 and 14, but increased at term. We conclude that cGMP concentration in the rat LV is influenced by both NOS and natriuretic peptide systems and may be involved in the changes of LV contractility and hypertrophy that occur during rat gestation.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Northern; Blotting, Western; Cardiomegaly; Cyclic GMP; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Heart Ventricles; Immunohistochemistry; Myocardium; Natriuretic Peptide, Brain; Natriuretic Peptides; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pregnancy; Rats; Receptors, Oxytocin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.

Topics: 14-3-3 Proteins; Active Transport, Cell Nucleus; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Cells, Cultured; DNA-Binding Proteins; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Models, Biological; Myocytes, Cardiac; NFATC Transcription Factors; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Biosynthesis; Protein Isoforms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Transcription Factors

Although increased levels of circulating interleukin (IL)-18 have been demonstrated in patients with cardiovascular diseases, the functional consequences of chronically increased circulating IL-18 with respect to myocardial function have not been defined. Thus we aimed to examine the effects of chronic IL-18 exposure on left ventricular (LV) function in healthy mice. Moreover, to clarify whether IL-18 has direct effects on the cardiomyocyte, we examined effects of IL-18 on cardiomyocytes in vitro. After 7 days of daily intraperitoneal injections of 0.5 microg IL-18 in healthy mice, a 40% (P < 0.05) reduction in the LV maximal positive derivative, a 25% (P < 0.05) reduction in the LV maximal rate of pressure decay, and a 2.8-fold (P < 0.001) increase in the LV end-diastolic pressure were measured, consistent with myocardial dysfunction. Furthermore, we measured a 75% (P < 0.05) reduction in beta-adrenergic responsiveness to isoproterenol. IL-18 induced myocardial hypertrophy, and there was a 2.9-fold increase (P < 0.05) in atrial natriuretic peptide mRNA expression in the LV myocardium. In vitro examinations of isolated adult rat cardiomyocytes being stimulated with IL-18 (0.1 microg/ml) exhibited an increase in peak Ca2+ transients (P < 0.05) and in diastolic Ca2+ concentrations (P < 0.05). In conclusion, this study shows that daily administration of IL-18 in healthy mice causes LV myocardial dysfunction and blunted beta-adrenergic responsiveness to isoproterenol. A direct effect of IL-18 on the cardiomyocyte in vitro was demonstrated, suggesting that IL-18 reduces the responsiveness of the myofilaments to Ca2+. Finally, induction of myocardial hypertrophy by IL-18 indicates a role for this cytokine in myocardial remodeling.

Topics: Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Cardiomegaly; Cardiomyopathies; Drug Administration Schedule; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interleukin-18; Isoproterenol; Male; Mice; Mice, Inbred BALB C; Myocardium; Myocytes, Cardiac; Rats; Receptors, Adrenergic, beta; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tumor Necrosis Factor-alpha; Ventricular Function, Left

Recent evidence indicates that the GTPase activated Rho/Rho-kinase pathway contributes angiotensin II-induced cardiac hypertrophy and vascular remodeling. We tested this hypothesis in vivo by determining the effects of fasudil, a Rho-kinase inhibitor, on angiotensin II-induced cardiac hypertrophy, coronary vascular remodeling, and ventricular dysfunction. Six-month-old apolipoprotein E deficient (apoE-KO) mice were subcutaneously infused with angiotensin II (1.44 mg/kg/day) using an osmotic mini-pump. Mice were randomly assigned to either vehicle or fasudil (136 or 213 mg/kg/day in drinking water) group. Infusion of angiotensin II for 4 weeks resulted in cardiac enlargement, myocyte hypertrophy, and myocardial interstitial and coronary artery perivascular fibrosis. These changes were accompanied by reduced aortic flow velocity and acceleration rate. Cardiac gene expression levels of atrial natriuretic peptide (ANP) and collagen type III detected by real-time reverse transcriptase polymerase chain reaction were significantly increased in angiotensin II-infused mice. Treatment with fasudil dose-dependently attenuated angiotensin II-induced cardiac hypertrophy, prevented perivascular fibrosis, blunted the increase in ANP and collagen type III expression, and improved cardiac function, without changing blood pressure. These data are consistent with a role for Rho-kinase activation in angiotensin II-induced cardiac remodeling and vascular wall fibrosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Angiotensin II; Animals; Apolipoproteins E; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Collagen Type III; Coronary Vessels; Dose-Response Relationship, Drug; Fibrosis; Gene Expression; Heart Rate; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Knockout; Myocardium; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; RNA, Messenger; Up-Regulation

To investigate the role of MyD88-dependent nuclear factor-kappaB (NF-(B) activation signaling pathway in the development of cardiac hypertrophy in vivo.. Dominant negative myeloid differentiation protein (dn-MyD) 88 fragment was inserted into pShuttle plasmid and then fused into adenovirus so as to construct Ad5-dn-MyD88. Some Sprague-Dawley (SD) rats underwent banding of aorta (aorta binding group) and some SD rats underwent sham operation (sham operation group). Part of the rats in the aorta banding group were transfected with Ad5-dn-MyD88 into the myocardium tissue (Ad5-dn-MyD88 transfection group) or adenovirus expressing dnMyD88 (Ad5-GFP) (control group) so as to determine the effect of blocking down stream of MyD88 signaling on the development of cardiac hypertrophy. Three days after the hearts of some rats from the 4 groups were collected and Western blotting was used to detect the expression of dn-MyD88 protein and fluorescent microscopy was used to detect the expression of GFP. Three weeks after the beginning of experiment the hearts were collected to calculate the heart weight/body weight (HW/BW) ratio and extract the plasma protein and nuclear protein. Electrophoretic mobility shift assay (EMSA) was used to determine the NFkappaB binding activity. Western blotting was used to examine the phosphorylation of IkappaBalpha and IKKalpha/beta with appropriate specific anti-phospho antibodies.. Flag and dn-MyD88 were effectively expressed 3 days after the transfection of Ad5-dn-MyD88 into the myocardium. Three weeks after the HW/BW ratio was 0.47 +/- 0.01 in the aorta banding group, significantly higher, by 37.8%, than that of the sham operation group (0.34 +/- 0.01, P < 0.01), and was 0.41 +/- 0.02 in the Ad5-dn-MyD88 transfection group, significantly lower, by 11.58%, than that of the aorta banding group (P < 0.01); the myocardial ANP protein expression level of the aorta binding group was significantly higher, by 43.5%, than that of the sham operation group (P < 0.01) and 36.2% higher than that of the Ad5-dnMyD88 transfection group (P < 0.01); the ANP/GAPDH in the aorta binding group was significantly higher than that of the sham operation group (P < 0.01) and that of the Ad5-dn-MyD88 transfection group (P < 0.01); the NF-kappaB binding activity in the myocardium of the aorta banding group was 9.94 +/- 1.58, significantly higher, by 144.8%, than that of the sham operation group (4.06 +/- 0.52, P < 0.01) and significantly lower, by 41.8%, than that of the Ad5-dn-MyD88 transfection group (5.79 +/- 0.52, P < 0.05); the phospho-(p-) IkappaBalpha level and p-IkappaBalpha/IkappaBalpha of the aorta binding group were significantly higher than those of the sham operation group (P < 0.01) and significantly higher, by 26.7%, than that of the Ad5-dn-MyD88 transfection group (P < 0.05); the p-IKKalphabeta/IKKalphabeta in the myocardium of the aorta binding group was significantly higher, by 318.0%, than that of the sham operation group (P < 0.01), and significantly higher, by 77.4%, than that of the Ad5-dn-MyD88 transfection group (P < 0.01).. MyD88-dependent NFkappaB signaling is a novel pathway for inducing the development of cardiac hypertrophy in vivo and blocking MyD88 mediated signaling pathway attenuates the development of cardiac hypertrophy.

Topics: Adaptor Proteins, Signal Transducing; Adenoviridae; Animals; Antigens, Differentiation; Atrial Natriuretic Factor; Cardiomegaly; Male; Myeloid Differentiation Factor 88; Myocardium; NF-kappa B; Rats; Rats, Sprague-Dawley; Receptors, Immunologic; Signal Transduction; Transfection; Xenopus Proteins

To study the effect of Danshensu (DSS) and Ligustrazine (TMZ), the extracts of Chinese herbs for promoting blood circulation, on angiotensin II (Ang II) induced myocardial hypertrophy and its related genes, and to explore the mechanisms of inhibitory effect.. Adopting one-step method, the total RNA of myocardial cells was extracted by TRIzol reagent. Then the expression of ANP and beta-actin mRNA, as symbol of myocardial cells, were detected by RT-PCR.. Molecular biological research showed that Ang II could significantly increase the expression of ANP mRNA in myocardial cells (P < 0.01), which could be significantly inhibited by Losartan (P < 0.01), both DSS and TMZ had the inhibitory effect (P < 0.05). Ang II could increase beta-actin mRNA expression in myocardial cells simultaneously, Losartan, DSS and TMZ could also significantly inhibit it (P < 0.05).. The effective ingredients of Chinese herbs for promoting blood circulation, DSS and TMZ, have the effect of inhibiting the hyper-expression of ANP and beta-actin induced by Ang II, and preventing myocardial hypertrophy, therefore, it could be used to prevent and treat cardiomegaly.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Drugs, Chinese Herbal; Female; Lactates; Male; Myocytes, Cardiac; Pyrazines; Rats; Rats, Wistar; RNA, Messenger

CGS 26303 is a vasopeptidase inhibitor that simultaneously inhibits endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP). We compared the effects of chronic treatment with CGS 26303 to the selective inhibition of angiotensin-converting enzyme (ACE) and NEP during the transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF) in hypertensive rats. LV geometry and function were assessed in Dahl salt-sensitive rats placed on a high-salt diet from age 6 weeks (hypertensive rats) and in control rats fed a low-salt diet. The hypertensive rats were randomized into groups that received no treatment or were treated with an ACE inhibitor (temocapril), an ECE/NEP inhibitor (CGS 26303), or a NEP inhibitor (CGS 24592) from the LVH stage (11 weeks) to the CHF stage (17 weeks). All treatments decreased the systolic blood pressure equally and significantly improved LV fractional shortening. Both temocapril and CGS 26303 ameliorated LV perivascular fibrosis, reduced mRNA levels of types I and III collagen, and decreased the heart weight/body weight ratio. CHF rats had increased plasma ET-1 levels compared with control rats. Only CGS 26303 reduced ET-1 to normal levels. ET-1 levels were found to correlate with heart/body weight, right ventricle/body weight and perivascular fibrosis ratios. During the transition to CHF, CGS 26303 produces effects that are comparable to temocapril and superior to CGS 24592. The beneficial effects of CGS 26303 are likely caused in part by the greater reduction of plasma ET-1. Dual ECE/NEP inhibitor may provide a new strategy for the treatment of human heart failure.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Aspartic Acid Endopeptidases; Atrial Natriuretic Factor; Cardiac Output, Low; Cardiomegaly; Collagen; Echocardiography; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Fibrosis; Heart; Male; Metalloendopeptidases; Myocardium; Neprilysin; Neurotransmitter Agents; Organophosphonates; Phenylalanine; Rats; Rats, Inbred Dahl; RNA, Messenger; Tetrazoles; Thiazepines

Atrial natriuretic peptide (ANP), via its vasodilating and diuretic effects, has an important physiological role in the maintenance of arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is highly expressed in vascular endothelium, but the functional relevance of this is controversial. To dissect the endothelium-mediated actions of ANP in vivo, we inactivated the GC-A gene selectively in endothelial cells by homologous loxP/Tie2-Cre-mediated recombination. Notably, despite full preservation of the direct vasodilating effects of ANP, mice with endothelium-restricted deletion of the GC-A gene (EC GC-A KO) exhibited significant arterial hypertension and cardiac hypertrophy. Echocardiographic and Doppler flow evaluations together with the Evan's blue dilution technique showed that the total plasma volume of EC GC-A KO mice was increased by 11-13%, even under conditions of normal dietary salt intake. Infusion of ANP caused immediate increases in hematocrit in control but not in EC GC-A KO mice, which indicated that ablation of endothelial GC-A completely prevented the acute contraction of intravascular volume produced by ANP. Furthermore, intravenous ANP acutely enhanced the rate of clearance of radio-iodinated albumin from the circulatory system in control but not in EC GC-A KO mice. We conclude that GC-A-mediated increases in endothelial permeability are critically involved in the hypovolemic, hypotensive actions of ANP.

Topics: Animals; Arteries; Atrial Natriuretic Factor; Capillary Permeability; Cardiomegaly; Endothelium, Vascular; Guanylate Cyclase; Hematocrit; Humans; Hypotension; Hypovolemia; Integrases; Mice; Mice, Knockout; Myocytes, Cardiac; Receptors, Atrial Natriuretic Factor; Vasodilation; Viral Proteins

Although disruption of guanylyl cyclase (GC) A, a natriuretic peptide receptor, induces cardiac hypertrophy and fibrosis, the molecular mechanism underlying these effects are not well understood. In this study, we examined the role of calcineurin, a calcium-dependent phosphatase, in cardiac remodeling in GCA-knockout (GCA-KO) mice.. At 14 weeks of age, calcineurin activity, nuclear translocation of nuclear factor of activated T cells c3 (NFATc3), and modulatory calcineurin-interacting protein 1 (MCIP1) gene expressions were increased in the hearts of GCA-KO mice compared with wild-type (WT) mice. Blockade of calcineurin activation by FK506 (6 mg/kg body weight administered subcutaneously once a day from 10 to 14 weeks of age) significantly decreased the heart-to-body weight ratio, cardiomyocyte size, and collagen volume fraction in GCA-KO mice, whereas FK506 did not affect these parameters in WT mice. Overexpression of atrial and brain natriuretic peptides, collagen, and fibronectin mRNAs in GCA-KO mice was also attenuated by FK506. Electrophoretic mobility shift assays demonstrated that GATA4 DNA-binding activity was increased in GCA-KO mice, and this increase was inhibited by calcineurin blockade. In neonatal cultured cardiac myocytes, inhibition of GCA by HS142-1 (100 microg/mL) increased basal and phenylephrine (10(-6) mol/L)-stimulated calcineurin activity, nuclear translocation of NFATc3, and MCIP1 mRNA expression. In contrast, activation of GCA by atrial natriuretic peptide (10(-6) mol/L) inhibited phenylephrine (10(-6) mol/L)-stimulated nuclear translocation of NFATc3.. These results suggest that activation of cardiac GCA by locally secreted natriuretic peptides protects the heart from excessive cardiac remodeling by inhibiting the calcineurin-NFAT pathway.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Calcineurin Inhibitors; Cardiomegaly; DNA-Binding Proteins; Enzyme Activation; Fibrosis; Gene Expression Regulation; Guanylate Cyclase; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Muscle Proteins; Myocardium; Natriuretic Peptide, Brain; NFATC Transcription Factors; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Tacrolimus

Cardiac mass increases with age and with declining estradiol serum levels in postmenopausal women. Although the non-selective estrogen receptor-alpha and -beta agonist 17beta-estradiol attenuates cardiac hypertrophy in animal models and in observational studies, it remains unknown whether activation of a specific estrogen receptor subtype (ERalpha or ERbeta) might give similar or divergent results. Therefore, we analyzed myocardial hypertrophy as well as cardiac function and gene expression in ovariectomized, spontaneously hypertensive rats (SHR) treated with the subtype-selective ERalpha agonist 16alpha-LE2 or 17beta-estradiol.. Long-term administration of 16alpha-LE2 or 17beta-estradiol did not affect elevated blood pressure, but both agonists efficiently attenuated cardiac hypertrophy and increased cardiac output, left ventricular stroke volume, papillary muscle strip contractility, and cardiac alpha-myosin heavy chain expression. The observed effects of E2 and 16alpha-LE2 were abrogated by the ER antagonist ZM-182780. Improved left ventricular function upon 16alpha-LE2 treatment was also observed in cardiac MRI studies. In contrast to estradiol and 16alpha-LE2, tamoxifen inhibited cardiac hypertrophy but failed to increase alpha-myosin heavy chain expression and cardiac output.. These results support the hypothesis that activation of ERalpha favorably affects cardiac hypertrophy, myocardial contractility, and gene expression in ovariectomized SHR. Further studies are required to determine whether activation ERbeta mediates redundant or divergent effects.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression; Hemodynamics; Hypertension; In Vitro Techniques; Magnetic Resonance Imaging; Models, Animal; Myocardium; Myosin Heavy Chains; Organ Size; Ovariectomy; Papillary Muscles; Random Allocation; Rats; Rats, Inbred SHR; Tamoxifen; Ventricular Myosins

Serum response factor (SRF) is a transcription factor required for the regulation of genes important for cardiac structure and function. Notably, the "fetal gene expression profile" that is characteristic of cardiac hypertrophy consists of genes known to be regulated by SRF. Transgenic animal studies suggest that cardiac-specific overexpression of SRF induces this pattern of hypertrophic genes and subsequently causes the progression of pathologic adaptations. Furthermore, studies examining cardiac tissues from patients with severe heart failure indicate significant alterations in SRF expression that correspond with alterations in expression of SRF-dependent genes. Based on these observations, it has been postulated that SRF may be critical for stimulating pathologic gene expression at the onset of hypertrophic adaptation. To address the role of SRF in cardiac hypertrophy we investigated whether SRF is necessary and sufficient for the expression of genes associated with the hypertrophic response. We used isolated cardiomyocytes from both neonatal rats, and transgenic mice containing floxed SRF alleles, to examine cardiac gene expression in response to overexpression and absence of SRF. Using this approach, we demonstrate that SRF is required for the induction of atrial naturetic factor (ANF), c-fos, NCX1, BNP, alpha-actins, alpha-myosin heavy chain, and beta-myosin heavy chain genes. However, overexpression of exogenous SRF in isolated cardiomyocytes is only sufficient to induce NCX1 and alpha-myosin heavy chain. These results indicate that SRF is critical for the regulation and induction of genes associated with the progression of pathologic cardiac hypertrophy, however, the pattern of genes induced by overexpression of SRF in isolated cardiomyocytes is different from those genes expressed in hypertrophic transgenic hearts. This suggests that SRF-dependent gene expression is modulated in a complex manner by in vivo physiologic systems prior to and during heart failure as the organism adapts to cardiac stress.

Topics: Actins; Adenoviridae; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Developmental; Genes, Reporter; Green Fluorescent Proteins; Heart Ventricles; Luciferases; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Serum Response Factor; Sodium-Calcium Exchanger

The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and alpha-smooth muscle actin (alpha-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25+/-1%, P<0.01 using biochemical analysis and 3-fold; P<0.01 using quantitative histology), enhanced expression of PCNA (by 70+/-8%; P<0.01), alpha-SMA (by 32+/-2%; P<0.05), and the collagen-degrading enzyme MMP-9 (by 70+/-6%; P<0.05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25+/-2%, P<0.05 using biochemical analysis and 2.4-fold; P<0.01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV (P<0.01) and kidney (P<0.05), completely inhibited cell proliferation (P<0.01) and fibroblast differentiation (P<0.05) in the LV, and increased MMP-2 expression (by 25+/-1%; P<0.05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Collagen; Fibroblasts; Fibrosis; Heart; Humans; Hypertension; Kidney; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Myocardium; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Recombinant Proteins; Relaxin

Peroxisome proliferator-activated receptor (PPAR)-gamma is required for adipogenesis but is also found in the cardiovascular system, where it has been proposed to oppose inflammatory pathways and act as a growth suppressor. PPAR-gamma agonists, thiazolidinediones (TZDs), inhibit cardiomyocyte growth in vitro and in pressure overload models. Paradoxically, TZDs also induce cardiac hypertrophy in animal models. To directly determine the role of cardiomyocyte PPAR-gamma, we have developed a cardiomyocyte-specific PPAR-gamma-knockout (CM-PGKO) mouse model. CM-PGKO mice developed cardiac hypertrophy with preserved systolic cardiac function. Treatment with a TZD, rosiglitazone, induced cardiac hypertrophy in both littermate control mice and CM-PGKO mice and activated distinctly different hypertrophic pathways from CM-PGKO. CM-PGKO mice were found to have increased expression of cardiac embryonic genes (atrial natriuretic peptide and beta-myosin heavy chain) and elevated nuclear factor kappaB activity in the heart, effects not found by rosiglitazone treatment. Rosiglitazone increased cardiac phosphorylation of p38 mitogen-activated protein kinase independent of PPAR-gamma, whereas rosiglitazone induced phosphorylation of extracellular signal-related kinase 1/2 in the heart dependent of PPAR-gamma. Phosphorylation of c-Jun N-terminal kinases was not affected by rosiglitazone or CM-PGKO. Surprisingly, despite hypertrophy, Akt phosphorylation was suppressed in CM-PGKO mouse heart. These data show that cardiomyocyte PPAR-gamma suppresses cardiac growth and embryonic gene expression and inhibits nuclear factor kappaB activity in vivo. Further, rosiglitazone causes cardiac hypertrophy at least partially independent of PPAR-gamma in cardiomyocytes and through different mechanisms from CM-PGKO.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Myocytes, Cardiac; Myosin Heavy Chains; NF-kappa B; Phosphorylation; PPAR gamma; Rosiglitazone; Systole; Thiazolidinediones

The membrane protease, corin, has been shown to be the proatrial natriuretic peptide (ANP)-converting enzyme. Like mice lacking ANP, corin-deficient animals are hypertensive and have cardiac hypertrophy.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; DNA, Complementary; Hormones; Humans; Mice; Molecular Sequence Data; Peptides; Serine Endopeptidases; Signal Transduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals are potent cardiovascular teratogens in developing piscine and avian species. In the present study we investigated the effects of TCDD on murine cardiovascular development. Pregnant mice (C57Bl6N) were dosed with 1.5-24 microg TCDD/kg on gestation day (GD) 14.5. At GD 17.5, fetal mice exhibited a dose-related decrease in heart-to-body weight ratio that was significantly reduced at a maternal dose as low as 3.0 microg TCDD/kg. In addition, cardiocyte proliferation was reduced in GD 17.5 fetal hearts at the 6.0-microg TCDD/kg maternal dose. To determine if this reduction in cardiac weight was transient, or if it continued after birth, dams treated with control or 6.0 microg TCDD/kg were allowed to deliver, and heart weight of offspring was determined on postnatal days (P) 7 and 21. While no difference was seen on P 7, on P 21 pups from TCDD-treated litters showed an increase in heart-to-body weight ratio and in expression of the cardiac hypertrophy marker atrial natriuretic factor. Additionally, electrocardiograms of P 21 offspring showed that the combination of in utero and lactational TCDD exposure reduced postnatal heart rate but did not alter cardiac responsiveness to isoproterenol stimulation of heart rate. These results demonstrate that the fetal murine heart is a sensitive target of TCDD-induced teratogenicity, resembling many of TCDD-induced effects observed in fish and avian embryos, including reduced cardiocyte proliferation and altered fetal heart size. Furthermore, the combination of in utero and lactational TCDD exposure can induce cardiac hypertrophy and bradycardia postnatally, which could increase the risk of cardiovascular disease development.

Topics: Animals; Animals, Suckling; Atrial Natriuretic Factor; Cardiomegaly; Cell Proliferation; Dose-Response Relationship, Drug; Environmental Pollutants; Female; Fetal Development; Gene Expression Profiling; Heart; Heart Rate; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Organ Size; Polychlorinated Dibenzodioxins; Pregnancy; Prenatal Exposure Delayed Effects; RNA, Messenger; Teratogens

Several reports have suggested that the TAK1-MKK3/6-p38MAPK signaling axis is important for TGF-beta-related cardiac hypertrophy. Despite this, the effects of exogenous TGF-beta on cardiac hypertrophy and associated signaling mechanisms have not been demonstrated directly. Moreover, the roles of the signaling mechanisms involved in cardiac hypertrophy (TAK1 upstream and p38MAPK downstream) remain unclear. In this study, we investigated the potential involvement of protein kinase C and activating transcription factor-2 in TGF-beta1-induced cardiac hypertrophic responses in cultured neonatal rat ventricular cardiomyocytes. TGF-beta1 treatment resulted in upregulation of mRNA expression or promoter activities of beta-myosin heavy chain, atrial natriuretic factor, and brain natriuretic peptide, and increased myocyte protein content, cell size, and sarcomeric organization. These are all characteristic hallmarks of cardiac hypertrophy. PKC was found to be involved throughout the signaling system, and it was shown that it acts by mediating upstream TAK1 activation and leads to ATF-2 activation. PKC-dependent ATF-2 activation was shown to be involved in TGF-beta1-induced cardiac hypertrophic responses. The PKC inhibitors, GO6976 and GF109203X, completely blocked TGF-beta1-induced TAK1 kinase activity and subsequent downstream signaling pathways including ATF-2 phosphorylation, leading to suppression of ATF-2 transcriptional activity. This inhibitory effect was reflected in cardiac hypertrophic responses such as inhibitions of beta-MHC gene induction and ANF promoter activity. Our results suggest that PKC is involved in TGF-beta1-induced cardiac hypertrophic responses in our cell culture system and that ATF-2 activation plays a role.

Topics: Activating Transcription Factor 2; Animals; Atrial Natriuretic Factor; Carbazoles; Cardiomegaly; Cyclic AMP Response Element-Binding Protein; Gene Expression; Gene Expression Regulation; Indoles; Maleimides; MAP Kinase Kinase Kinases; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Promoter Regions, Genetic; Protein Kinase C; Rats; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1

In this study we examined diabetes- and hypertension-induced changes in cardiac structure and function in an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We hypothesized that treatment with omapatrilat, a vasopeptidase inhibitor, which causes simultaneous inhibition of angiotensin converting enzyme and neutral endopeptidase, provides additional cardioprotective effects, during normal- as well as high sodium intake, compared to treatment with enalapril, a selective inhibitor of angiotensin converting enzyme. Fifty-two GK rats were randomized into 6 groups to receive either normal-sodium (NaCl 0.8%) or high-sodium (NaCl 6%) diet and enalapril, omapatrilat or vehicle for 12 weeks. The GK rats developed hypertension, cardiac hypertrophy and overexpression of cardiac natriuretic peptides and profibrotic connective tissue growth factor compared to nondiabetic Wistar rats. The high dietary sodium further increased the systolic blood pressure, and changed the mitral inflow pattern measured by echocardiography towards diastolic dysfunction. Enalapril and omapatrilat equally decreased the systolic blood pressure compared to the control group during normal- as well as high-sodium diet. Both drugs had beneficial cardioprotective effects, which were blunted by the high dietary sodium. Compared to enalapril, omapatrilat reduced the echocardiographically measured left ventricular mass during normal-sodium diet and improved the diastolic function during high-sodium diet in GK rats. Furthermore, omapatrilat reduced relative cardiac weight more effectively than enalapril during high sodium intake. Our results suggest that both the renin-angiotensin and the neutral endopeptidase system are involved in the pathogenesis of diabetic cardiomyopathy since vasopeptidase inhibition was shown to provide additional benefits in comparison with selective angiotensin converting enzyme inhibition alone.

Topics: Aldosterone; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Blood Glucose; Blood Pressure; Body Weight; Cardiomegaly; Collagen; Diabetes Mellitus, Type 2; Echocardiography; Enalapril; Fibrosis; Heart; Insulin; Male; Metalloendopeptidases; Myocardium; Natriuretic Peptide, Brain; Organ Size; Protease Inhibitors; Pyridines; Random Allocation; Rats; Rats, Wistar; RNA, Messenger; Sodium Chloride, Dietary; Thiazepines

Increased cardiovascular mortality is an unresolved problem of chronic renal failure. Cardiac hypertrophy, observed in many patients with chronic renal failure, is a major risk factor for cardiovascular death. The purpose of the present study was to examine the effects of pitavastatin on cardiac hypertrophy in a progressive renal injury rat model by subtotal nephrectomy (SNx). Because we previously reported that angiotensin II played a pivotal role in cardiac hypertrophy of SNx rats, we first investigated the effects of pitavastatin on angiotensin II-induced activation of extracellular signal-regulated kinase (ERK) and serum response element (SRE) DNA-binding activity using neonatal rat cardiomyocytes. Angiotensin II-induced ERK activation was attenuated by pretreatment with pitavastatin. Luciferase assay revealed that angiotensin II-induced increase in SRE DNA-binding activity was inhibited by pitavastatin. We next examined the effect of pitavastatin on cardiac hypertrophy of SNx rats in vivo. Treatment with pitavastatin prevented ERK activation and cardiac hypertrophy in SNx rats without changes in blood pressure. The increased expression of atrial natriuretic factor mRNA in SNx rat hearts was significantly attenuated by the treatment with pitavastatin. These results suggest that pitavastatin has a beneficial effect on cardiac hypertrophy in renal failure through preventing the activation of ERK.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Disease Progression; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Kidney; Male; Myocytes, Cardiac; Nephrectomy; Phosphorylation; Quinolines; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasoconstrictor Agents

cAMP is one of the most important second messenger in the heart. The discovery of Epac as a guanine exchange factor (GEF), which is directly activated by cAMP, raises the question of the role of this protein in cardiac cells. Here we show that Epac activation leads to morphological changes and induces expression of cardiac hypertrophic markers. This process is associated with a Ca2+-dependent activation of the small GTPase, Rac. In addition, we found that Epac activates a prohypertrophic signaling pathway, which involves the Ca2+ sensitive phosphatase, calcineurin, and its primary downstream effector, NFAT. Rac is involved in Epac-induced NFAT dependent cardiomyocyte hypertrophy. Blockade of either calcineurin or Rac activity blunts the hypertrophic response elicited by Epac indicating these signaling molecules coordinately regulate cardiac gene expression and cellular growth. Our results thus open new insights into the signaling pathways by which cAMP may mediate its biological effects and identify Epac as a new positive regulator of cardiac growth.

Topics: Active Transport, Cell Nucleus; Adenoviridae; Animals; Atrial Natriuretic Factor; Calcineurin; Calcium; Cardiomegaly; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Genes, fos; Guanine Nucleotide Exchange Factors; NFATC Transcription Factors; Promoter Regions, Genetic; rac GTP-Binding Proteins; Rats; Signal Transduction; Thionucleotides

There is great interest in deciphering mechanisms of maladaptive remodeling in cardiac hypertrophy in the hope of affording clinical benefit. Potential targets of therapeutic intervention include the cytoplasmic phosphatase calcineurin and small guanosine triphosphate-binding proteins, such as Rac1 and RhoA, all of which have been implicated in maladaptive hypertrophy. However, little is known about the interaction-if any-between these important signaling molecules in hypertrophic heart disease. In this study, we examined the molecular interplay among these molecules, finding that Rho family guanosine triphosphatase signaling occurs either downstream of calcineurin or as a required, parallel pathway. It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition blocks hypertrophy, and we report here that "statin" therapy effectively suppresses small G protein activation and blunts hypertrophic growth in vitro and in vivo. Importantly, despite significant suppression of hypertrophy, clinical and hemodynamic markers remained compensated, suggesting that the hypertrophic growth induced by this pathway is not required to maintain circulatory performance.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cells, Cultured; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Rats; Signal Transduction

Lysophosphatidylcholine (LPC) is an endogenous phospholipid released from the cell membrane during ischemia, and it has potent cardiac effects, including inhibition of atrial natriuretic peptide (ANP) release. The aim of this study was to investigate the effects of LPC on hemodynamics and ANP release in hypertrophied atria and to define its mechanism. Isolated, perfused, beating, hypertrophied atria from monocrotaline-treated rats were used. LPC (30 micromol/L), a mixture of stearoyl-LPC, palmitoyl-LPC, and oleoyl-LPC, caused suppression of ANP release, which was markedly attenuated in hypertrophied atria compared with nonhypertrophied atria. Suppression of ANP release by stearoyl-LPC, palmitoyl-LPC, or oleoyl-LPC was also attenuated in hypertrophied atria. The potency appeared to be dependent on the species of fatty acid residue of LPC. Changes in ANP release by LPC, palmitoyl-LPC, and oleoyl-LPC were positively correlated with the degree of cardiac hypertrophy, but that by stearoyl-LPC was not. Changes in ANP release by LPC also were negatively correlated with changes in pulse pressure. Stearoyl-LPC caused an increase in intracellular Ca2+ in single, atrial myocytes in a concentration-dependent manner, which was markedly attenuated in hypertrophied atrial myocytes. These results suggest that attenuation of LPC-induced suppression of ANP release from hypertrophied atria might partly be related to changes in pulse pressure in terms of cardiac hypertrophy and/or disturbance of intracellular Ca2+ regulation.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Calcium; Cardiomegaly; Cells, Cultured; Culture Techniques; Heart Atria; Lysophosphatidylcholines; Male; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley

In neonatal rat ventricular myocytes, activation of receptors that couple to the G(q) family of heterotrimeric G proteins causes hypertrophic growth, together with expression of "hypertrophic marker" genes, such as atrial natriuretic peptide (ANP) and myosin light chain 2 (MLC2). As reported previously for other G(q)-coupled receptors, stimulation of alpha(1)-adrenergic receptors with phenylephrine (50 microM) caused phosphorylation of epidermal growth factor (EGF) receptors as well as activation of ERK1/2, cellular growth, and ANP transcription. These responses depended on EGF receptor activation. In marked contrast, stimulation of G(q)-coupled purinergic receptors with UTP caused EGF receptor phosphorylation, ERK1/2 activation, and cellular growth but minimal increases in ANP transcription. UTP inhibited phenylephrine-dependent transcription from ANP and MLC2 promoters but not transcription from myoglobin promoters or from AP-1 elements. Myocardin is a muscle-specific transcription enhancer that activates transcription from ANP and MLC2 promoters but not myoglobin promoters or AP-1 elements. UTP inhibited ANP and MLC2 responses to overexpressed myocardin but did not inhibit responses to c-Jun, GATA4, or serum response factor, all of which are active in nonmuscle cells. Thus, UTP inhibits transcriptional responses to phenylephrine only at cardiac-specific promoters, and this may involve the muscle-specific transcription enhancer, myocardin. These studies show that EGF receptor activation is necessary but not sufficient for ANP and MLC2 responses to activation of G(q)-coupled receptors in ventricular myocytes, because inhibitory mechanisms can oppose such stimulation. ANP is a compensatory and protective factor in cardiac hypertrophy, and mechanisms that reduce its generation need to be defined.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; ErbB Receptors; Myocytes, Cardiac; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Transcriptional Activation; Uridine Triphosphate

Topics: Aged; Aged, 80 and over; Atrial Natriuretic Factor; Cachexia; Cardiomegaly; Cardiomyopathy, Dilated; Fatal Outcome; Female; Heart Atria; Humans

Imidazoline receptors are divided into I(1) and I(2) subtypes. I(1)-imidazoline receptors are distributed in the heart and are upregulated during hypertension or heart failure. The aim of this study was to define the possible role of I(1)-imidazoline receptors in the regulation of atrial natriuretic peptide (ANP) release in hypertrophied atria. Experiments were performed on isolated, perfused, hypertrophied atria from remnant-kidney hypertensive rats. The relatively selective I(1)-imidazoline receptor agonist moxonidine caused a decrease in pulse pressure. Moxonidine (3, 10, and 30 micromol/l) also caused dose-dependent increases in ANP secretion, but clonidine (an alpha(2)-adrenoceptor agonist) did not. Pretreatment with efaroxan (a selective I(1)-imidazoline receptor antagonist) or rauwolscine (a selective alpha(2)-adrenoceptor antagonist) inhibited the moxonidine-induced increases in ANP secretion and interstitial ANP concentration and decrease in pulse pressure. However, the antagonistic effect of efaroxan on moxonidine-induced ANP secretion was greater than that of rauwolscine. Neither efaroxan nor rauwolscine alone has any significant effects on ANP secretion and pulse pressure. In hypertrophied atria, the moxonidine-induced increase in ANP secretion and decrease in pulse pressure were markedly augmented compared with nonhypertrophied atria, and the relative change in ANP secretion by moxonidine was positively correlated to atrial hypertrophy. The accentuation by moxonidine of ANP secretion was attenuated by efaroxan but not by rauwolscine. These results show that moxonidine increases ANP release through (preferentially) the activation of atrial I(1)-imidazoline receptors and also via different mechanisms from clonidine, and this effect is augmented in hypertrophied atria. Therefore, we suggest that cardiac I(1)-imidazoline receptors play an important role in the regulation of blood pressure.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Antihypertensive Agents; Atrial Function; Atrial Natriuretic Factor; Benzofurans; Blood Pressure; Cardiomegaly; Clonidine; Heart Atria; Hemodynamics; Imidazoles; Imidazoline Receptors; Male; Rats; Rats, Sprague-Dawley; Receptors, Drug; Yohimbine

Previous studies suggest that atrial natriuretic peptide (ANP) may act as an autocrine/paracrine factor to modulate cardiac hypertrophy in response to various stimuli. The effect of ANP deficiency on the response to volume overload has not previously been studied. We hypothesised that ANP deficient mice would develop excess cardiac hypertrophy in response to volume overload stress.. Male homozygous ANP deficient (Nppa(-/-)) and wildtype (Nppa(+/+)) male mice maintained on either a normal salt (0.55% NaCl) or low salt (0.05% NaCl) diet from weaning were studied after 2 weeks of volume overload from an aorto-caval fistula (ACF). Unoperated littermates served as controls. Left ventricular (LV) structure and function was evaluated by echocardiography. Heart, LV, and lung weights were determined at sacrifice. Myocyte diameter was measured by morphometric analysis of fixed sections of the left ventricle.. BP, heart weight, and LV weight were increased in Nppa(-/-) vs. Nppa(+/+) unoperated mice. Nppa(-/-) mice developed exaggerated heart and LV weight compared to Nppa(+/+) mice following ACF. Increased myocyte diameter paralleled increased echo LV wall thickness following ACF in Nppa(+/+) but not Nppa(-/-) mice fed with 0.55% NaCl, indicating that an alternate mechanism contributed to increased wall thickness in Nppa(-/-) mice. Mid-wall shortening was mildly depressed in the Nppa(-/-) vs. Nppa(+/+) genotype following ACF with fed 0.55% NaCl. A 0.05% NaCl diet from weaning normalized BP, but did not prevent exaggerated cardiac enlargement and LV hypertrophy following ACF in Nppa(-/-) mice.. ANP-deficient mice exhibited an exaggerated increase in heart and LV weight in response to volume overload, which was not prevented by normalization of blood pressure. The findings suggest that ANP is an important physiologic modulator of the cardiac hypertrophy induced by volume overload.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Echocardiography; Lung; Male; Mice; Mice, Knockout; Models, Animal; Myocardium; Organ Size

Vasopeptidase inhibitors simultaneously inhibit both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The aim of this study was to determine the cardiorenal effects of the vasopeptidase inhibitor omapatrilat in the transgenic m(Ren-2)27 rat which exhibits fulminant hypertension and severe organ pathology. At 6 weeks of age, male Ren-2 rats were randomized to receive no treatment (N = 10), the ACE inhibitor fosinopril 10 mg/kg/day (N = 10), or omapatrilat 10 mg/kg/day (N = 10) or 40 mg/kg/day (N = 10) by daily gavage for 24 weeks. Various cardiorenal functional and structural parameters were assessed. Compared to controls, all treatment groups reduced hypertension in control Ren-2 rats, with both doses of omapatrilat reducing systolic blood pressure significantly more than fosinopril (control, 178 +/- 3 mmHg; fosinopril 10 mg/kg/day, 130 +/- 4 mmHg; omapatrilat 10 mg/kg/day, 110 +/- 3 mmHg; omapatrilat 40 mg/kg/day, 91 +/- 3 mmHg). Omapatrilat dose-dependently reduced cardiac hypertrophy, caused a greater inhibition of renal ACE than fosinopril, and was the only treatment to inhibit renal NEP. Attenuation of albuminuria, glomerulosclerosis and cardiorenal fibrosis occurred to a similar degree with omapatrilat and fosinopril. Omapatrilat confers cardiorenal protection in the hypertensive Ren-2 rat. Although inhibition of tissue NEP may contribute to the superior blood pressure reduction by omapatrilat, overall, the results are consistent with the central role that angiotensin II plays in renal and cardiac fibrosis in this model of hypertension.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Animals, Genetically Modified; Antihypertensive Agents; Atrial Natriuretic Factor; Biomarkers; Blood Pressure; Body Weight; Cardiomegaly; Disease Models, Animal; Dose-Response Relationship, Drug; Fosinopril; Hypertension; Kidney; Male; Models, Cardiovascular; Neprilysin; Pyridines; Rats; Renin; Statistics as Topic; Survival Analysis; Systole; Thiazepines; Time Factors; Treatment Outcome

Glycogen synthase kinase 3beta (GSK-3beta) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3beta is eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon). Overexpression of GSK-3beta increased the cellular kinase activity toward GST-eIF2Bepsilon in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 micromol/L), a treatment known to inhibit GSK-3beta, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bepsilon antibody showed that S535 phosphorylation of endogenous eIF2Bepsilon was decreased by LiCl or ISO, suggesting that GSK-3beta is the predominant kinase regulating phosphorylation of eIF2Bepsilon-S535 in cardiac myocytes. Decreases in eIF2Bepsilon-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bepsilon alone moderately increased cell size (+31+/-11%; P<0.05 versus control), whereas treatment of eIF2Bepsilon-transduced myocytes with LiCl (+73+/-22% versus eIF2Bepsilon only; P<0.05) or ISO (+84+/-33% versus eIF2Bepsilon only; P<0.05) enhanced the effect of eIF2Bepsilon. Overexpression of eIF2Bepsilon-S535A, which is not phosphorylated by GSK-3beta, increased cell size (+107+/-35%) as strongly as ISO (+95+/-25%), and abolished antihypertrophic effects of GSK-3beta, indicating that S535 phosphorylation of eIF2Bepsilon critically mediates antihypertrophic effects of GSK-3beta. Furthermore, expression of eIF2Bepsilon-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2Bepsilon is required for beta-adrenergic hypertrophy. Interestingly, expression of eIF2Bepsilon-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3beta is the predominant kinase mediating phosphorylation of eIF2Bepsilon-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.

Topics: Adenoviridae; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Eukaryotic Initiation Factor-2B; Gene Expression Regulation; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heart Ventricles; Hypertrophy; Isoproterenol; Lithium Chloride; Male; Myocardial Infarction; Myocytes, Cardiac; Myosin Heavy Chains; Peptide Chain Initiation, Translational; Phosphoserine; Propranolol; Protein Processing, Post-Translational; Rats; Rats, Wistar; Recombinant Fusion Proteins; Transduction, Genetic; Ventricular Remodeling

Osteopontin (OPN) is upregulated in left ventricular hypertrophy and is stimulated by angiotensin II (AngII). Our objective was to determine whether mice deficient in OPN would be protected from AngII-induced cardiac fibrosis.. Interstitial fibrosis can lead to myocardial dysfunction and ultimately heart failure. Osteopontin activates integrins that regulate cell adhesion, migration, and growth, thus implicating OPN in the process of cardiac fibrosis.. Osteopontin null (OPN(-/-)) mice (n = 18) and wild-type controls (n = 20) were infused with AngII (2.5 or 3.0 microg/kg/min) for four days or three weeks via osmotic mini-pumps. Hearts were assessed morphometrically and histologically, including quantitative assessment of fibrosis via optical microscopic imaging analysis. Cardiac fibroblasts derived from these mice were evaluated for adhesion and proliferation. Cardiac transcript expression for cytokines, extracellular matrix (ECM), integrin, and atrial natriuretic peptide were assessed.. Osteopontin(-/-) mice exhibited less cardiac fibrosis (0.7%) than wild-type mice (8.0%) (p < 0.01) and lowered heart/body weight ratios (0.10% vs. 0.23%) (p < 0.01) after three weeks of AngII infusion. Expression of transforming growth factor-beta, fibronectin, and collagen was not different between OPN(-/-) and wild-type mice, despite the decrease in ECM accumulation in the OPN(-/-) mice. Adhesion to ECM substrates decreased by 30% to 50% in cardiac fibroblasts of OPN(-/-) mice but was restored in OPN(-/-) cells by the addition of recombinant osteopontin.. Osteopontin mediates cardiac fibrosis, probably through the modulation of cellular adhesion and proliferation. Because OPN is increased in cardiac hypertrophy and its lack attenuates fibrosis, understanding of OPN function is essential to extend our knowledge about molecular determinants of cardiac hypertrophy and failure.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Pressure; Blotting, Northern; Cardiomegaly; Cell Adhesion; Cell Division; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Matrix; Fibroblasts; Fibrosis; Mice; Mice, Knockout; Models, Cardiovascular; Muscle Proteins; Myocardium; Nuclear Proteins; Osteopontin; Repressor Proteins; Sialoglycoproteins; Up-Regulation; Vasoconstrictor Agents

Atrial natriuretic peptide (ANP) enhances cardiac vagal baroreflexes in normotensive animals. In spontaneously hypertensive rats (SHRs) this effect of ANP was absent. The reflex actions of ANP were preserved if hypertrophy was completely prevented in SHRs. However even a small amount of cardiac hypertrophy, with no hypertension, in SHRs was accompanied by a loss of the reflex bradycardic actions of ANP. In the present study, we investigated whether pathophysiological cardiac hypertrophy, induced by one-kidney, one-clip renovascular hypertension (1K-1C; n = 6), or physiological cardiac hypertrophy induced by chronic spontaneous, wheel-running exercise training (n = 7), similarly prevented vagal reflex actions of ANP. Cardiac baroreceptor-activated bradycardia was measured during rapid ramp increases ( approximately 5 s) in blood pressure after bolus doses of methoxamine or vehicle in conscious, chronically instrumented rats during infusions of ANP (50 pmol kg(-1) min(-1)). Compared with uninephrectomised control rats (n = 10), rats with 1K-1C had cardiac hypertrophy (approximately 55% increase in left ventricle:body weight (LV:BW) ratio; P < 0.05) and blunted vagal baroreflex gain (-0.93 +/- 0.18 versus-0.50 +/- 0.13 beats min(-1) mmHg(-1); P < 0.05). ANP did not augment baroreflex function in 1K-1C. Compared with their sedentary controls (n = 7), exercise-trained rats with cardiac hypertrophy ( approximately 20% increase LV:BW ratio; P < 0.05) also had blunted ramp baroreflex bradycardia (-1.28 +/- 0.23 versus-0.57 +/- 0.09 beats min(-1) mmHg(-1); P < 0.05). In contrast, ANP more than doubled baroreflex bradycardia in exercise-trained rats (P < 0.05). The aetiology of cardiac hypertrophy therefore influenced whether ANP retained its vagal baroreflex enhancing properties.

Topics: Animals; Atrial Natriuretic Factor; Baroreflex; Cardiomegaly; Hypertension, Renal; Male; Nephrectomy; Physical Conditioning, Animal; Physical Exertion; Rats; Rats, Wistar; Vagus Nerve

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.

Topics: Adenoviridae; Animals; Antioxidants; Aorta; Atrial Natriuretic Factor; Cardiomegaly; I-kappa B Kinase; I-kappa B Proteins; Ligation; Male; Myocardium; Natriuretic Peptide, Brain; NF-kappa B; NF-KappaB Inhibitor alpha; Proline; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Thiocarbamates; Transfection

Stored cardiac pro-atrial natriuretic peptide (pro-ANP) is converted to ANP and released upon stretch from the atria into the circulation. Corin is a serin protease with pro-ANP-converting properties and may be the rate-limiting enzyme in ANP release. This study was aimed to clone and sequence corin in the rat and to analyze corin mRNA expression in heart failure when ANP release upon stretch is blunted. Full-length cDNA of rat corin was obtained from atrial RNA by RT-PCR and sequenced. Tissue distribution as well as regulation of corin mRNA expression in the atria were determined by RT-PCR and RNase protection assay. Heart failure was induced by an infrarenal aortocaval shunt. Stretch was applied to the left atrium in a working heart modus, and ANP was measured in the perfusates. The sequence of rat corin cDNA was found to be 93.6% homologous to mouse corin cDNA. Corin mRNA was expressed almost exclusively in the heart with highest concentrations in both atria. The aortocaval shunt led to cardiac hypertrophy and heart failure. Stretch-induced ANP release was blunted in shunt animals (control 1,195 +/- 197 fmol.min(-1).g(-1); shunt: 639 +/- 99 fmol.min(-1).g(-1), P < 0.05). Corin mRNA expression was decreased in both atria in shunt animals [right atrium: control 0.638 +/- 0.004 arbitrary units (AU), shunt 0.566 +/- 0.014 AU, P < 0.001; left atrium: control 0.564 +/- 0.009 AU, shunt 0.464 +/- 0.009 AU, P < 0.001]. Downregulation of atrial corin mRNA expression may be a novel mechanism for the blunted ANP release in heart failure.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Cardiomegaly; Central Venous Pressure; Cloning, Molecular; Gene Expression; Heart Failure; Male; Molecular Sequence Data; Myocardium; Organ Size; Rats; Rats, Wistar; RNA, Messenger; Serine Endopeptidases; Ventricular Pressure

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression; Heart Failure; Myocytes, Cardiac; Natriuretic Peptide, Brain; Rats; RNA, Messenger; Serine Endopeptidases; Up-Regulation

Cardiac hypertrophy is characterized by increased cardiomyocyte size, mRNA levels for atrial natriuretic factor (ANF), and protein synthesis. Although activation of the phosphoinositide-specific phospholipase C (PLC) leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the involvement of PLC in hypertrophic response remains to be fully understood. The present study was therefore undertaken to examine if the inhibition of PLC activity is associated with a decrease in ANF expression and protein synthesis in cardiomyocytes, due to norepinephrine (NE), a known hypertrophic agent. NE resulted in an increase in ANF gene expression and protein synthesis in adult rat cardiomyocytes, these effects of NE were attenuated by a PLC inhibitor, U73122. The NE-induced increase in ANF gene expression and protein synthesis was also inhibited by an alpha-adrenoceptor blocker, prazosin. Both U73122 and prazosin depressed the NE-induced increase in DAG production in cardiomyocytes. These results indicate that the alpha-adrenoceptor mediated PLC activation may be involved in the process of NE-induced cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Dose-Response Relationship, Drug; Heart Ventricles; Male; Myocytes, Cardiac; Norepinephrine; Rats; Rats, Sprague-Dawley; Type C Phospholipases

The success of any gene-therapy approach depends on the survival of the genetically engineered cells that are implanted in the patient to deliver the therapeutic product. Immunoisolation of nonautologous cells within a microcapsule is a unique approach for gene therapy.. We employed an immunoisolation device that protects nonautologous cells from destruction, to implant human atrial natriuretic peptide (hANP)-producing Chinese hamster ovary (CHO) cells in two-kidney, one-clip (2K1C) hypertensive rats. CHO cells transfected with the plasmid CMV-cANP were encapsulated in biocompatible polycaprolactone (PCL) capsules, and then the PCL capsules were implanted into 2K1C hypertensive rats intraperitoneally.. The implantation of encapsulated hANP-producing cells caused a significant delay of blood pressure (BP) increase 2 weeks post-implantation and the effect lasted for more than 5 months. The implantation of encapsulated hANP-producing cells also caused significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), sodium output, urine excretion, and urinary cGMP levels. These beneficial effects were reflected morphologically by an attenuation of the glomerular sclerotic lesions, reduction in cardiomyocyte size, tubular injury and renal arterial thickening. Immunoreactive hANP can be detected in the blood of 2K1C rats after implantation of the PCL capsules containing hANP-producing cells.. This study demonstrates the usefulness of encapsulated ANP gene transfected cells as a new tool for ANP gene delivery in studying renovascular hypertension and cardiovascular diseases. Thus, our results may have important implications for clinical use of transgene cells as therapeutic agents in the treatment of cardiovascular diseases.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; CHO Cells; Cricetinae; Hypertension; Kidney; Kidney Function Tests; Male; Microscopy, Confocal; Myocardium; Rats; Rats, Wistar; Transfection; Transgenes

Cardiac hypertrophy is formed in response to hemodynamic overload. Although a variety of factors such as catecholamines, angiotensin II (AngII), and endothelin-1 (ET-1) have been reported to induce cardiac hypertrophy, little is known regarding the factors that inhibit the development of cardiac hypertrophy. Production of atrial natriuretic peptide (ANP) is increased in the hypertrophied heart and ANP has recently been reported to inhibit the growth of various cell types. We therefore examined whether ANP inhibits the development of cardiac hypertrophy. Pretreatment of cultured cardiomyocytes with ANP inhibited the AngII- or ET-1-induced increase in the cell size and the protein synthesis. ANP also inhibited the AngII- or ET-1-induced hypertrophic responses such as activation of mitogen-activated protein kinase (MAPK) and induction of immediate early response genes and fetal type genes. To determine how ANP inhibits cardiomyocyte hypertrophy, we examined the mechanism of ANP-induced suppression of the MAPK activation. ANP strongly induced expression of MAPK phosphatase-1 (MKP-1) and overexpression of MKP-1 inhibited AngII- or ET-1-induced hypertrophic responses. These growth-inhibitory actions of ANP were mimicked by a cyclic GMP analog 8-bromo-cyclic GMP. Taken together, ANP directly inhibits the growth factor-induced cardiomyocyte hypertrophy at least partly via induction of MKP-1. Our present study suggests that the formation of cardiac hypertrophy is regulated not only by positive but by negative factors in response to hemodynamic load.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Cycle Proteins; Cells, Cultured; Dose-Response Relationship, Drug; Dual Specificity Phosphatase 1; Immediate-Early Proteins; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Rats, Wistar; Signal Transduction

Obesity is a major risk factor for the development of cardiovascular disease. Emerging evidence indicates that leptin, a protein encoded by the obesity gene, is linked with cardiac hypertrophy in obese humans and directly induces cardiomyocyte hypertrophy in vitro. However, the mechanisms by which leptin induces cardiomyocyte hypertrophy are poorly understood.. This study investigated how leptin contributes to cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were used to evaluate the effects of leptin on hypertrophy. Both endothelin-1 (ET-1) and reactive oxygen species (ROS) levels were elevated in a concentration-dependent manner in cardiomyocytes treated with leptin for 4 hours compared with those cells without leptin treatment. ET-1 stimulated ROS production in a concentration-dependent manner in cardiomyocytes. The augmentation of ROS levels in cardiomyocytes treated with both leptin and ET-1 was reversed by a selective ET(A) receptor antagonist, ABT-627, and catalase, a hydrogen peroxide-decomposing enzyme. After treatment for 72 hours, leptin or ET-1 concentration-dependently increased total RNA levels, cell surface areas, and protein synthesis in cardiomyocytes, all of which were significantly inhibited by ABT-627 or catalase treatment.. These findings indicate that leptin elevates ET-1 and ROS levels, resulting in hypertrophy of cultured neonatal rat cardiac myocytes. The ET-1-ET(A)-ROS pathway may be involved in cardiomyocyte hypertrophy induced by leptin. ET(A) receptor antagonists and antioxidant therapy may provide an effective means of ameliorating cardiac dysfunction in obese humans.

Topics: Animals; Animals, Newborn; Antioxidants; Atrasentan; Atrial Natriuretic Factor; Cardiomegaly; Catalase; Cell Size; Cells, Cultured; Endothelin A Receptor Antagonists; Endothelin-1; Gene Expression Regulation; Leptin; Myocytes, Cardiac; Obesity; Oxidative Stress; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We hypothesized that a single copy of the proatrial natriuretic peptide gene (Nppa+/-) would not be adequate to protect heterozygous mice against exaggerated cardiac hypertrophy and remodeling after pressure-overload stress. Nppa+/+, Nppa+/-, and Nppa-/- mice were subjected to sham surgery or transverse aortic constriction and fed a basal salt diet. Heart weight varied inversely with Nppa gene load by 1 week after either surgery. Fractional shortening did not differ among genotypes at baseline and fell in Nppa-/- mice only after transverse aortic constriction. There was a graded response in collagen deposition related to atrial natriuretic peptide (ANP) expression after either surgery. A robust interstitial and perivascular fibrosis was noted in Nppa-/- and Nppa+/- but not in Nppa+/+ mice after transverse aortic constriction. Our findings are consistent with a growing body of evidence that ANP is an important modulator of cardiac hypertrophy and remodeling in response to hemodynamic stress. The observation that partial ANP deficiency results in exaggerated hypertrophy and remodeling after pressure overload suggests that genetic or environmental variation in ANP levels may play a role in the development of cardiac hypertrophy, remodeling, and failure in humans.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Collagen; Disease Models, Animal; Genotype; Heart Ventricles; Male; Mice; Mice, Knockout; Myocardium; Organ Size; Phenotype; Stress, Physiological; Ventricular Remodeling

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner.

Topics: Animals; Atrial Natriuretic Factor; Basic Helix-Loop-Helix Transcription Factors; Cardiomegaly; Cells, Cultured; Chlorocebus aethiops; COS Cells; DNA-Binding Proteins; GATA4 Transcription Factor; Gene Deletion; Gene Expression; Helix-Loop-Helix Motifs; Humans; Luciferases; Mice; Myocytes, Cardiac; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Recombinant Fusion Proteins; Repressor Proteins; RNA, Messenger; Signal Transduction; Transcription Factors; Transfection

cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.

Topics: Adrenergic Agonists; Adrenergic alpha-1 Receptor Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; CREB-Binding Protein; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Immunoprecipitation; Indoles; Isoquinolines; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Rats; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Messenger; Signal Transduction; Sulfonamides; Trans-Activators

Diverse pathological insults trigger a cardiac remodeling process during which myocytes undergo hypertrophy, with consequent decline in cardiac function and eventual heart failure. Multiple transcriptional regulators of pathological cardiac hypertrophy are controlled at the level of subcellular distribution. For example, prohypertrophic transcription factors belonging to the nuclear factor of activated T cells (NFAT) and GATA families are subject to CRM1-dependent nuclear export but are rapidly relocalized to the nucleus in response to cues for hypertrophic growth. Here, we demonstrate that the antihypertrophic chromatin-modifying enzyme histone deacetylase 5 (HDAC5) is shuttled out of the cardiomyocyte nucleus via a CRM1-mediated pathway in response to diverse signals for hypertrophy. CRM1 antagonists block the agonist-mediated nuclear export of HDAC 5 and repress pathological gene expression and associated hypertrophy of cultured cardiomyocytes. Conversely, CRM1 activity is dispensable for nonpathological cardiac gene activation mediated by thyroid hormone and insulin-like growth factor 1, agonists that fail to trigger the nuclear export of HDAC5. These results suggest a selective role for CRM1 in derepression of pathological cardiac genes via its neutralizing effects on antihypertrophic factors such as HDAC5. Pharmacological approaches targeting CRM1-dependent nuclear export in heart muscle may have salutary effects on cardiac function by suppressing maladaptive changes in gene expression evoked by stress signals.

Topics: Adenoviridae; Adenylate Kinase; Adhesins, Bacterial; Animals; Animals, Newborn; Antibodies, Monoclonal; Atrial Natriuretic Factor; Cardiomegaly; Cell Nucleus; Cell Size; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Exportin 1 Protein; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation; Green Fluorescent Proteins; Heart Ventricles; Histone Deacetylases; Immunoblotting; Karyopherins; Microscopy, Fluorescence; Myocytes, Cardiac; Precipitin Tests; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; RNA; Transcriptional Activation

Neonatal cardiac hypertrophy associated with diabetic pregnancy is transient and regresses naturally, but is associated with increased morbidity and mortality. This study was undertaken to analyse the changes in expression of 5 cardiac genes, including atrial natriuretic peptide, alpha- and beta-myosin heavy chain, and cardiac and skeletal alpha-actin genes, using a rat neonatal model, in which cardiac hypertrophy was induced via maternal diabetes. In the hypertrophied left ventricle of neonates from diabetic mothers, the levels of mRNA from all the above genes except skeletal alpha-actin were increased by between 1.8- and 12-fold compared with the controls at birth (p < 0.05). In the first 28 days, the level of mRNA for alpha-myosin heavy chain increased slightly, while that for atrial natriuretic peptide and beta-myosin heavy chain decreased continuously similar to the controls, but at a significantly faster rate. No significant difference between the two groups of neonates was observed in all 5 genes after 1 month, indicating complete regression. Expression of 5 cardiac genes in the neonatal cardiac hypertrophy was characterised in both hypertrophic and regressive phases. Hypertrophic regression provides a unique model for the testing of new drugs or genetic modifying factors in cardiac hypertrophy.

Topics: Actins; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Female; Pregnancy; Pregnancy in Diabetics; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Ventricular Myosins

Diabetes mellitus is associated with endothelial and cardiac dysfunction, and endothelin has been suggested to alter cardiac function by being a positive inotropic agent, modulating the Frank-Starling response, contracting the coronary arteries and inducing tissue proliferation. We investigated endothelin levels in diabetic and in healthy dogs, 1 and 3 days after placing arteriovenous shunts (8 weeks after diabetes induction) in the femoral regions. Right and left ventricular weight/body weight ratios and Nterminal- atrial natriuretic peptide were increased in shunted animals (P < 0.05). Plasma endothelin levels were comparable in healthy and diabetic dogs. Shunted circulation did not change systemic endothelin levels in healthy dogs but reduced endothelin levels in diabetic dogs. The functional significance of altered endothelin responses to acute hemodynamic burden in experimental diabetes needs further investigation.

Topics: Animals; Arteriovenous Shunt, Surgical; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Diabetes Mellitus, Experimental; Disease Models, Animal; Dogs; Endothelin-1; Female; Male; Protein Precursors; Time Factors

Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Caveolae; Caveolin 1; Caveolins; Cell Membrane; Extracellular Matrix; Female; Fibroblasts; Hypertrophy, Left Ventricular; Immunohistochemistry; Magnetic Resonance Imaging; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocardium; Myocytes, Cardiac; Ventricular Dysfunction, Right

Extracellular-signal-regulated protein kinases (ERKs) are activated rapidly and transiently in response to phenylephrine (PE) and endothelin-1 (ET-1) in cardiac myocytes, but whether this is linked to the subsequent development of the hypertrophic phenotype remains equivocal. To investigate this, we examined the dependence of the hypertrophic response on the length of exposure to PE in neonatal myocyte cultures. In addition to the initial transient activation of ERKs (maximum at 5-10 min), PE (10 microM) induced a second, more prolonged peak of activity several hours later. The activity of a transfected atrial natriuretic factor-luciferase reporter gene was increased 10- to 24-fold by PE. This response was inhibited by the alpha(1)-antagonist prazosin (100 nM) and by U0126 (10 microM) and PD184352 (1 microM), inhibitors of ERK activation, irrespective of whether these were added before or up to 24 h after the addition of PE. Prazosin had no effect on ET-1 (50 nM)-stimulated atrial natriuretic factor-luciferase activity. Protein synthesis was enhanced by 35+/-6% by PE, and this was blocked by prazosin added 1 h after the addition of PE, but decreased only by half when added 8 h after PE. Similarly, PE (48 h) increased myocyte area by 49% and this was prevented by prazosin added 1 h after PE, but decreased only by half when added at 24 h. These results demonstrate that prolonged exposure to PE is required to elicit alterations in gene expression, protein synthesis and cell size, characteristic of hypertrophied myocytes, and they confirm that the initial peak of ERK activity is insufficient to trigger hypertrophic responses.

Topics: Adrenergic alpha-Antagonists; Animals; Atrial Natriuretic Factor; Benzamides; Butadienes; Cardiomegaly; Cell Size; Cells, Cultured; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Luciferases; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Nitriles; Phenylephrine; Prazosin; Protein Biosynthesis; Proteins; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Time Factors

The present study was undertaken to determine tonin expression and activity in rat heart presenting isoproterenol-induced hypertrophy. Renin, angiotensin-converting enzyme (ACE), and angiotensinogen (AG) expression were also determined. Wistar rats were treated with isoproterenol for 7 days (5 mg x kg(-1) x day(-1) sc). For untreated animals, the levels of tonin-specific activity in the atrium were 2.6- and 5.5-fold higher than those of the left and right ventricle, respectively. After treatment, the levels of tonin-specific activity increased twofold in the atrium but did not change in the ventricles. Renin expression was not detectable in these structures, and ACE expression levels did not change with treatment. AG expression was detected in the left ventricle at very low levels compared with the atrium and increased significantly only in the hypertrophied atrium (1.8-fold). Tonin mRNA was not detected in the ventricle but was found at low levels in the atrium, which increased after isoproterenol treatment. Our results permit us to conclude that tonin may play a role in the process of heart hypertrophy in the rat.

Topics: Adrenergic beta-Agonists; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Progression; Gene Expression Regulation; Isoproterenol; Male; Myocardium; Organ Size; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA Probes; Tissue Kallikreins

Atrial natriuretic peptide (ANP) prevents hypertrophy of neonatal cardiomyocytes. However, whether this effect is retained in the adult phenotype or if other members of the natriuretic peptide family exhibit similar antihypertrophic properties, has not been elucidated.. Our objective was to examine whether the natriuretic peptides protect against adult cardiomyocyte hypertrophy in vitro.. Adult rat cardiomyocytes were incubated with angiotensin II (Ang II)+/-ANP, B-type (BNP) or C-type (CNP) natriuretic peptides for determination of [3H]phenylalanine incorporation, c-fos mRNA expression and cyclic GMP. The effects of 8-bromo-cyclic GMP (cyclic GMP analogue), HS-142-1 (particulate guanylyl cyclase inhibitor) and KT5823 (cyclic GMP-dependent protein kinase inhibitor) were also investigated.. Ang II-stimulated increases in markers of hypertrophy, [3H]phenylalanine incorporation (to 136+/-3% of control, n=9) and c-fos mRNA expression (4.3+/-1.4-fold, n=5), were completely prevented by each of ANP, BNP or CNP. This protective action was accompanied by increased cardiomyocyte cyclic GMP. Inhibitory actions on [3H]phenylalanine incorporation were mimicked by 8-bromo-cyclic GMP, and were abolished by HS-142-1. KT5823 blocked the response to BNP and CNP, but not to ANP.. ANP prevents hypertrophy of adult rat cardiomyocytes. This protective action is shared by BNP and CNP and involves activation of particulate guanylyl cyclase receptors. Antihypertrophic effects of BNP and CNP are mediated through cyclic GMP-dependent protein kinase, but ANP can activate additional pathways independent of cyclic GMP to prevent adult cardiomyocte hypertrophy. These novel findings are of interest particularly since BNP appears to exert antifibrotic rather than antihypertrophic actions in vivo, while CNP is thought to act at least in part via the endothelium.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Drug Interactions; Guanylate Cyclase; Male; Muscle Cells; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Collagen; Enzyme Inhibitors; Extracellular Matrix; Fibronectins; Integrins; Myocytes, Cardiac; Okadaic Acid; Phenylephrine; Protein Isoforms; Rats

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

Topics: Actins; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Gene Deletion; Guanylate Cyclase; Hemodynamics; Humans; Mice; Mice, Knockout; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Organ Size; Phenotype; Receptors, Atrial Natriuretic Factor; RNA, Messenger

This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Constriction; Extracellular Matrix Proteins; Gene Expression Profiling; Immunohistochemistry; Male; Mice; Mice, Knockout; Myocardium; Natriuretic Peptide, Brain; Oligonucleotide Array Sequence Analysis; Organ Size; Pressure; Protein Precursors; RNA, Messenger

Postnatal cardiac myocytes respond to stress signals by hypertrophic growth and activation of a fetal gene program. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy, and mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals. To further define the roles of HDACs in cardiac hypertrophy, we analyzed the effects of HDAC inhibitors on the responsiveness of primary cardiomyocytes to hypertrophic agonists. Paradoxically, HDAC inhibitors imposed a dose-dependent blockade to hypertrophy and fetal gene activation. We conclude that distinct HDACs play positive or negative roles in the control of cardiomyocyte hypertrophy. HDAC inhibitors are currently being tested in clinical trials as anti-cancer agents. Our results suggest that these inhibitors may also hold promising clinical value as therapeutics for cardiac hypertrophy and heart failure.

Topics: Adenylate Kinase; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Fluorescent Antibody Technique, Indirect; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Leucine; Myocardium; Rats; Rats, Sprague-Dawley; Tritium; Ventricular Myosins

Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. We previously reported that 5-HT2B receptor ablation in mice leads to dilated cardiomyopathy. In this study, we investigated the pathological consequence of overexpressing 5-HT2B receptors in heart in vivo.. We have generated transgenic mice overexpressing the Gq-coupled 5-HT2B receptor specifically in heart. We found that overexpression of 5-HT2B receptor in heart leads to ventricular hypertrophy as the result of increased cell number and size. Increased atrial natriuretic peptide and myosin heavy chain expression demonstrated activation of the molecular program for cardiac hypertrophy. Echocardiographic analysis indicated the presence of thickened ventricular free wall without alteration of the systolic function, showing that transgenic mice have compensated hypertrophy. Electron microscopic analysis revealed structural abnormalities including mitochondrial proliferation, as also manifested by histological staining. Transgenic mouse heart displayed a specific reduction in the expression levels of the adenine nucleotide translocator associated to increase in the succinate dehydrogenase and cytochrome C oxidase mitochondrial activities.. Our results constitute the first genetic evidence that overexpression of the 5-HT2B receptor in the heart leads to compensated hypertrophic cardiomyopathy associated with proliferation of the mitochondria. This observation suggests a role for mitochondria in the hypertrophic signaling that is regulated by serotonin. These transgenic mice provide a new genetic model for hypertrophic heart disease.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Count; Cell Size; Disease Models, Animal; Echocardiography; Electron Transport Complex IV; Gene Expression; GTP-Binding Protein alpha Subunits, Gq-G11; Heart Ventricles; Heterotrimeric GTP-Binding Proteins; Male; Mice; Mice, Transgenic; Mitochondria; Mitochondrial ADP, ATP Translocases; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Organ Specificity; Receptor, Serotonin, 5-HT2B; Receptors, Serotonin; Signal Transduction; Succinate Dehydrogenase

Cardiac hypertrophy is an adaptive response to increases in blood pressure. Recent studies indicate that the hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. We hypothesize that superoxide anion mediates the hypertrophic response and that antioxidant therapy may be effective in attenuating cardiac hypertrophy. Neonatal rat cardiac myocytes were stimulated with angiotensin II (AngII, 1 microM) with and without various antioxidants. N-acetylcysteine (NAC, 10 mM) and probucol (50 microM), and to a lesser extent, vitamin C (500 microM) and reduced glutathione (1 mM), inhibited AngII-induced [(3)H]-leucine uptake and atrial natriuretic factor (ANF) promoter activity. The hypertrophic response is mediated by superoxide anion (O(2)(-).) since cell-permeable polyethylene glycol (PEG)-conjugated superoxide dismutase (50 U/ml), but not PEG-catalase (500 U/ml), attenuated AngII-induced [(3)H]-leucine uptake and ANF promoter activity. Furthermore, NAC blocked AngII-induced increase in myocardial oxidative stress, decreased the expression of ANF and myosin light chain-2v, and inhibited the re-organization of cytoskeletal proteins, desmin and alpha-actinin. These effects of AngII were abolished by angiotensin type 1 receptor blocker, losartan, but not type 2 receptor blocker, PD123319. Indeed, co-administration of losartan (10 mg/kg/d, 14 d) or NAC (200 mg/kg/d, 14 d) inhibited AngII-induced O(2)(-). production and cardiac hypertrophy in rats without affecting blood pressure. These findings indicate that the generation of O(2)(-). contributes to oxidant-induced hypertrophic response and suggest that antioxidant therapy may have beneficial effects in cardiac hypertrophy.

Topics: Angiotensin II; Animals; Antioxidants; Atrial Natriuretic Factor; Cardiomegaly; Myocytes, Cardiac; NADPH Oxidases; Promoter Regions, Genetic; rac1 GTP-Binding Protein; Rats; Reactive Oxygen Species; Superoxides; Vasoconstrictor Agents

Recent in vivo and in vitro studies in animals have demonstrated that cytokines of the IL-6 family are involved in cardiac hypertrophy and in protection of cardiomyocytes against apoptosis. The present study aims to analyse the capacity of human atrial cardiac cells (i.e., cardiomyocytes and fibroblasts) to display the gp130 receptor subunit, and to evaluate its functionality.. Twenty human atrial biopsies were used for immunohistochemistry, in situ hybridisation, and western blot analysis or dissociated for isolation and primary culture of cardiac cells.. Fibroblasts present in tissue or maintained in primary culture clearly express gp130 whereas the signal in cardiomyocytes is weaker. Culture of cardiac cells with a gp130 agonist antibody enhances atrial natriuretic peptide (ANP), beta myosin heavy chain (beta-MHC) expression in cardiomyocytes, and significantly increases the cell surface area microm(2)). This process could involve STAT3 (signal transducer and activator of transcription 3) phosphorylation.. These results demonstrate that gp130 activation in human cardiac cells leads to cardiomyocyte hypertrophy. We discuss several hypotheses on the role of IL-6-type cytokines on cardiomyocyte functions.

Topics: Aged; Analysis of Variance; Antibodies, Blocking; Antigens, CD; AraC Transcription Factor; Atrial Natriuretic Factor; Bacterial Proteins; Blotting, Western; Cardiomegaly; Cell Size; Cells, Cultured; Cytokine Receptor gp130; DNA-Binding Proteins; Fibroblasts; Fluorescent Antibody Technique, Indirect; Heart Atria; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin-6; Membrane Glycoproteins; Middle Aged; Myocytes, Cardiac; Myosin Heavy Chains; Phosphorylation; Quaternary Ammonium Compounds; Receptors, Cytokine; Receptors, Interleukin-6; Repressor Proteins; STAT3 Transcription Factor; Trans-Activators; Transcription Factors

Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6-related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and beta-galactosidase (Adbeta-gal). Compared with cardiomyocytes infected with Adbeta-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Adbeta-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal alpha-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal-regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Adbeta-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Topics: Actins; Adenoviridae; Animals; Antigens, CD; Atrial Natriuretic Factor; Binding Sites; Cardiomegaly; Cells, Cultured; Cytokine Receptor gp130; Gene Expression Regulation; Genes, Reporter; Genetic Vectors; Growth Inhibitors; Interleukin-6; Intracellular Signaling Peptides and Proteins; Leukemia Inhibitory Factor; Lymphokines; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 7; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Binding; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Transfection

The ribosomal DNA transcription-specific factor, UBF, is a key target for the regulation of ribosomal RNA synthesis and hypertrophic growth of isolated neonatal cardiomyocytes. In this study, we have examined whether UBF expression is also an important determinant of cardiac growth rates in vivo. We show that rDNA transcription, rRNA synthesis and UBF expression in left ventricular myocytes isolated from mice 1-6 weeks following transverse aortic constriction were significantly increased (2.5-3.5-fold) compared to the levels in myocytes from the left ventricle of sham-operated mice.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart Ventricles; Hypertrophy, Left Ventricular; Mice; Muscle Cells; Pol1 Transcription Initiation Complex Proteins; RNA, Messenger; RNA, Ribosomal; Transcription, Genetic; Up-Regulation

Stimulation of cardiomyocyte guanosine 3',5'-cyclic monophosphate (cyclic GMP) via endothelial-derived nitric oxide (NO) is an important mechanism by which bradykinin and ACE inhibitors prevent hypertrophy. Endothelial NO dysfunction and cardiac hypertrophy are morbid features of diabetes not entirely prevented by ACE inhibitors. In cardiomyocyte/endothelial cell cocultures, bradykinin efficacy is abolished by high-glucose-induced endothelial NO dysfunction. We now demonstrate that antihypertrophic actions of natriuretic peptides, which stimulate cyclic GMP independently of NO, are preserved in cardiomyocytes despite high-glucose-induced endothelial dysfunction. Further, streptozotocin-induced diabetes significantly impairs the effectiveness of acute antihypertrophic strategies in isolated rat hearts. In hearts from citrate-treated control rats, angiotensin II-stimulated [(3)H]phenylalanine incorporation and atrial natriuretic peptide and beta-myosin heavy chain mRNA expression were prevented by B-type natriuretic peptide (BNP), bradykinin, the ACE inhibitor ramiprilat, and the neutral endopeptidase inhibitor candoxatrilat. These antihypertrophic effects were accompanied by increased left ventricular cyclic GMP. In age-matched diabetic hearts, the antihypertrophic and cyclic GMP stimulatory actions of bradykinin, ramiprilat, and candoxatrilat were absent. However, the blunting of hypertrophic markers and accompanying increases in cyclic GMP stimulated by BNP were preserved in diabetes. Thus BNP, which increases cyclic GMP independently of NO, is an important approach to prevent growth in the diabetic myocardium, where endothelium-dependent mechanisms are compromised.

Topics: Acute Disease; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Chronic Disease; Cyclic GMP; Diabetes Mellitus, Experimental; Gene Expression; Heart Ventricles; Male; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phenylalanine; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tritium

Electrical conductance is greatly altered in end-stage heart failure, but little is known about the underlying events. We therefore investigated the expression of genes coding for major inward and outward ion channels, calcium binding proteins, ion receptors, ion exchangers, calcium ATPases, and calcium/calmodulin-dependent protein kinases in explanted hearts (n=13) of patients diagnosed with end-stage heart failure. With the exception of Kv11.1 and Kir3.1 and when compared with healthy controls, major sodium, potassium, and calcium ion channels, ion transporters, and exchangers were significantly repressed, but expression of Kv7.1, HCN4, troponin C and I, SERCA1, and phospholamban was elevated. Hierarchical gene cluster analysis provided novel insight into regulated gene networks. Significant induction of the transcriptional repressor m-Bop and the translational repressor NAT1 coincided with repressed cardiac gene expression. The statistically significant negative correlation between repressors and ion channels points to a mechanism of disease. We observed coregulation of ion channels and the androgen receptor and propose a role for this receptor in ion channel regulation. Overall, the reversal of repressed ion channel gene expression in patients with implanted assist devices exemplifies the complex interactions between pressure load/stretch force and heart-specific gene expression.

Topics: Action Potentials; Adenosine Triphosphatases; Adrenergic beta-Antagonists; Antiporters; Atrial Natriuretic Factor; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Cardiac Output, Low; Cardiomegaly; Digitalis Glycosides; Electric Conductivity; Gene Expression Regulation; Heart-Assist Devices; Humans; Ion Channels; Myocytes, Cardiac; Natriuretic Peptide, Brain; Promoter Regions, Genetic; Repressor Proteins

The aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, is the receptor for the polycyclic aromatic hydrocarbons found in tobacco smoke, polychlorinated biphenyls, and the environmental pollutant, dioxin. To better understand the role of the AhR in the heart, echocardiography, invasive measurements of aortic and left ventricular pressures, isolated working heart preparations, as well as morphological and molecular analysis were used to investigate the impact of AhR inactivation on the mouse heart using the AhR knockout as a model. Cardiac hypertrophy is an early phenotypic manifestation of the AhR knockout. Although the knockout animals were not hypertensive at the ages examined, cardiomyopathy accompanied by diminished cardiac output developed. Despite the structural left ventricular remodeling, the hearts of these animals exhibit minimal fibrosis and do not have the expected increases in surrogate molecular markers of cardiac hypertrophy. The anatomic remodeling without typical features of molecular remodeling is not consistent with hypertrophic growth secondary to pressure or volume overload, suggesting that increased cardiomyocyte size may be a direct consequence of the absence of the AhR in this cell type.

Topics: Actins; Animals; Aorta; Atrial Natriuretic Factor; Biomarkers; Blood Pressure; Cardiomegaly; Cardiovascular Physiological Phenomena; Echocardiography; Hypertrophy; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Myocytes, Cardiac; Organ Size; Receptors, Aryl Hydrocarbon; RNA, Messenger; Ventricular Function, Left; Ventricular Remodeling

Cardiac hypertrophy is an early landmark during the clinical course of heart failure, and is an important risk factor for subsequent morbidity and mortality. The hypertrophy response to different types of cardiac overload is distinguished both at the molecular and cellular levels. These changes have been extensively characterized for pressure load hypertrophy; however, similar information for volume load hypertrophy is still needed. This study was undertaken to improve the existing method of producing experimental cardiac volume load. Previous investigators have employed surgical aorto-caval shunt (ACS) as a model for volume load hypertrophy (VO) in rats. The procedure is relatively simple and involves glue to seal the aortic hole after ACS. However, it has several limitations mostly related to the use of glue e.g. poor visualization due to hardening of tissues, imperfect sealing of the puncture site and glue seeping through the aortic hole resulting in shunt occlusion. We have modified the procedure using aortic adventitial suture instead of glue and 18G angiocatheter instead of 16G needle, which eliminated the technical difficulties from the former method. The ACS was visually confirmed at sacrifice, and the VO demonstrated by time-related changes in the heart weight/body weight ratio which increased from 78% at 4 weeks to 87% at 10 weeks and increased liver/body weight ratio by 22% at 10 weeks of post aorto-caval shunt. Cardiac expression of atrial natriuretic peptide (ANF) also demonstrated time-related increase in ANF mRNA (+275% increase at 4 weeks, p < 0.05, and +370% increase at 10 weeks, p < 0.001). This modified technique of aorto-caval shunt offers simpler, reproducible and consistent model for VO hypertrophy in rats.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Body Weight; Cardiac Volume; Cardiomegaly; Catheters, Indwelling; Gene Expression; Male; Models, Cardiovascular; Organ Size; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sutures; Time Factors; Vascular Surgical Procedures; Venae Cavae

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg x h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg x h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Degranulation; Female; Fibrosis; Hemodynamics; Interleukin-6; Mast Cells; Norepinephrine; Rats; Rats, Sprague-Dawley; RNA, Messenger

Previous studies have demonstrated that integrins link the extracellular matrix to the hypertrophic response pathway of cardiac myocytes in vitro. To examine the direct relation between integrin beta1 and cardiac hypertrophy in vivo, we studied the effects of a newly developed angiotensin II type 1 (AT1) blocker, CS866 (ARB; 10 mg/kg/day), an angiotensin-converting enzyme inhibitor, temocapril (ACEI, 10 mg/kg/day), or both on modulation of integrin beta1 in the hypertrophied hearts of stroke-prone spontaneously hypertensive rats (SHRSP) 6 to 12 weeks of age. Treatments with ARB, ACEI, and combination therapy significantly reduced systolic blood pressure. However, the reduction in cardiac hypertrophy was greater in SHRSP treated with ARB or combination therapy than in those treated with ACEI. Multiplex reverse transcription-polymerase chain reaction revealed significantly higher mRNA expression of atrial natriuretic factor, AT1 receptor, and integrin beta1 in untreated SHRSP than in normotensive Wistar-Kyoto rats (WKY). The mRNA levels of ANP, AT1 receptor, and integrin B1 in SHRSP were significantly decreased by treatment with ARB, ACEI, or combination therapy. Decreased mRNA expression of ANP, AT1 receptor, and integrin beta1 in the treated SHRSP was associated with reductions in blood pressure; ARB and combination therapy produced greater decreases in expression than did ACEI. These observations suggest that CS866 has a beneficial effect on myocyte hypertrophy and that down-regulation of AT1 receptor and suppression of integrin beta1 participate in the regression of pressure-induced cardiac hypertrophy in vivo. The correlation between the expression of integrin beta1 and AT1 receptor was significant. Our results also suggest that integrin expression by myocytes might be modulated by angiotensin II via AT1 receptor.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Down-Regulation; Drug Therapy, Combination; Imidazoles; Integrin beta1; Male; Myocytes, Cardiac; Olmesartan Medoxomil; Polymerase Chain Reaction; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; RNA, Messenger; Tetrazoles; Thiazepines

1. The natriuretic peptide precursor A (Nppa) and B (Nppb) genes are candidate genes for hypertension and cardiac hypertrophy in the spontaneously hypertensive rat (SHR). The purpose of the present study was to determine the role of the Nppa and Nppb genes in the development of hypertension in the SHR. 2. A cohort (n = 162) of F2 segregating intercross animals was established between strains of hypertensive SHR and normotensive Wistar-Kyoto rats. Blood pressure and heart weight were measured in each rat at 12-16 weeks of age. Rats were genotyped using 11 informative microsatellite markers, distributed in the vicinity of the Nppa marker on rat chromosome 5 including an Nppb marker. The phenotype values were compared with genotype using the computer package mapmaker 3.0 (Whitehead Institute, Boston, MA, USA) to determine whether there was a link between the genetic variants of the natriuretic peptide family and blood pressure or cardiac hypertrophy. 3. A strong correlation was observed between the Nppa marker and blood pressure. A quantitative trait locus (QTL) for blood pressure on chromosome 5 was identified between the Nppa locus and the D5Mgh15 marker, less than 2 cM from the Nppa locus. The linkage score for the blood pressure QTL on chromosome 5 was 3.8 and the QTL accounted for 43% of the total variance of systolic blood pressure, 54% of diastolic blood pressure and 59% of mean blood pressure. No association was found between the Nppb gene and blood pressure. This is the first report of linkage between the Nppa marker and blood pressure in the rat. There was no correlation between the Nppa or Nppb genes or other markers in this region and either heart weight or left ventricular weight in F2 rats. 4. These findings suggest the existence of a blood pressure-dependent Nppa marker variant or a gene close to Nppa predisposing to spontaneous hypertension in the rat. It provides a strong foundation for further detailed genetic studies in congenic strains, which may help to narrow down the location of this gene and lead to positional cloning.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Chromosome Segregation; Chromosomes, Mammalian; Crosses, Genetic; Female; Gene Expression; Genotype; Male; Organ Size; Phenotype; Polymorphism, Genetic; Quantitative Trait Loci; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that regulate blood pressure and volume, and exert their biological actions via the natriuretic peptide receptor-A gene (Npr1). Mice lacking Npr1 (Npr(-/-)) have marked cardiac hypertrophy and fibrosis disproportionate to their increased blood pressure. This study examined the relationships between ANP and BNP gene expression, immunoreactivity and fibrosis in cardiac tissue, circulating ANP levels, and ANP and BNP mRNA during embryogenesis in Npr1(-/-) mice. Disruption of the Npr1 signaling pathway resulted in augmented ANP and BNP gene and ANP protein expression in the cardiac ventricles, most pronounced for ANP mRNA in females [414 +/- 57 in Npr1(-/-) ng/mg and 124 +/- 25 ng/mg in wild-type (WT) by Taqman assay, P < 0.001]. This increased expression was highly correlated to the degree of cardiac hypertrophy and was localized to the left ventricle (LV) inner free wall and to areas of ventricular fibrosis. In contrast, plasma ANP was significantly greater than WT in male but not female Npr1(-/-) mice. Increased ANP and BNP gene expression was observed in Npr1(-/-) embryos from 16 days of gestation. Our study suggests that cardiac ventricular expression of ANP and BNP is more closely associated with local hypertrophy and fibrosis than either systemic blood pressure or circulating ANP levels.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathies; Embryo, Mammalian; Female; Fibrosis; Guanylate Cyclase; Heart Ventricles; Hypertension; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Natriuretic Peptide, Brain; Receptors, Atrial Natriuretic Factor; Reference Values; RNA, Messenger

Differentiated cardiomyocytes have little capacity to proliferate and show the hypertrophic growth in response to alpha1-adrenergic stimuli via the Ras/MEK pathway. In this study, we investigated a role of cyclin D1 and CDK4, a positive regulator of cell cycle, in cultured neonatal rat cardiomyocyte hypertrophy. D-type cyclins including cyclin D1 were induced in cells stimulated by phenylephrine. This induction was inhibited by MEK inhibitor PD98059 and the dominant negative RasN17, but mimicked by expression of the constitutive active Ras61L. Over-expression of cyclin D1 and CDK4 using adenovirus gene transfer caused the hypertrophic growth of cardiomyocytes, as evidenced by an increase of the cell size as well as the amount of cellular protein and its rate of synthesis. However, the cyclin D1/CDK4 kinase activity was not up-regulated in cells treated by hypertrophic stimuli or in cells over-expressing the cyclin D1 and CDK4. Furthermore, a CDK inhibitor, p16, did not inhibit the hypertrophic growth of cardiomyocytes. These results clearly indicated that cyclin D1 and CDK4 have a role in hypertrophic growth of cardiomyocytes through a novel mechanism(s) which appears not to be related to its activity required for cell cycle progression.

Topics: Actins; Adenoviridae; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Culture Media, Serum-Free; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Heart; Myocardium; Phenylephrine; Proto-Oncogene Proteins; ras Proteins; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins

Guanylyl cyclase (GC)-A, a natriuretic peptide receptor, lowers blood pressure and inhibits the growth of cardiac myocytes and fibroblasts. Angiotensin II (Ang II) type 1A (AT1A), an Ang II receptor, regulates cardiovascular homeostasis oppositely. Disruption of GC-A induces cardiac hypertrophy and fibrosis, suggesting that GC-A protects the heart from abnormal remodeling. We investigated whether GC-A interacts with AT1A signaling in the heart by target deletion and pharmacological blockade or stimulation of AT1A in mice.. We generated double-knockout (KO) mice for GC-A and AT1A by crossing GC-A-KO mice and AT1A-KO mice and blocked AT1 with a selective antagonist, CS-866. The cardiac hypertrophy and fibrosis of GC-A-KO mice were greatly improved by deletion or pharmacological blockade of AT1A. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, transforming growth factors beta1 and beta3, were also strongly inhibited. Furthermore, stimulation of AT1A by exogenous Ang II at a subpressor dose significantly exacerbated cardiac hypertrophy and dramatically augmented interstitial fibrosis in GC-A-KO mice but not in wild-type animals.. These results suggest that cardiac hypertrophy and fibrosis of GC-A-deficient mice are partially ascribed to an augmented cardiac AT1A signaling and that GC-A inhibits AT1A signaling-mediated excessive remodeling.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensinogen; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Collagen; Fibrosis; Gene Targeting; Guanylate Cyclase; Heart Rate; Heart Ventricles; Hypertension; Imidazoles; Mice; Mice, Knockout; Myocardium; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Organ Size; Peptidyl-Dipeptidase A; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Tetrazoles; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Ventricular Remodeling

Left ventricular (LV) hypertrophy increases susceptibility to reperfusion arrhythmias and the angiotensin-converting enzyme inhibitor, ramipril, may reduce that susceptibility via regression of LV hypertrophy. Rats (n=12 per group) were subjected to abdominal aortic constriction (AC) or sham-operation (SH) and from 3 to 6 weeks after surgery, 3 AC groups received ramipril (0.01, 0.1, or 1 mg/kg per day p.o.) while the SH and 1 AC group received vehicle. Six weeks after surgery (ie after 3 weeks of treatment), the hearts were excised and subjected to independent Langendorf perfusion of left and right coronary beds. The left coronary bed was then subjected to ischemia (7 min) and reperfusion (5 min). Hypertrophied hearts from the vehicle AC group showed a significant increase in the incidence of reperfusion-induced ventricular fibrillation (VF) compared with control hearts from the SH group (92%* vs 33%: *p<0.05); this difference was abolished by ramipril (42%, 50%, and 42%, at 0.01, 0.1, or 1 mg/kg per day, respectively). The LV weight/body weight ratio was significantly increased in all AC groups (regardless of ramipril treatment) relative to the SH group. At the cellular level, myocyte length was significantly increased in the vehicle AC group, but was normalized by ramipril treatment (1 mg/kg per day). At the molecular level, atrial natriuretic factor (ANF) mRNA expression was also significantly increased in the vehicle AC group, but was again normalized by ramipril treatment (1 mg/kg per day). In conclusion, short-term treatment with ramipril reduced susceptibility to severe ventricular arrhythmias in hypertrophied rat hearts. This protection was achieved in the absence of a significant reduction in LV weight, but was accompanied by regression of myocyte hypertrophy, as reflected by reductions in cell size and ANF expression.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Heart Ventricles; Male; Myocardial Reperfusion Injury; Myocytes, Cardiac; Ramipril; Rats; Rats, Wistar; RNA, Messenger; Ventricular Fibrillation

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC alpha, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKCalpha oligonucleotides resulted in a marked reduction in both PKCalpha mRNA and protein levels. PKCalpha antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKCalpha. Antisense inhibition of PKCalpha led to reduction of PE-induced increase in skeletal alpha-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced beta-myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKCalpha treatment attenuated endothelin-1-induced increase in ANP and BNP peptide secretion, whereas endothelin-1-induced gene expression of ANP and BNP remained unchanged. The hypertrophic agonist-induced growth of cardiomyocytes, characterized by increased [(3)H]leucine incorporation, was not affected with antisense PKCalpha treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKCalpha treatment, implicating ERK as a downstream mediator for PKCalpha signaling. These results indicate that PKCalpha isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKCalpha or other PKC isozymes.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression; Heart; Isoenzymes; Myocardium; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Rats; Rats, Sprague-Dawley

Large myocardial infarction (MI) causes substantial cardiac remodeling and often leads to heart failure. The genetically engineered mouse is believed to provide a powerful tool for investigating the underlying pathophysiological mechanisms and for developing new therapeutic strategies. The present study investigates the functional parameters and expression levels of transforming growth factor (TGF) beta isoforms, interleukin-6 (IL-6) and tumor necrosis factor (TNF) alpha, which may be involved in the remodeling mechanisms, in a mouse model of MI; comparisons with data from rats were also made. Female Sprague-Dawley rats ( n=10-12 at each time point) and female Balb/c mice ( n=6-8 at each time point) were used. In both mice and rats MI induced a time-dependent reduction in heart function with subsequent development of heart failure. The hemodynamic consequences after 4 weeks are characterized by reduced left ventricular (LV) developed pressure and increased right ventricular (RV) developed pressure. The pattern of increased expression of most, but not all, of the analyzed cytokines and growth factors is comparable. This emphasizes the important role of these factors in the remodeling processes. However, TNFalpha was more strongly expressed in both the infarct and the non-infarcted area of mice. Since functional and molecular biological parameters can readily be measured in mice with advanced technologies, this qualifies this species as a powerful experimental model, particularly in view of the various transgenic and knock-out mice that are available.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Body Weight; Cardiomegaly; Cytokines; Female; Heart; Hemodynamics; Mice; Mice, Inbred BALB C; Myocardial Infarction; Protein Isoforms; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Ventricular Function, Left

Calcium-dependent mechanisms and the renin angiotensin system (RAS) are critically involved in the hypertrophic growth of the myocardium. The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator in calcium signaling and modulates calcium handling and growth mechanisms in cardiomyocytes. Here we present data on expression of cardiac isoforms of CaMKIIdelta, the dominant form in the myocardium, in compensatory hypertrophy of stroke-prone spontaneously hypertensive rats (SHRSP) compared to the normotensive Wistar-Kyoto (WKY) control strain. Cardiac hypertrophy in SHRSP was documented by an increased heart weight/body weight ratio (HW/BW) of 31% (p < 0.05) and a more than six-fold elevated atrial natriuretic factor (ANF) transcript level (p < 0.05). Compensatory hypertrophic growth in SHRSP produced a specific phenotype of CaMKIIdelta isoforms characterized by increased transcript levels of the embryonic/neonatal isoform delta4 (48%, p < 0.05) and the isoform delta9 (31%, p < 0.05) with no changes in delta2 and delta3. Inhibition of angiotensin converting enzyme (ACE) by cilazapril completely regressed myocardial hypertrophy, normalized ANF transcript levels, and restored the normal phenotype of CaMKIIdelta by reducing transcripts for delta4 and delta9 to levels present in WKY controls. Our data suggest the importance of specific changes in the CaMKII isoform composition for growth processes in the myocardium.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Body Weight; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cilazapril; Genetic Predisposition to Disease; In Vitro Techniques; Isoenzymes; Male; Myocardium; Organ Size; Phenotype; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reverse Transcriptase Polymerase Chain Reaction; Stroke; Transcription, Genetic

Integrins are heterodimeric cell-surface receptors that link the extracellular matrix and the intracellular cytoskeleton and function as mechanotransducers. Signaling through integrins is important for cell growth, migration, and survival. Extracellular matrix is altered in the myocardium during hypertrophic induction and the transition to heart failure. The role of integrins in this process is poorly understood. Recently, integrin subunits have been identified that are dominantly expressed in striated muscle. We tested the hypothesis that since integrins are mechanotransducers, their expression and signaling would be modulated with murine left ventricular hemodynamic loading. The acute and chronic effects of pressure overload on changes in the expression of integrins, as well as related integrin-mediated signaling events were studied. Acute pressure loading increased phosphorylation of focal adhesion kinase, p42 and p44 extracellular signal-regulated kinase. Chronic loading: (1) increased expression of alpha1, alpha5, and beta1 integrin transcripts and (2) increased protein expression of integrin subunits which are dominantly expressed in striated muscle (alpha7 and beta1D) both by western blotting and immunofluorescent microscopy. These results show that adaptive responses of the myocardium to pressure overload include acute modulation of integrin-related signaling molecules and more chronic changes effect expression of integrin subunits, including ones dominantly expressed in muscle.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blotting, Northern; Blotting, Western; Cardiomegaly; Constriction, Pathologic; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression; Heart Ventricles; In Vitro Techniques; Integrins; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Phosphorylation; Pressure; Protein-Tyrosine Kinases; Signal Transduction

The cardiac-specific sodium-calcium exchanger (NCX1) is a GATA-4 dependent gene that is upregulated during cardiac hypertrophy and heart failure. To date, lack of an appropriate inhibitor of NCX1 and embryonic lethality of NCX1 knockout mice have slowed investigation of the relation between NCX1 upregulation and cardiac hypertrophy. Recently, in vitro studies have shown that cyclosporin A (CSA), a calcineurin inhibitor, significantly downregulated expression of the hypertrophic genes atrial natriuretic factor and beta-myosin heavy chain and protected against cardiac hypertrophy and heart failure in calcineurin overexpressing mice. This suggested that CSA might play an important role in the treatment of hypertrophy and heart failure. In an in vitro model of cardiac hypertrophy, we showed that CSA is a potent inhibitor of NCX1 basal expression and NCX1 promoter activity. Female homozygous transgenic mice that overexpress NCX1 develop heart failure and die prematurely after two or more pregnancies. Others have demonstrated that pressure overloaded wild-type mice treated with CSA do not develop cardiac hypertrophy and downregulate expression of NCX1. We investigated the effect of CSA on NCX1 expression and transverse aortic constriction-induced cardiac hypertrophy in NCX1 overexpressing mice. We found that CSA blunted these responses.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin Inhibitors; Cardiomegaly; Cyclosporine; Gene Expression Regulation; Humans; In Vitro Techniques; Mice; Mice, Transgenic; Myosin Heavy Chains; Promoter Regions, Genetic; Rats; Recombinant Proteins; Sequence Deletion; Sodium-Calcium Exchanger

The aryl hydrocarbon receptor (AhR) is a member of the basic helix loop helix PAS (Per-ARNT-SIM) transcription family, which also includes hypoxiainducible factor-1alpha (HIF-1alpha) and its common dimerization partner AhR nuclear translocator (ARNT). Following ligand activation or hypoxia, AhR or HIF-1alpha, respectively, translocate into the nucleus, dimerize with ARNT, and regulate gene expression. Mice lacking the AhR have been shown previously to develop cardiac enlargement. In cardiac hypertrophy, it has been suggested that the myocardium becomes hypoxic, increasing HIF-1alpha stabilization and inducing coronary neovascularization, however, this mechanism has not been demonstrated in vivo. The purpose of this study was to investigate the cardiac enlargement reported in AhR(-/-) mice and to determine if it was associated with myocardial hypoxia and subsequent activation of the HIF-1alpha pathway. We found that AhR(-/-) mice develop significant cardiac hypertrophy at 5 mo. However, this cardiac hypertrophy was not associated with myocardial hypoxia. Despite this finding, cardiac hypertrophy in AhR(-/-) mice was associated with increased cardiac HIF-1alpha protein expression and increased mRNA expression of the neovascularization factor vascular endothelial growth factor (VEGF). These data demonstrate that the development of cardiac hypertrophy in AhR(-/-) mice not associated with myocardial hypoxia, but is correlated with increased cardiac HIF-1alpha protein and VEGF mRNA expression.

Topics: Age Factors; Animals; Atrial Natriuretic Factor; Biomarkers; Body Weight; Cardiac Myosins; Cardiomegaly; Disease Models, Animal; Endothelin-1; Gene Expression Regulation; Heart Ventricles; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Mutant Strains; Models, Cardiovascular; Myocardium; Myosin Heavy Chains; Myosin Light Chains; Nonmuscle Myosin Type IIB; Organ Size; Phenotype; Rats; Receptors, Aryl Hydrocarbon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics as Topic; Time Factors; Transcription Factors; Vascular Endothelial Growth Factor A

Targeted disruption of the gene for natriuretic peptide receptor-A (NPR-A) worsens pulmonary hypertension and right ventricular hypertrophy during hypoxia, but its effect on left ventricular mass and systemic pressures is not known. We examined the effect of 3 wk of hypobaric hypoxia (0.5 atm) on right and left ventricular pressure and mass in mice with 2 (wild type), 1, or 0 copies of Npr1, the gene that encodes for NPR-A in mice. Under normoxic conditions, right ventricular peak pressure (RVPP) was greater in 0 than in 2 copy mice, but there were no genotype-related differences in carotid artery PP (CAPP). The left ventricular free wall weight-to-body weight (LV/body wt) ratio was greater in 0 than in 2 copy mice and there was a trend toward a greater right ventricular weight-to-body weight (RV/body wt) ratio. Three weeks of hypoxia increased RVPP and RV/body wt in all genotypes. The increase in RVPP was similar in all genotypes (11-14 mmHg), but the hypoxia-induced increase in RV/body wt was more than twice as great in 0 copy mice than in 2 copy mice (1.11 +/- 0.06 to 2.65 +/- 0.46 vs. 0.96 +/- 0.04 to 1.4 +/- 0.09, P < 0.05). Chronic hypoxia had no effect on CAPP in any genotype and did not effect LV/body wt in 1 or 2 copy mice, but increased LV/body wt 41% in 0 copy mice. We conclude that absent expression of NPR-A worsens right ventricular hypertrophy and causes left ventricular hypertrophy during exposure to chronic hypoxia without increasing pulmonary or systemic arterial pressure responses.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Carotid Arteries; Disease Progression; Guanylate Cyclase; Hydroxyproline; Hypoxia; Lung; Mice; Mice, Knockout; Pulmonary Circulation; Receptors, Atrial Natriuretic Factor; Systole; Ventricular Function, Left; Ventricular Function, Right

The aims of the present study were to determine the effects of endothelin ET(A) receptor antagonism on carbon monoxide (CO)-induced cardiac hypertrophy and endothelin-1 (ET-1) expression and to compare myocardial effects of chronic nicotine with CO exposure. Female Sprague-Dawley rats (n = 84) were randomized to three groups exposed 20 h/day to CO (200 ppm), nicotine (500 microg/m3), or air for 14 consecutive days. In each exposure group, animals were randomized to ET(A) receptor antagonist LU 135252 in drinking water (0.5 mg/ml) or placebo. Myocardial ET-1 and atrial natriuretic peptide (ANP) expression was measured by competitive RT-PCR and plasma ET-1 by immunoassay. Carboxyhemoglobin was 22.1 +/- 0.3% in CO-exposed animals and 2.8 +/- 0.3% in controls. Plasma nicotine was 57 +/- 7 ng/ml and plasma cotinine was 590 +/- 23 ng/ml in nicotine-exposed animals and below detection levels in controls. CO exposure induced a 21% increase in right ventricular hypertrophy (p < 0.01), a 7% increase in left ventricular hypertrophy (p < 0.01), a 25% increase in right ventricular ET-1 expression (p < 0.05), and an eightfold increase in ANP expression (p = 0.08). ET(A) receptor antagonism reduced right ventricular hypertrophy by 60% (p < 0.05) with no significant effect on left ventricular hypertrophy or myocardial ET-1 expression. Chronic nicotine exposure did not significantly affect cardiac weights or ANP and ET-1 expression. We conclude that ET(A) receptor antagonism reduces right ventricular hypertrophy induced by chronic CO exposure, whereas CO-induced myocardial ET-1 expression remains unchanged.

Topics: Animals; Atmosphere Exposure Chambers; Atrial Natriuretic Factor; Body Weight; Carbon Monoxide; Cardiomegaly; Endothelin Receptor Antagonists; Endothelin-1; Female; Nicotine; Nicotinic Agonists; Organ Size; Phenylpropionates; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; RNA, Messenger

The activator protein 1 (AP-1) transcriptional complex, containing Jun and Fos proteins, is involved in regulating many cellular processes such as proliferation and differentiation. However, little is known about a direct relationship between AP-1 activities and cardiomyocyte hypertrophy. To elucidate the roles of myocardial AP-1 activities, dominant negative mutant of c-Jun (DNJun) was overexpressed in cultured rat neonatal ventricular myocytes by adenovirus vector to abrogate endogenous AP-1 activation. Cardiomyocytes were treated with 100 nmol/L endothelin 1 (ET) and 10 micromol/L phenylephrine (PE) to induce myocardial cell hypertrophy. Both ET and PE significantly enhanced AP-1 DNA binding activities (3.4-fold by ET and 4.8-fold by PE at 3 hours, P<0.01). At 48 hours after stimulation, ET and PE significantly increased incorporation of (3)H-phenylalanine (1.4-fold by ET and 1.5-fold by PE, P<0.01), cell size (2.3-fold and 2.5-fold, P<0.01), and mRNA expression of atrial natriuretic peptide (ANP; 1.9-fold and 1.8-fold, P<0.01) and brain natriuretic peptide (BNP; 1.6-fold and 1.6-fold, P<0.01). Adenovirus carrying DNJun prevented the transcriptional activation of the AP-1 by ET and PE, using AP-1 reporter enzyme firefly luciferase assay. Moreover, DNJun prevented the increase in incorporation of (3)H-phenylalanine, cell size, and the mRNA expression of ANP and BNP by ET and PE. In conclusion, we provide the first evidence that DNJun inhibits cardiomyocyte hypertrophy through inhibition of AP-1 transcriptional activity.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; DNA, Complementary; Endothelin-1; Genes, Dominant; Genes, jun; Heart Ventricles; Lac Operon; Mutation; Myocardium; Natriuretic Peptide, Brain; Phenylephrine; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Transcription Factor AP-1; Transcriptional Activation; Transfection

Angiotensin II induces both cardiac and vascular smooth muscle (VSM) hypertrophy. Recent studies suggest a central role for a phagocyte-type NADPH oxidase in angiotensin II-induced VSM hypertrophy. The possible involvement of an NADPH oxidase in the development of cardiac hypertrophy has not been studied. Methods and Results- Mice with targeted disruption of the NADPH oxidase subunit gp91(phox) (gp91(phox-/-)) and matched wild-type mice were subjected to subcutaneous angiotensin II infusion at a subpressor dose (0.3 mg/kg/day) for 2 weeks. Systolic blood pressure was unaltered by angiotensin II in either group. Angiotensin II significantly increased heart/body weight ratio, atrial natriuretic factor and beta-myosin heavy chain mRNA expression, myocyte area, and cardiac collagen content in wild-type but not gp91(phox-/-) mice. Angiotensin II treatment increased myocardial NADPH oxidase activity in wild-type but not gp91(phox-/-) mice.. A gp91(phox)-containing NADPH oxidase plays an important role in the development of angiotensin II-induced cardiac hypertrophy, independent of changes in blood pressure.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Collagen; Membrane Glycoproteins; Mice; Mice, Knockout; Myocardium; Myosin Heavy Chains; NADPH Oxidase 2; NADPH Oxidases; Organ Size; RNA, Messenger; Superoxides

Angiotensin II (Ang II) may cause cardiac hypertrophy via type 1 Ang II receptors (AT(1)) on cardiomyocytes and through growth factors released from cardiac fibroblasts. Whereas cardiomyocyte-specific AT(1) receptor expression produces cardiac hypertrophy and remodeling in vivo, delineation of the signals that mediate growth to Ang II is challenging because the prevailing in vitro model (cultured neonatal cardiomyocytes) expresses low levels of AT(1) receptor and responds inconsistently to Ang II. In this study, when AT(1A) receptors were expressed using adenovirus in cultured neonatal cardiomyocytes, Ang II stimulated a robust hypertrophy that was not secondary to the release of cardiac fibroblast-derived factors, specifically endothelin-1. Hypertrophy was accompanied by the induction of the immediate-early response genes, c-fos and c-jun, and reexpression of atrial natriuretic peptide (ANP). Ang II-induced activation of an ANP promoter-reporter was inhibited by the dominant/negative mutants, GalphaqI and N17Ras, indicating that hypertrophic signaling by the AT(1A) receptor is via heterotrimeric G protein coupling and downstream Ras pathways. AT(1A)-mediated cardiomyocyte hypertrophy and mitogen-activated protein kinase (MAPK) activation were inhibited by the MAPK kinase inhibitor, PD98059, and the epidermal growth factor (EGF) receptor kinase antagonist, AG1478, but not by PKC inhibitor, bisindolylmaleimide-1. Moreover, Ang II-induced MAPK activation was prevented by treatment with a matrix metalloproteinase inhibitor, consistent with the tyrosine phosphorylation of the EGF receptor in response to AT(1A) receptor activation. These data unequivocally demonstrate that Ang II can directly promote cardiac myocyte growth via AT(1A) receptors expressed on these cells and reveal for the first time the important contribution of EGF receptor-transactivated MAPK signaling to this process.

Topics: Adenoviridae; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Enzyme Inhibitors; ErbB Receptors; Gene Expression Regulation; Genetic Vectors; MAP Kinase Signaling System; Myocardium; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Signal Transduction; Transcriptional Activation; Transfection

It remains unclear how mineralocorticoids induce cardiac hypertrophy and fibrosis. Recently, activation of the calcium-dependent phosphatase, calcineurin, has been shown to induce cardiac hypertrophy. In the present study, we examine the role of calcineurin in mineralocorticoid-induced cardiac hypertrophy and fibrosis.. Uninephrectomized Wistar-Kyoto rats were placed on a 1.0% NaCl diet and treated with aldosterone (0.75 microg x h(-1)) for 6 weeks with or without the calcineurin inhibitors, FK506 (0.5 mg x kg(-1) x d(-1)) or cyclosporine A (10 mg x kg(-1) x d(-1)). The effect of the angiotensin II type 1 receptor antagonist, losartan (10 mg x kg(-1) x d(-1))on aldosterone-induced cardiac hypertrophy was also studied. Treatment with aldosterone increased the heart weight/body weight ratio, cardiomyocyte size, and collagen amount. The expression of mRNA of both type-III collagen and atrial natriuretic peptide in the heart were increased by aldosterone administration. Both calcineurin activity and its mRNA expression were also increased in aldosterone-induced hypertrophic heart. Treatment with losartan, FK506, or cyclosporine partially prevented aldosterone-induced cardiac hypertrophy and fibrosis.. These results suggest that calcineurin is involved in the development of cardiac hypertrophy and fibrosis induced by mineralocorticoid excess. Inhibition of calcineurin may therefore prevent cardiac hypertrophy and fibrosis in mineralocorticoid hypertension.

Topics: Aldosterone; Animals; Anti-Arrhythmia Agents; Antihypertensive Agents; Atrial Natriuretic Factor; Body Weight; Calcineurin; Calcineurin Inhibitors; Cardiomegaly; Collagen Type III; Cyclosporine; Enzyme Inhibitors; Fibrosis; Heart; Immunosuppressive Agents; Losartan; Male; Mineralocorticoids; Myocardium; Nephrectomy; Organ Size; Rats; Rats, Inbred WKY; RNA, Messenger; Sodium Chloride, Dietary; Tacrolimus

Recent studies suggest that female gender is associated with a lower prevalence and a more benign prognosis of heart failure. In the current population-based study, it was our objective to evaluate the implications of gender on the association between impaired left ventricular (LV) function and mass as well as neurohumoral activation.. A total of 1883 subjects (992 female, 891 male) of two MONICA surveys in Augsburg, Germany, were analyzed. Participants of one of these surveys were additionally characterized with respect to neurohormonal activation. As compared to men, women were characterized by a slightly higher LV ejection fraction (EF, Teichholz-Method, 65.4 +/- 0.3% vs. 63.4 +/- 0.3, P<0.01) and a markedly lower LV mass index (LVMI 81 +/- 1 g/m(2) vs. 96 +/- 1, P<0.01). As compared to men with normal LV function, those with LV dysfunction (EF below mean minus two standard deviations, S.D.) were characterized by significantly increased LV mass (LVMI +48%, P<0.01), plasma BNP (+373%, P<0.01) and ANP (+57%, P<0.01), while no significant changes were observed in women (LVMI +3%, BNP +48%, ANP +27%, all P=n.s). Only a small subgroup of women with severe LVD (EF below mean - 3 S.D.) was characterized by significantly increased LV mass (LVMI +23%, P<0.05 vs. control and LVD), however, this increase was less pronounced as compared to men with severe LVD (LVMI +46%, P<0.01 vs. control). Gender-specific differences between LV function and structure were also confirmed by multivariate analysis. While LVMI was independently and significantly correlated with EF in male subjects in addition to systolic blood pressure, age, and body mass index (all P<0.01), these parameters displaced EF as a predictor of LVMI in female subjects.. Men with moderate or severe LV dysfunction are characterized by an increase in both LV mass and cardiac natriuretic peptide plasma concentrations. In contrast, LV mass and natriuretic peptide concentrations increase to a lesser extent and only with severe LV dysfunction in women. These observational data suggest gender-specific control of myocardial adaptations to hemodynamic overload and a more rapid induction of LV hypertrophy during myocardial dysfunction in male subjects.

Topics: Atrial Natriuretic Factor; Cardiomegaly; Cohort Studies; Cyclic GMP; Echocardiography; Female; Humans; Male; Regression Analysis; Renin; Sex; Ventricular Dysfunction, Left; Ventricular Remodeling

GATA-4 transcription factor is required for normal cardiac development. However, it is unknown whether GATA-4 is an essential mediator of hypertrophic responses in the heart. Rat B-type natriuretic peptide (BNP) gene promoter contains a region of two adjacent GATA binding sites (between -68 and -97) with high affinity for GATA-4. In order to block GATA-4 dependent signaling in cultured neonatal rat ventricular myocytes we administered a synthetic 30-bp phosphorothioated double-stranded DNA complementary to the rat BNP promoter region (between -68 and -97) as a "decoy" cis-element to bind GATA-4. GATA decoy oligodeoxynucleotide treatment of cardiomyocytes blocked GATA-4 DNA binding activity in electrophoretic mobility shift analysis and decreased baseline expression of cardiac natriuretic peptides and GATA-dependent promoter activity. In contrast, blocked GATA-4 DNA binding did not prevent endothelin-1 or phenylephrine induced expression of cardiac natriuretic peptides. Mutation of GATA binding sites at -80 and -91 rat BNP promoter downregulated baseline but did not affect endothelin-1 or angiotensin II induced promoter activity. Additively, GATA decoy oligodeoxynucleotide treatment was insufficient to block endothelin-1 induced activation of protein synthesis or sarcomeric protein assembly. In conclusion, a targeted disruption of GATA-4 DNA binding activity is insufficient to prevent hypertrophic agonist induced responses of ventricular myocytes.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Binding Sites; Cardiomegaly; DNA-Binding Proteins; Endothelin-1; GATA4 Transcription Factor; Gene Expression Regulation; Myocardium; Natriuretic Peptide, Brain; Oligodeoxyribonucleotides; Phenylephrine; Promoter Regions, Genetic; Protein Binding; Rats; Transcription Factors

Peroxisome proliferator-activated receptors (PPARs) are transcription factors of the nuclear receptor superfamily. It has been reported that the thiazolidinediones, which are antidiabetic agents and high-affinity ligands for PPARgamma, regulate growth of vascular cells. In the present study, we examined the role of PPARgamma in angiotensin II (Ang II)-induced hypertrophy of neonatal rat cardiac myocytes and in pressure overload-induced cardiac hypertrophy of mice.. Treatment of cultured cardiac myocytes with PPARgamma ligands such as troglitazone, pioglitazone, and rosiglitazone inhibited Ang II-induced upregulation of skeletal alpha-actin and atrial natriuretic peptide genes and an increase in cell surface area. Treatment of mice with a PPARgamma ligand, pioglitazone, inhibited pressure overload-induced increases in the heart weight-to-body weight ratio, wall thickness, and myocyte diameter in wild-type mice and an increase in the heart weight-to-body weight ratio in heterozygous PPARgamma-deficient mice. In contrast, pressure overload-induced increases in the heart weight-to-body weight ratio and wall thickness were more prominent in heterozygous PPARgamma-deficient mice than in wild-type mice.. These results suggest that the PPARgamma-dependent pathway is critically involved in the inhibition of cardiac hypertrophy.

Topics: Actins; Angiotensin II; Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Cell Size; Cells, Cultured; Chromans; Disease Models, Animal; Gene Expression; Heart; Heterozygote; Hypoglycemic Agents; Ligands; Mice; Mice, Knockout; Myocardium; Organ Size; Pioglitazone; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone

Topics: Atrial Natriuretic Factor; Cardiomegaly; Humans; Natriuretic Peptide, Brain; Receptors, Atrial Natriuretic Factor

Members of the Rho GTPase family, Rac1 and RhoA have been suggested to be mediators of cardiac hypertrophy in mice. Rho proteins are posttranslationally isoprenylated. In addition to cholesterol-lowering, statins inhibit the isoprenylation of small G proteins. Therefore, it was tested if these drugs inhibit Rac1 and RhoA activity in cardiomyocytes and, thereby, prevent angiotensin II-mediated expression of atrial natriuretic factor (ANF) and myosin light chain (MLC)-2 in the heart.. Western and Northern analysis of rat neonatal cardiomyocytes and H9C2 cells showed inhibition of basal and angiotensin-stimulated Rac1 expression, membrane-translocation and activity after statin treatment. Similarly, basal and stimulated RhoA membrane expression was inhibited. Statins concentration- and time-dependently downregulated basal as well as angiotensin-induced expression of ANF by 86+/-2.3% and 89+/-1.7%, as well as MLC-2 by 75+/-4.1% and 84+/-6%, respectively. Direct inhibition of Rac GTPase by overexpression of the dominant negative mutant RacN17 or by Clostridium sordellii lethal toxin in rat H9C2 cells inhibited ANF expression by 70+/-4.9% and 78+/-10%, respectively. Inhibition of RhoA by Clostridium botulinum C3 transferase or the dominant negative mutant RhoN19 reduced ANF mRNA by 19+/-11% and 23+/-8%, respectively. To test these findings in vivo, spontaneously hypertensive rats were treated with atorvastatin, leading to a decrease in cardiac Rac1 and RhoA activity as determined by [35S]-GTP gamma S-binding assays by 61+/-16% and 72+/-24%, and downregulation of MLC-2 as well as ANF mRNA expression by 31+/-16% and 80+/-24%, respectively.. (1) Statins downregulate the activity of small G proteins in cardiomyocytes in culture as well as in vivo. (2) Inhibition of Rac1 and RhoA by statins reduces myocardial expression of ANF and MLC-2. (3) Targeting myocardial Rho GTPases by statins may be a novel treatment strategy to prevent cardiac hypertrophy.

Topics: Angiotensin II; Animals; Atorvastatin; Atrial Natriuretic Factor; Cardiac Myosins; Cardiomegaly; Cell Membrane; Cells, Cultured; Gene Expression Regulation; GTP Phosphohydrolases; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypolipidemic Agents; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Myocardium; Myosin Light Chains; Pyrroles; rac1 GTP-Binding Protein; Rats; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; RNA, Messenger; Simvastatin

We examined whether Ca2+ channel blockers inhibit the activation of the Ca2+-dependent phosphatase calcineurin and the development of cardiac hypertrophy in spontaneously hypertensive rats (SHR). We randomly divided 12-week-old SHR into three groups, one each receiving vehicle, bolus injection or continuous infusion of nifedipine (10 mg/kg/day) from 12 to 24 weeks of age. Systolic blood pressure (BP) and heart rate were measured every week after the treatment using the tail-cuff plethysmography method. After 4, 8 and 12 weeks of treatment, 6 rats of each group were subjected to examinations that included an assay for calcineurin activity in the heart, magnetic resonance imaging (MRI), histology and Northern blot analysis. Continuous infusion of nifedipine consistently reduced BP, whereas bolus injection resulted in a fluctuation of BP. Continuous infusion of nifedipine not only reduced left ventricular mass but also decreased the transverse diameter of cardiomyocytes, interstitial fibrosis and the expression of the atrial natriuretic peptide and brain natriuretic peptide genes in the heart, while bolus injection of nifedipine did not significantly attenuate any of these hypertrophic responses in SHR. The activity of calcineurin in the heart was strongly suppressed by continuous but not bolus infusion of nifedipine in SHR. The results indicate that continuous blockade of Ca2+ channels with nifedipine effectively suppresses the development of cardiac hypertrophy in SHR, possibly through inhibition of the calcineurin activity.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Calcineurin Inhibitors; Calcium Channel Blockers; Calcium Channels, L-Type; Cardiomegaly; Fibrosis; Gene Expression; Heart Rate; Hypertension; Male; Myocardium; Natriuretic Peptide, Brain; Nifedipine; Rats; Rats, Inbred SHR

The alpha1-adrenergic receptors (alpha1-ARs) play a key role in cardiovascular homeostasis. However, the functional role of alpha1-AR subtypes in vivo is still unclear. The aim of this study was to evaluate the cardiovascular influences of alpha1b-AR.. In transgenic mice lacking alpha1-AR (KO) and their wild-type controls (WT), we evaluated blood pressure profile and cardiovascular remodeling induced by the chronic administration (18 days via osmotic pumps) of norepinephrine, angiotensin II, and subpressor doses of phenylephrine. Our results indicate that norepinephrine induced an increase in blood pressure levels only in WT mice. In contrast, the hypertensive state induced by angiotensin II was comparable between WT and KO mice. Phenylephrine did not modify blood pressure levels in either WT or KO mice. The cardiac hypertrophy and eutrophic vascular remodeling evoked by norepinephrine was observed only in WT mice, and this effect was independent of the hypertensive state because it was similar to that observed during subpressor phenylephrine infusion. Finally, the cardiac hypertrophy induced by thoracic aortic constriction was comparable between WT and KO mice.. Our data demonstrate that the lack of alpha1b-AR protects from the chronic increase of arterial blood pressure induced by norepinephrine and concomitantly prevents cardiovascular remodeling evoked by adrenergic activation independently of blood pressure levels.

Topics: Angiotensin II; Animals; Aorta; Arterioles; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Cardiovascular System; Echocardiography; Heart Rate; Heart Ventricles; Hypertension; Male; Mesentery; Mice; Mice, Transgenic; Norepinephrine; Organ Size; Phenylephrine; Receptors, Adrenergic, alpha-1; RNA, Messenger; Vasoconstrictor Agents; Ventricular Remodeling

Our studies have established that a single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense gene causes prolonged antihypertensive actions in the spontaneously hypertensive rat. These results suggest that antisense gene therapy is a conceptually valid strategy for the control of hypertension at the genetic level. To evaluate whether attenuation of the pathophysiological aspects of hypertension are dependent on the blood pressure lowering actions of antisense gene therapy, we chose the renin transgenic rat as a hypertensive animal model and cardiac hypertrophy as the hypertension-associated pathophysiology. A single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense in the neonatal rat resulted in long-term expression of the antisense transgene in various cardiovascular-relevant tissues, including the heart. This expression was associated with a significant attenuation of cardiac hypertrophy despite its failure to normalize high blood pressure. Developmental studies indicated that cardiac hypertrophy was evident as early as 16 days of age in viral vector-treated control transgenic rats, despite these animals exhibiting normal blood pressure. These observations demonstrate that, in the renin-transgenic rat, the onset of cardiac hypertrophy occurs during development and is prevented without normalization of high blood pressure. Collectively, these results provide further proof of the concept and indicate that antisense gene therapy could successfully target the local tissues' renin-angiotensin system to produce beneficial cardiovascular outcomes.

Topics: Actins; Animals; Animals, Genetically Modified; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; DNA, Antisense; Female; Gene Expression; Genetic Therapy; Genetic Vectors; Male; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Renin; Retroviridae; RNA, Messenger; Treatment Outcome

To investigate how cardiac hypertrophy and heart failure develop, we isolated and characterized a candidate initiator, the soluble 12-kDa protein myotrophin, from rat and human hearts. Myotrophin stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes. Recombinant myotrophin from the cloned gene showed structural/functional motifs, including ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and casein kinase II. One repeat, homologous with I kappaB, interacts with rel/NF-kappaB in vitro. We analyzed the interaction of recombinant myotrophin and nuclear extracts prepared from neonatal and adult cardiomyocytes; gel mobility shift assay showed that myotrophin bound to kappaB DNA. To define PKC's role in myotrophin-induced myocyte growth, we incubated neonatal rat myocytes (normal and stretch) with specific inhibitors and found that myotrophin inhibits [3H]leucine incorporation into myocytes and different hypertrophic gene expression in neonatal myocytes. Using confocal microscopy, we observed that a basal level of myotrophin was present in both cytoplasm and nucleus under normal conditions, but under cyclic stretch, myotrophin levels became elevated in the nucleus. Myotrophin gene levels were upregulated when myocytes underwent cyclic stretch or were treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta and also when excised beating hearts were exposed to high pressure. Our data showed that the myotrophin-kappaB interaction was increased with age in spontaneously hypertensive rats (SHRs) only. Our data provide evidence that myotrophin-kappaB DNA interaction may be an important step in initiating cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Binding Sites; Blotting, Northern; Cardiomegaly; DNA; Electrophoretic Mobility Shift Assay; Escherichia coli; Growth Substances; Humans; I-kappa B Proteins; Intercellular Signaling Peptides and Proteins; Microscopy, Confocal; Myocardium; Naphthalenes; Pressure; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction; Tumor Necrosis Factor-alpha

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) regulate cardiac hypertrophy. We investigated ventricular alterations of ANP and BNP in interleukin-6 (IL-6) transgenic mice (TG) and wild type (WT) mice with or without viral infection. The ANP and BNP mRNA/GAPDH mRNA ratios in the ventricles of IL-6 TG mice were twice that of WT mice, but were not increased significantly by viral inoculation. In WT mice, both ANP and BNP responses were significantly increased in the ventricles of mice 10 days after encephalomyocarditis (EMC) viral inoculation. Cardiac weight in IL-6 TG mice was significantly greater than in WT 10 days after viral inoculation. Left ventricular wall thickness and the diameter of ventricular myocytes also were greater in IL-6 TG than WT after viral infection. Primary cultures of neonatal rat cardiac myocyte showed that IL-6 increased ANP and BNP mRNA expression in a dose-responsive fashion. In summary, overexpression of ANP and BNP occurs in the ventricles of IL-6 TG mice, along with increased cardiac weight after infection with EMC virus, and impaired responses in the expression of ANP and BNP.

Topics: Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; DNA Probes; Encephalomyocarditis virus; Interleukin-6; Mice; Mice, Inbred C57BL; Mice, Transgenic; Natriuretic Peptide, Brain; Organ Size; Rats

The present study investigated the role of calcineurin in angiotensin II(AngII) -induced cardiac myocyte hypertrophy of rats.. The primary cardiac myocytes were cultured under the standard conditions. The calcineurin activity in AngII-treated cardiomyocytes was tested by using PNPP; protein synethsis rate was assessed by 3H-leucine incorporation; atrial natriuretic factor(ANF) mRNA level was determined by Northern blot analysis. Cell viability was estimated by lactate dehydrogenase (LDH) levels in cultured medium and by dyed cell numbers.. After stimulation of 10, 100 and 1 000 nmol/L of AngIi, calcineurin activities in the cardiomyocytes were increased by 13%, 57% (P < 0.05) and 228% (P < 0.01) respectively, compared with control group. Cyclosporin A(CsA), a specific inhibitor of calcineurin, markedly inhibited the calcineurin activity and decreased the 3H-leucine incorporation in AngII-treated cardiomyocytes in a dose-dependent manner. It was also found that CsA slightly reduced the mRNA level of ANF gene in AngII-stimulated cardiomyocytes.. During AngII-induced cardiac myocyte hypertrophy, calcineurin signal pathway is activated, and inhibition of the pathway can attenuate AngII-induced cardiac myocyte hypertrophy, which suggests that the calcineurin signal pathway may play an important role in AngII-induced myocardial hypertrophy of rats.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cells, Cultured; Cyclosporine; Myocytes, Cardiac; Rats; Rats, Wistar; RNA, Messenger

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcium-Binding Proteins; Calsequestrin; Cardiomegaly; Heart; Immunohistochemistry; Isoproterenol; Mice; Mice, Knockout; Myocardial Contraction; Myocardium; Patch-Clamp Techniques; Sarcoplasmic Reticulum

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.

Topics: Adenoviridae; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Catecholamines; Cells, Cultured; DNA, Recombinant; Endothelin-1; Female; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Mice; Mice, Transgenic; Myocardium; Phenylephrine; Phosphorylation; Protein Tyrosine Phosphatases; Rats; RNA, Messenger

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Endothelin-1; Enzyme Activation; Gene Expression Regulation; Guanosine Triphosphate; Humans; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Myocardium; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; rac1 GTP-Binding Protein; Rats; rhoA GTP-Binding Protein; Transfection

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-epsilon, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-epsilon did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-epsilon also increased atrial natriuretic factor (ANF) and beta-myosin heavy chain (MHC) mRNA levels 2.5 +/- 0.3- and 2.1 +/- 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector (P < 0.05 for both). Conversely, Adv-dnPKC-epsilon did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of beta-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-epsilon overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-epsilon activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.

Topics: Adenoviridae; Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Endothelin-1; Gene Expression Regulation, Enzymologic; Heart Ventricles; Isoenzymes; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Muscle Fibers, Skeletal; Mutagenesis; Myocardium; Myosin Heavy Chains; Protein Kinase C; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; RNA, Messenger

The intrinsic GTPase activity of Galpha q is low, and RGS proteins which activate GTPase are expressed in the heart; however, their functional relevance in vivo is unknown. Transgenic mice with cardiac-specific overexpression of Galpha q in myocardium exhibit cardiac hypertrophy, enhanced PKC xi membrane translocation, embryonic gene expression, and depressed cardiac contractility. We recently reported that transgenic mice with cardiac-specific expression of RGS4, a Galpha q and Galpha i GTPase activator, exhibit decreased left ventricular hypertrophy and ANF induction in response to pressure overload. To test the hypothesis that RGS4 can act as a Galpha q-specific GTPase activating protein (GAP) in the in vivo heart, dual transgenic Galpha q-40xRGS4 mice were generated to determine if RGS4 co-expression would ameliorate the Galpha q-40 phenotype. At age 4 weeks, percent fractional shortening was normalized in dual transgenic mice as was left ventricular internal dimension and posterior and septal wall thicknesses. PKC xi membrane translocation and ANF and alpha -skeletal actin mRNA levels were also normalized. Compound transgenic mice eventually developed depressed cardiac contractility that was evident by 9 weeks of age. These studies establish for the first time a role for RGS4 as a GAP for Galpha q in the in vivo heart, and demonstrate that its regulated expression can have pathophysiologic consequences.

Topics: Actins; Animals; Atrial Natriuretic Factor; Blotting, Northern; Blotting, Western; Cardiomegaly; Cell Nucleus; Echocardiography; GTPase-Activating Proteins; Isoenzymes; Mice; Mice, Transgenic; Muscle, Skeletal; Myocardial Contraction; Phenotype; Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; RGS Proteins; RNA, Messenger; Time Factors

Myocardial beta-adrenergic receptors (beta -ARs) consist of beta(1)- and beta(2)-subtypes, which mediate distinct signaling mechanisms. We examined which beta-AR subtype mediates cardiac hypertrophy. The beta(2)-subtype is predominant in neonatal rat cardiac myocytes (beta(1), 36%vbeta(2), 64%), while the beta(1)-subtype predominates in the adult rat heart (59%v 41%). Stimulation of cultured cardiac myocytes in vitro with isoproterenol (ISO), an agonist for beta(1)- and beta(2)-ARs, caused hypertrophy of myocytes along with increases in transcription of atrial natriuretic factor (ANF) and actin reorganization. All of these ISO-mediated myocyte responses in vitro were inhibited by a beta(1)-AR antagonist, betaxolol, but not by a beta(2)-AR antagonist, ICI 118551. Pertussis toxin failed to affect ISO-induced increases in total protein/DNA content and ANF transcription in vitro. ISO increased LV weight/body weight and ANF transcription in the adult rat in vivo, which were also inhibited by betaxolol but not by ICI 118551. These results suggest that beta -AR stimulated hypertrophy is mediated by the beta(1)-subtype and by a pertussis toxin-insensitive mechanism

Topics: Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-2 Receptor Antagonists; Adrenergic beta-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Heart; Heart Ventricles; Isoproterenol; Proteins; Rats; Rats, Wistar; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2

The relationship was investigated between coronary flow reserve and Doppler echocardiographic parameters, hemodynamic parameters and plasma natriuretic peptide concentrations in the hypertrophied heart.. The subjects were 19 patients with hypertrophied heart due to various etiologies and no significant coronary artery stenosis. All patients were in sinus rhythm. The left ventricular wall thickness, the E/A ratio in transmitral flow velocity pattern and the Doppler index were determined by Doppler echocardiography, and the plasma atrial and brain natriuretic peptide concentrations were measured. At cardiac catheterization, pulmonary capillary wedge pressure and left ventricular end-diastolic pressure were measured, and the coronary flow reserve was obtained by injecting intracoronary adenosine triphosphate into the left anterior descending artery using a Doppler guidewire.. Coronary flow reserve in the patients was significantly lower than in 11 normal control subjects (2.50 +/- 0.76 vs 3.90 +/- 0.64, p < 0.001). There were no significant correlations between coronary flow reserve and the left ventricular wall thickness or the E/A ratio. The mean value of the Doppler index in the patients was 0.48 +/- 0.10 and there was a significant negative correlation between coronary flow reserve and the Doppler index (r = -0.73, p < 0.001). The correlation between coronary flow reserve and left ventricular end-diastolic pressure was not significant, but there was a significant negative correlation between coronary flow reserve and pulmonary capillary wedge pressure (r = -0.64, p < 0.01). There were significant negative correlations between coronary flow reserve and atrial (r = -0.62, p < 0.01), or brain natriuretic peptide concentrations (r = -0.56, p < 0.05).. Coronary flow reserve may reflect overall cardiac performance evaluated by the Doppler index and plasma natriuretic peptide concentrations in the hypertrophied heart, and the measurement of coronary flow reserve may be useful for evaluating disease severity in patients with hypertrophied heart.

Topics: Aged; Atrial Natriuretic Factor; Blood Flow Velocity; Cardiomegaly; Coronary Circulation; Echocardiography, Doppler; Female; Heart Function Tests; Hemodynamics; Humans; Male; Middle Aged; Natriuretic Peptide, Brain; Severity of Illness Index

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Guanylate Cyclase; Heart Ventricles; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Models, Genetic; Receptors, Atrial Natriuretic Factor; RNA, Messenger

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.

Topics: Animals; Atrial Natriuretic Factor; Calcium; Calcium-Transporting ATPases; Cardiomegaly; Cardiotonic Agents; Disease Models, Animal; Gene Expression; Heart Rate; Isoproterenol; Mice; Mice, Inbred Strains; Myocardial Contraction; Myocardium; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Organ Size; Ryanodine; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases

Studies in human and rodent models have shown that activation of protein kinase C-beta (PKC-beta) is associated with the development of pathological hypertrophy, suggesting that ablation of the PKC-beta pathway might prevent or reverse cardiac hypertrophy. To explore this, we studied mice with targeted disruption of the PKC-beta gene (knockout, KO). There were no detectable differences in expression or distribution of other PKC isoforms between the KO and control hearts as determined by Western blot analysis. Baseline hemodynamics were measured using a closed-chest preparation and there were no differences in heart rate and arterial or left ventricular pressure. Mice were subjected to two independent hypertrophic stimuli: phenylephrine (Phe) at 20 mg x kg(-1) x day(-1) sq infusion for 3 days, and aortic banding (AoB) for 7 days. KO animals demonstrated an increase in heart weight-to-body weight ratio (Phe, 4.3 +/- 0.6 to 6.1 +/- 0.4; AoB, 4.0 +/- 0.1 to 5.8 +/- 0.7) as well as ventricular upregulation of atrial natriuretic factor mRNA analogous to those seen in control animals. These results demonstrate that PKC-beta expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response.

Topics: Animals; Aorta; Atrial Natriuretic Factor; beta-Galactosidase; Biomarkers; Blood Pressure; Cardiomegaly; Carotid Arteries; Gene Expression Regulation, Enzymologic; Isoenzymes; Lac Operon; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Protein Kinase C; Protein Kinase C beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Ventricular Remodeling

Eprosartan is a selective angiotensin II type I receptor antagonist approved for the treatment of hypertension. In the present studies, eprosartan's ability to provide end-organ protection was evaluated in a model of cardiomyopathy and renal failure in stroke-prone rats (SP).. SP were fed a high fat (24.5% in food) and high salt (1% in water) diet (SFD). Eprosartan (60 mg/kg/day) or vehicle (saline control) (n = 25/group) was administered by intraperitoneally-implanted minipumps to these SP on the SFD for 12 weeks. Normal diet fed SP and WKY rats (n = 25/group) were also included for comparison (i.e. served as normal controls). Mortality, hemodynamics, and both renal and cardiac function and histopathology were monitored in all treatment groups.. Eprosartan decreased the severely elevated arterial pressure (-12%; P < 0.05) produced by SFD but did not affect heart rate. Vehicle-treated SP-SFD control rats exhibited significant weight loss (-13%; P < 0.05) and marked mortality (50% by week 6 and 95% by week 9; P < 0.01). Eprosartan-treated SP-SFD rats maintained normal weight, and exhibited zero mortality at week 12 and beyond. Eprosartan prevented the increased urinary protein excretion (P < 0.05) that was observed in vehicle-treated SP-SFD rats. Echocardiographic (i.e. 2-D guided M-mode) evaluation indicated that SP-SFD vehicle control rats exhibited increased septal (+22.2%) and posterior left ventricular wall (+30.0%) thickness, and decreased left ventricular chamber diameter (-15.9%), chamber volume (-32.7%), stroke volume (-48.7%) and ejection fraction (-22.3%), and a remarkable decrease in cardiac output (-59.3%) compared to controls (all P < 0.05). These same parameters in eprosartan-treated SP-SFD rats were normal and differed markedly and consistently from vehicle-treated SP-SFD rats (i.e. treatment prevented pathology; all P < 0.05). Cardiac-gated MRI data confirmed the ability of eprosartan to prevent cardiac pathology/remodeling (P < 0.05). Histopathological analysis of hearts and kidneys indicated that eprosartan treatment significantly reduced end-organ damage (P < 0.01) and provided corroborative evidence that eprosartan reduced remodeling of these organs. Vehicle-treated SP-SFD rats exhibited a 40% increase in the plasma level of pro-atrial natiuretic factor that was reduced to normal by eprosartan (P < 0.05).. These data demonstrate that eprosartan, at a clinically relevant dose, provides significant end-organ protection in the severely hypertensive stroke-prone rat. It preserves cardiac and renal structural integrity, reduces cardiac hypertrophy and indices of heart failure, maintains normal function of the heart and kidneys, and eliminates premature mortality due to hypertension-induced end-organ failure.

Topics: Acrylates; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Heart Rate; Hypertension; Imidazoles; Kidney; Magnetic Resonance Imaging; Male; Myocardium; Natriuresis; Organ Size; Peptide Fragments; Protein Precursors; Proteinuria; Rats; Rats, Inbred SHR; Stroke; Survival Rate; Thiophenes; Ventricular Remodeling

The transcription factor NF-kappaB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-kappaB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-kappaB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-kappaB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-kappaB was inhibited by expression of a "supersuppressor" IkappaBalpha mutant that is resistant to stimulation-induced degradation and a dominant negative IkappaB kinase (IKKbeta) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IkappaBalpha degradation in an IKK-dependent manner, suggesting that NF-kappaB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IkappaBalpha mutant or the dominant negative IKKbeta mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-kappaB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-kappaB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; DNA-Binding Proteins; I-kappa B Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Phenylephrine; Rats; Rats, Sprague-Dawley

Cardiac hypertrophy is one of the serious complications which increase mortality due to cardiovascular diseases. However, only a partial reduction of cardiac hypertrophy has been successful using current drug therapy. We demonstrate here reduction of cardiac hypertrophy in vitro and in vivo using an adenovirus vector encoding cyclin-dependent kinase (cdk) inhibitor p16INK4a. Adenovirus-mediated overexpression of cdk inhibitor p16INK4a completely inhibited cardiac myocyte hypertrophy induced by endothelin (ET)-1, as evaluated by [3H]leucine incorporation into the cells and mRNA levels of skeletal alpha -actin (SK-A) or atrial natriuretic peptide (ANP) as well as by morphometric analyses. We then evaluated whether p16INK4a can suppress left-ventricular (LV) hypertrophy induced by aortic banding (AOB) in rats. Catheter-mediated gene transfer of AxCAp16 was performed according to the method reported by Hajjar et al. LV overload was produced by coarctation of the ascending aorta immediately after inoculation of the heart with adenovirus. Two weeks after the procedure, the left ventricular weight/body weight ratio (LVW/BW) increased in the AOB+LacZ group in comparison to that in controls. However, LVW/BW was identical in the AOB+p16 group and controls. Histologic analysis revealed that p16INK4a inhibited hypertrophy of cardiac myocytes. These results suggest that G1 cell cycle regulators may restrict cardiac hypertrophy, and offer a novel strategy for the gene therapy of cardiac hypertrophy.

Topics: Actins; Adenoviridae; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Cyclin-Dependent Kinases; Endothelin Receptor Antagonists; Endothelin-1; Gene Expression; Gene Transfer Techniques; Genes, p16; Genetic Therapy; Genetic Vectors; Myocardium; Rats; Rats, Inbred Strains; RNA, Messenger; Transfection

Adrenomedullin is a potent hypotensive, natriuretic and diuretic peptide that is coexpressed in the heart with its receptor, suggesting that it may have localized actions as a modulator of cardiac function. Although expression of adrenomedullin is upregulated in the pathological heart, its cardiac function has not been clearly elucidated and it is not known whether this represents a common feature of cardiac hypertrophy, nor whether this is restricted to cardiac myocytes. We have determined the direct effects of hypertrophic agents on cardiomyocyte adrenomedullin gene expression and peptide secretion and have examined the effects of adrenomedullin on biochemical markers of cardiomyocyte hypertrophy.. Regulation of adrenomedullin expression and its effects on the hypertrophic response were studied in cultured rat neonatal ventricular cardiomyocytes.. Incubation with phenylephrine or endothelin for 48 h led to a hypertrophic response with an associated fivefold stimulation of ANP gene expression. In contrast, adrenomedullin mRNA was inhibited by 30-50% in response to phenylephrine or endothelin-mediated hypertrophy, and this was associated with a 35-45% reduction in secretion of immunoreactive adrenomedullin. Phorbol ester mediated activation of protein kinase C and increasing intracellular Ca(2+) with ionomycin led to significant downregulation of adrenomedullin gene expression in cardiomyocytes. Co-incubation with 100 nM adrenomedullin for 48 h inhibited phenylephrine-induced cardiomyocyte hypertrophy as determined by protein:DNA ratio. Adrenomedullin partially blocked phenylephrine-mediated transcriptional activation of ANP and MLC-2 reporter gene expression in cardiomyocytes and this effect was mimicked by 2 microM forskolin, suggesting that this response was mediated via the activation of adenylate cyclase.. These data demonstrate that the cardiomyocyte adrenomedullin gene is repressed by phenylephrine or endothelin-mediated hypertrophy. The inhibitory effects of adrenomedullin on the cardiomyocyte hypertrophic response suggests that this peptide acts as a regulated autocrine or paracrine modulator of cardiomyocyte function and that downregulation of adrenomedullin expression may play a role in induction and maintenance of cardiomyocyte hypertrophy.

Topics: Adenylyl Cyclases; Adrenomedullin; Analysis of Variance; Animals; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Cells, Cultured; Colforsin; Endothelin-1; Enzyme Activation; Gene Expression Regulation; Myocardium; Peptides; Phenylephrine; Protein Kinase C; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

In vivo, the normal heart obtains at least 60% of its energy from lipids and the remainder from glucose. Several lines of evidence indicate that an increase in the utilization of glucose [at the expense of fatty acids (FA)] may play a role in the genesis of hypertrophy. Primary cultures of neonatal cardiomyocytes have been used extensively to study the phenotype of these cells as well as their responses to hormonal hypertrophic agents. Unfortunately, such cultures are most typically cultured in glucose-rich FA-free media, and thus might be hypertrophied to start with. We therefore tested the effects of FA-albumin complexes on three different surrogate end points of hypertrophy of cardiomyocytes. Oleate-albumin complexes decreased the baseline values of all three variables, and increased the relative response of these variables to administration of norepinephrine. Oleate:palmitate-albumin complexes also affected all three variables and their responses to norepinephrine, but the effects differed somewhat from that of oleate-albumin complexes. Our results suggest that addition of long-chain FA, by providing conditions that more closely resemble physiological situations, may optimize the expression of hypertrophic responses in such cells. However, the differences between the effects of oleate and oleate:palmitate also suggest that the precise composition of FA may affect the phenotype of cardiomyocytes and how these cells respond to hypertrophic agents.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Culture Media; Fatty Acids; Heart; Myocardium; Norepinephrine; Oleic Acid; Palmitic Acid; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine

Activation of the alpha(1A)-adrenergic receptor (alpha(1A)-AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the alpha(1A)-AR (4- to 170-fold) were generated, using the rodent alpha-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt(max) (80%, P<0.0001), dP/dt(max)/LVP(inst) (76%, P<0.001), dP/dt(max):dP/dt(min) (104%, P<0.0001), and fractional shortening (33%, P<0.05). Moreover, changes in the dP/dt(max)-end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (-)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute alpha(1A)- but not beta-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the alpha(1A)-AR.

Topics: Adenylyl Cyclases; Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Echocardiography; Electrocardiography; Gene Expression; Gene Targeting; Heart Rate; Heterozygote; Inositol Phosphates; Mice; Mice, Transgenic; Myocardial Contraction; Myosin Heavy Chains; Organ Size; Organ Specificity; Promoter Regions, Genetic; Receptors, Adrenergic, alpha-1; RNA, Messenger; Transgenes; Type C Phospholipases

Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Fractionation; Cells, Cultured; Crk-Associated Substrate Protein; Culture Media, Serum-Free; Cytoskeletal Proteins; Endothelins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Genes, Reporter; Immunoblotting; Immunohistochemistry; Microfilament Proteins; Myocardium; Myosin Heavy Chains; Paxillin; Phosphoproteins; Phosphorylation; Promoter Regions, Genetic; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proteins; Rats; Recombinant Fusion Proteins; Retinoblastoma-Like Protein p130; Sarcomeres; Signal Transduction; src-Family Kinases; Tensins

The present study examined the role of calcineurin in insulin-like growth factor (IGF)-1-induced hypertrophy in primary cultures of adult rat ventricular myocytes (ARVM), prepared from the ventricles of 14-16-week-old male Sprague-Dawley rats. The effects of several humoral factors, including phenylephrine, angiotensin II, endothelin-1, IGF-1 and interleukin-6, on the morphology of ARVM were studied. Myocyte surface area was significantly increased by IGF-1 (2,268 +/- 571 to 3,018 +/- 836 microm2, p < 0.01), but not by other humoral factors. This hypertrophic effect of IGF-1 was blocked by genistein (tyrosine kinase inhibitor), PD98059 (MEK inhibitor). These findings suggest that IGF-1 produces ARVM hypertrophy by a tyrosine kinase-MEK mediated pathway as has been reported in neonatal cardiomyocytes. IGF-1-mediated ARVM hypertrophy was also attenuated by cyclosporine A (calcineurin inhibitor), and staurosporine and chelerythrine (protein kinase C inhibitors). IGF-1 markedly increased calcineurin activity (8.7 +/- 1.2 to 98.0 +/- 54.3 pmol x h(-1) mg(-1), p < 0.01), and this activation was completely blocked by pre-treatment with cyclosporine A (8.5 +/- 11.4pmol x h(-1) x mg(-1), p < 0.01) and chelerythrine (2.3 +/- 2.7 pmol x h(-1) mg(-1), p < 0.01). It appears that IGF-1 activates calcineurin by a protein kinase C-dependent pathway. Increased mRNA expression of atrial natriuretic factor by IGF-1 was inhibited by cyclosporine A (p < 0.01). The findings indicate that IGF-1 induces ARVM hypertrophy by protein kinase C and calcineurin-related mechanisms. The fact that elevated calcineurin activity and induced atrial natriuretic factor mRNA expression by IGF-1 were blocked by cyclosporine A further supports the hypothesis that calcineurin is critically involved in IGF-1-induced ARVM hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cell Size; Cells, Cultured; Enzyme Inhibitors; Heart Ventricles; Insulin-Like Growth Factor I; Male; Protein Kinase C; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction

Cardiac hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in men and in women. Epidemiological studies indicate that estrogen replacement therapy is cardioprotective; the mechanisms involved in this process, however, are poorly understood. We therefore studied the effect of 17beta-estradiol (E(2)) on the development of pressure-overload hypertrophy.. Ovariectomized mice receiving E(2) or placebo underwent transverse aortic constriction (TAC) or sham operation. TAC led to a significant increase in ventricular mass compared with sham operation. E(2) treatment reduced cardiac hypertrophy by 31% and 26% compared with placebo 4 and 8 weeks after TAC, whereas it had no effect on the degree of pressure overload, as determined by hemodynamic measurements. Furthermore, E(2) blocked the increased phosphorylation of p38-mitogen-activated protein kinase (MAPK) observed in the placebo-treated animals with TAC. No differences were observed in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 between the groups. E(2) had no effect on the expression of angiotensin-converting enzyme (ACE) or the angiotensin II type 1 receptor. Ventricular atrial natriuretic peptide (ANP) expression was detected only in the animals with TAC. Compared with placebo, E(2) treatment led to an increased expression of ANP in animals with pressure overload.. Here, we show that E(2) attenuates the hypertrophic response to pressure overload in mice. This observation demonstrates that hormone replacement therapy with E(2) has direct effects on the heart and may be beneficial in the treatment of postmenopausal women to reduce cardiac hypertrophy.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Disease Models, Animal; Estradiol; Estrogen Replacement Therapy; Female; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Organ Size; Ovariectomy; p38 Mitogen-Activated Protein Kinases; Peptidyl-Dipeptidase A; Phosphorylation; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Signal Transduction

In pressure overload-induced hypertrophy, the heart increases its reliance on glucose as a fuel while decreasing fatty acid oxidation. A key regulator of this substrate switching in the hypertrophied heart is peroxisome proliferator-activated receptor alpha (PPARalpha). We tested the hypothesis that down-regulation of PPARalpha is an essential component of cardiac hypertrophy at the levels of increased mass, gene expression, and metabolism by pharmacologically reactivating PPARalpha. Pressure overload (induced by constriction of the ascending aorta for 7 days in rats) resulted in cardiac hypertrophy, increased expression of fetal genes (atrial natriuretic factor and skeletal alpha-actin), decreased expression of PPARalpha and PPARalpha-regulated genes (medium chain acyl-CoA dehydrogenase and pyruvate dehydrogenase kinase 4), and caused substrate switching (measured ex vivo in the isolated working heart preparation). Treatment of rats with the specific PPARalpha agonist WY-14,643 (8 days) did not affect the trophic response or atrial natriuretic factor induction to pressure overload. However, PPARalpha activation blocked skeletal alpha-actin induction, reversed the down-regulation of measured PPARalpha-regulated genes in the hypertrophied heart, and prevented substrate switching. This PPARalpha reactivation concomitantly resulted in severe depression of cardiac power and efficiency in the hypertrophied heart (measured ex vivo). Thus, PPARalpha down-regulation is essential for the maintenance of contractile function of the hypertrophied heart.

Topics: Actins; Animals; Aorta; Atrial Natriuretic Factor; Cardiomegaly; Down-Regulation; Glucose; Isoenzymes; Male; Muscle, Skeletal; Myocardium; Oleic Acid; Oxygen; Oxygen Consumption; Perfusion; Protein Kinases; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transcription Factors

Recently, deletion of specific genes by so called knock-out techniques has become important for investigating the pathogenesis of various diseases. This form of genetic engineering is widely performed in murine models. There are, however, only a limited number of mouse models available in cardiovascular pathology. The objective of this study, therefore, was to develop a new model of overt congestive heart failure associated with myocardial hypertrophy in the mouse.. Female C57/BL6 mice weighing 19-20 g were anesthetized with ether. After abdominal incision, the aorta was temporarily clamped proximal to the renal arteries. The aorta was then punctured with a needle (outer diameter 0.6 mm) and the needle was further advanced into the adjacent vena cava. After withdrawal of the needle, the aortic puncture site was sealed with cyanoacrylate glue. The clamp was removed, and the patency of the shunt was visually verified as swelling and mixing of venous and arterial blood in the vena cava. Sham-operated mice served as controls.. Perioperative mortality of mice with aortocaval shunt was 42%. Four weeks after shunt induction, mice showed a significant cardiac hypertrophy with a relative heart weight of 7.5+/-0.2 mg/100 g body weight (vs. 5.1+/-0.7 mg/100 g in control mice, P<0.001). While no changes in blood pressure and heart rate occurred, left ventricular enddiastolic pressure was significantly increased in mice with shunt, and left ventricular contractility was impaired from 6331+/-412 to 4170+/-296 mmHg/s (P<0.05). Plasma concentrations of atrial natriuretic peptide (ANP) and its second messenger cGMP as humoral markers of heart failure as well as ventricular expression of ANP- and brain natriuretic peptide (BNP)-mRNA were significantly increased in mice with shunt compared to control mice.. The aortocaval shunt in the mouse constitutes a new model of overt congestive heart failure with impaired hemodynamic parameters and may be a useful tool to investigate the role of particular genes in the development of heart failure.

Topics: Animals; Arteriovenous Shunt, Surgical; Atrial Natriuretic Factor; Blood Pressure; Cardiac Volume; Cardiomegaly; Disease Models, Animal; Female; Heart Failure; Heart Rate; Mice; Mice, Inbred C57BL; Natriuretic Peptide, Brain

To investigate modulation of ANP secretion by atrial hypertrophy, the secretion of ANP in response to stretch and endothelin-1 was studied using isolated perfused quiescent atria from rats treated with monocrotaline (MCT). Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg MCT or saline and were sacrificed at 6 weeks. Rats with right heart hypertrophy showed an increase in ANP mRNA and decrease in tissue concentration of ANP in hypertrophied atria and a marked increase in plasma concentration of ANP. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. Changes in atrial volume and mechanically stimulated ECF translocation in hypertrophied right atria were not different from those in control right atria. The stretch-activated ANP secretion was suppressed without significant difference in basal ANP secretion, as compared to control right atria. Therefore, the stretch-activated ANP secretion from hypertrophied right atria into the atrial lumen in relation to the ECF translocation (ANP concentration in the interstitium) was lower than that from control atria. A positive correlation between the stretch-activated ANP secretion in relation to the ECF translocation and tissue ANP content was found in control atria but not in hypertrophied atria. Endothelin-1 caused increases in stretch-activated ANP secretion in a dose-dependent manner, which were accentuated in hypertrophied right atria. Therefore, we suggest that atrial hypertrophy causes an attenuated response to stretch and accentuated response to endothelin-1 of ANP secretion.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Extracellular Space; Male; Monocrotaline; Rats; Rats, Sprague-Dawley; RNA, Messenger; Statistics as Topic; Stress, Mechanical; Time Factors

It has been shown that atrial natriuretic peptide (ANP) influences proliferation of cardiac cells. To define the possible role of C-type natriuretic peptide (CNP) in cardiac hypertrophy, the influence of CNP on the secretion of ANP was studied with the use of perfused nonbeating atria from monocrotaline-treated rats. Increases in atrial volume caused proportional increases in ANP secretion that were markedly suppressed by CNP (10(-6) M) in nonhypertrophied left atria and control right atria but not in hypertrophied right atria. However, increases in atrial volume and mechanically stimulated extracellular fluid (ECF) translocation by CNP were similar to those in the control group. Therefore, the secretion of ANP in terms of ECF translocation was decreased by CNP in nonhypertrophied left and control right atria but not in hypertrophied atria. However, the inhibitory effect of 8-bromo-cGMP on the secretion of ANP was observed in both atria. The cGMP productions from perfused hypertrophied atria and their membranes exposed to CNP were significantly lower than those from nonhypertrophied atria. No significant difference in natriuretic peptide receptor-B transcript was found. Therefore, attenuation of the inhibitory effect of CNP on the ANP secretion in hypertrophied atria may be due to lack of cGMP production. The results showing the relief of CNP-induced negative inhibition of ANP secretion by atrial hypertrophy suggest that CNP may be a contributing factor to delay the development of cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic GMP; Extracellular Space; Guanylate Cyclase; Heart Atria; In Vitro Techniques; Male; Monocrotaline; Myocardium; Natriuretic Peptide, C-Type; Perfusion; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor

Topics: Amlodipine; Antihypertensive Agents; Atenolol; Atrial Natriuretic Factor; Bendroflumethiazide; Cardiomegaly; Endothelin-1; Female; Heart Failure; Humans; Hypertension; Male; Middle Aged; Natriuretic Peptide, Brain; Perindopril; Procollagen; Randomized Controlled Trials as Topic

Urotensin II (UII), a cyclic neuropeptide, functions not only in the central nervous system but also in non-neural systems including cardiovascular systems. In the present study we examined whether UII regulates hypertrophy in cardiomyocytes. The exposure of cultured cardiomyocytes from neonatal rats to UII dose-dependently activated extracellular signal-regulated kinases (ERKs), important molecules in the development of cardiac hypertrophy. ERK activation by UII at 100 nM peaked at 8 min after stimulation. UII markedly induced expression of specific genes encoding atrial natriuretic peptide and brain natriuretic peptide, and significantly increased amino acid incorporation into proteins. Incubation of cardiomyocytes with UII increased cell size and myofibril organisation. UII, then, might participate in cardiomyocyte hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Enzyme Activation; Gene Expression; Heart; Humans; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Myocardium; Natriuretic Peptide, Brain; Phenylalanine; Protein Biosynthesis; Rats; Rats, Wistar; Urotensins

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by small G proteins of the Rho family. We hypothesized that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, inhibit cardiac hypertrophy by blocking Rho isoprenylation. We treated neonatal rat cardiac myocytes with angiotensin II (AngII) with and without simvastatin (Sim) and found that Sim decreased AngII-induced protein content, [3H] leucine uptake, and atrial natriuretic factor (ANF) promoter activity. These effects were associated with decreases in cell size, membrane Rho activity, superoxide anion (O2*-) production, and intracellular oxidation, and were reversed with L-mevalonate or geranylgeranylpyrophosphate, but not with farnesylpyrophosphate or cholesterol. Treatments with the Rho inhibitor C3 exotoxin and with cell-permeable superoxide dismutase also decreased AngII-induced O2*- production and myocyte hypertrophy. Overexpression of the dominant-negative Rho mutant N17Rac1 completely inhibited AngII-induced intracellular oxidation and ANF promoter activity, while N19RhoA partially inhibited it, and N17Cdc42 had no effect. Indeed, Sim inhibited cardiac hypertrophy and decreased myocardial Rac1 activity and O2*- production in rats treated with AngII infusion or subjected to transaortic constriction. These findings suggest that statins prevent the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1.

Topics: Angiotensin II; Animals; Antioxidants; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Heart; Mice; Myocardium; Oxidation-Reduction; Promoter Regions, Genetic; rac1 GTP-Binding Protein; Rats; Rats, Sprague-Dawley; Simvastatin; Superoxides

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCbeta and skeletal alpha-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1-2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP.

Topics: Actins; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Calcitonin Gene-Related Peptide; Calcium-Transporting ATPases; Capsaicin; Cardiomegaly; Cells, Cultured; Constriction, Pathologic; Glucose Transporter Type 4; Histocompatibility Antigens Class I; Monosaccharide Transport Proteins; Muscle Proteins; Myocardium; Organ Size; Rats; Rats, Wistar; Sarcoplasmic Reticulum Calcium-Transporting ATPases

We examined whether adrenomedullin (AM), a vasoactive peptide with significant expression and binding sites in the heart, modulates the hypertrophic response in cultured neonatal rat ventricular myocytes. Myocyte hypertrophy was induced by treating the cells with angiotensin II (Ang II), endothelin-1 (ET-1) or alpha-adrenergic agonist, L-phenylephrine (PHE). All treatments resulted in a hypertrophic response as reflected by increased protein synthesis and expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) genes. AM treatment resulted in a complete inhibition of the Ang II-induced increase in ANP and BNP gene expression and secretion. In contrast, no inhibitory effect was seen in either ET-1-induced natriuretic peptide gene expression or PHE-induced ANP and BNP gene expression and secretion. AM had only a modest effect on basal levels of natriuretic peptide secretion and gene expression. When AM mRNA levels in isolated neonatal rat myocytes treated for 48 h with Ang II, ET-1 or PHE were measured, only Ang II induced a consistent increase in AM gene expression. These results indicate that AM is not invariably associated with attenuation of the hypertrophic response but its effect is dependent on the stimulus activating myocyte hypertrophy. AM may form an important autocrine/paracrine growth-inhibitory loop in Ang II-induced myocyte hypertrophy.

Topics: Adrenomedullin; Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclic AMP; Cyclic GMP; Drug Interactions; Endothelin-1; Gene Expression; Heart; Heart Ventricles; In Vitro Techniques; Myocardium; Natriuretic Peptide, Brain; Peptides; Phenylephrine; Rats; Sarcomeres

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3 in vivo, transgenic mice with cardiac-specific overexpression of the Stat3 gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of the atrial natriuretic factor (ANF), beta-myosin heavy chain (MHC), and cardiotrophin (CT)-1 genes. The animals were injected i.p. with 15 mg/kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5/20) for control littermates (WT), but 80% (16/20) for STAT3-TG (P < 0.01). WT showed increased expression of beta-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of the cardiac alpha-actin and Stat3 genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors.

Topics: Actins; Animals; Antineoplastic Agents; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathies; Cytokines; DNA-Binding Proteins; Doxorubicin; Gene Expression Regulation; Mice; Mice, Transgenic; Myocardium; Myosin Heavy Chains; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Trans-Activators

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.

Topics: 1-Sarcosine-8-Isoleucine Angiotensin II; Angiotensin Receptor Antagonists; Animals; Atrial Natriuretic Factor; Binding, Competitive; Blotting, Northern; Cardiomegaly; Gene Expression Regulation; Heart Atria; Heart Ventricles; Humans; Imidazoles; Immunohistochemistry; Losartan; Mice; Mice, Transgenic; Myocardium; Phenotype; Pyridines; Radioligand Assay; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; RNA, Messenger; Tissue Distribution; Transgenes; Ventricular Remodeling

Atrial natriuretic peptide (ANP) may function as an endogenous regulator of cardiac hypertrophy, because the natriuretic peptide receptor has been found in the heart and because mice lacking its receptor have been shown to have a markedly elevated ventricular mass. We examined the role of endogenous ANP in cardiac hypertrophy in vitro. The effects of the blockade of endogenous ANP by its receptor antagonist, HS-142-1, on cell hypertrophy were investigated with the use of cultured neonatal rat ventricular myocytes. HS-142-1 increased the basal and phenylephrine (PE, 10(-5) mol/L)-stimulated protein syntheses in a concentration-dependent manner (1 to 300 microg/mL). A significant increase in the cell size of myocytes was also induced by this antagonist. In addition, the expression levels of skeletal alpha-actin, beta-myosin heavy chain, and ANP genes, markers of hypertrophy, were partially elevated by treatment with HS-142-1 (100 microg/mL) under nonstimulated or PE-stimulated conditions. A cGMP-specific phosphodiesterase inhibitor, zaprinast (5x10(-4) mol/L), and a cGMP analogue (10(-4) mol/L) suppressed the basal and PE-stimulated protein syntheses. Our observations suggest that endogenous ANP inhibits cardiac myocyte hypertrophy under basal and PE-stimulated conditions, probably through a cGMP-dependent process. ANP may play a role as an autocrine factor in the regulation of cardiac myocyte growth.

Topics: Actins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Gene Expression; Mice; Myocardium; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phenylephrine; Polysaccharides; Protein Biosynthesis; Rats; RNA, Messenger

The potential involvement of the brain renin-angiotensin system in the hypertension induced by subpressor doses of angiotensin II was tested by the use of newly developed transgenic rats with permanent inhibition of brain angiotensinogen synthesis [TGR(ASrAOGEN)]. Basal systolic blood pressure monitored by telemetry was significantly lower in TGR(ASrAOGEN) than in Sprague-Dawley rats (parent strain) (122.5+/-1.5 versus 128.9+/-1.9 mm Hg, respectively; P<0.05). The increase in systolic blood pressure induced by 7 days of chronic angiotensin II infusion was significantly attenuated in TGR(ASrAOGEN) in comparison with control rats (29.8+/-4.2 versus 46. 3+/-2.5 mm Hg, respectively; P<0.005). Moreover, an increase in heart/body weight ratio was evident only in Sprague-Dawley (11.1%) but not in TGR(ASrAOGEN) rats (2.8%). In contrast, mRNA levels of atrial natriuretic peptide (ANP) and collagen III in the left ventricle measured by ribonuclease protection assay were similarly increased in both TGR(ASrAOGEN) (ANP, x2.5; collagen III, x1.8) and Sprague-Dawley rats (ANP, x2.4; collagen III, x2) as a consequence of angiotensin II infusion. Thus, the expression of these genes in the left ventricle seems to be directly stimulated by angiotensin II. However, the hypertensive and hypertrophic effects of subpressor angiotensin II are at least in part mediated by the brain renin-angiotensin system.

Topics: Angiotensin II; Angiotensinogen; Animals; Animals, Genetically Modified; Atrial Natriuretic Factor; Blood Pressure; Brain Chemistry; Cardiomegaly; Collagen; Gene Expression; Heart Ventricles; Hypertension, Renal; Male; Myocardium; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; RNA, Messenger

Cardiac hypertrophy is a major predictor of future morbidity and mortality. Recent investigation has centered around identifying the molecular signaling pathways that regulate cardiac myocyte reactivity with the goal of modulating pathologic hypertrophic programs. One potential regulator of cardiomyocyte hypertrophy is the calcium-sensitive phosphatase calcineurin. We show here that calcineurin enzymatic activity, mRNA, and protein levels are increased in cultured neonatal rat cardiomyocytes by hypertrophic agonists such as angiotensin II, phenylephrine, and 1% fetal bovine serum. This induction of calcineurin activity was associated with an increase in calcineurin Abeta (CnAbeta) mRNA and protein, but not in CnAalpha or CnAgamma. Agonist-dependent increases in calcineurin enzymatic activity were specifically inhibited with an adenovirus expressing a noncompetitive peptide inhibitor of calcineurin known as cain [Lai, M. M., Burnett, P. E., Wolosker, H., Blackshaw, S. & Snyder, S. H. (1998) J. Biol. Chem. 273, 18325-18331]. Targeted inhibition of calcineurin with cain or an adenovirus expressing only the calcineurin inhibitory domain of AKAP79 attenuated cardiomyocyte hypertrophy and atrial natriuretic factor expression in response to angiotensin II, phenylephrine, and 1% fetal bovine serum. These data demonstrate that calcineurin is an important regulator of cardiomyocyte hypertrophy in response to certain agonists and suggest that cyclosporin A and FK506 function to attenuate cardiac hypertrophy by specifically inhibiting calcineurin.

Topics: Adenoviridae; Angiotensin II; Animals; Apoptosis Regulatory Proteins; Atrial Natriuretic Factor; Calcineurin; Calcineurin Inhibitors; Cardiomegaly; Carrier Proteins; Cattle; Cells, Cultured; DNA-Binding Proteins; Fetal Blood; Gene Expression Regulation; Genes; Genetic Therapy; Genetic Vectors; Hypertrophy; Myocardium; NFATC Transcription Factors; Nuclear Proteins; Phenylephrine; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transfection

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar heart-to-body weight ratios but express lower amounts of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice. alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF, beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1, GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+ mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump removal caused the agonist-treated hearts and gene expression to regress to control or near-control levels. Thus Egr-1 deficiency caused a blunted catecholamine-induced hypertrophy response and increased sensitivity to stress.

Topics: Actins; Adrenergic alpha-Agonists; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression; Genes, fos; Genes, jun; Immediate-Early Proteins; Isoproterenol; Male; Mice; Mice, Mutant Strains; Myosin Heavy Chains; Neoplasm Proteins; Phenylephrine; Receptors, Adrenergic; Repressor Proteins; Sp1 Transcription Factor; Transcription Factors

The incidence of heart failure in the population increases steeply among older people. Overactivation of the sympathetic nervous system is associated to and responsible for worsening of heart failure. This study describes the influence of aging on short-term left ventricular (LV) adaptation to b-adrenergic stimulation in Wistar rats. In controls at 18 mo, interstitial fibrosis was increased with respect to 3-mo-old rats, whereas myocytes dimension and the messenger RNA (mRNA) abundance of atrial natriuretic peptide (ANP), a2(I) procollagen, transforming growth factor (TGF-b1, TGF-b3), and secreted protein, acidic and rich in cysteine (SPARC) were not different. To determine how aging affects LV adaptation to adrenergic stimulation, two groups of animals received isoproterenol (ISO, 1 mg/kg/d) for 3 days. There was no significant difference between young and older rats with respect to increase in LV weight, myocytes dimension, and mRNA abundance of all the genes considered, except a1(III) procollagen. These findings indicate that despite limited compensatory hypertrophy and higher fibrosis, LV from aged nonsenescent rats preserves the capacity to adapt to b-adrenergic stimulation through the upregulation of several genes encoding extracellular matrix-related proteins.

Topics: Adaptation, Physiological; Adrenergic beta-Agonists; Aging; Analysis of Variance; Animals; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Collagen; Gene Expression Regulation; Heart Rate; Isoproterenol; Male; Myocardium; Rats; Rats, Wistar; Transforming Growth Factor beta; Up-Regulation; Ventricular Function, Left

Dietary copper (Cu) restriction causes a hypertrophic cardiomyopathy similar to that induced by work overload in rodent models. However, a possible change in the program of hypertrophic gene expression has not been studied in the Cu-deficient heart. This study was undertaken to fill that gap. Dams of mouse pups were fed a Cu-deficient diet (0.35 mg/kg diet) or a Cu-adequate control diet (6.10 mg/kg) on the fourth day after birth, and weanling mice continued on the dams' diet until they were sacrificed. After 5 weeks of feeding, Cu concentrations were dramatically decreased in the heart and the liver of the mice fed the Cu-deficient diet. Corresponding to these changes, serum ceruloplasmin concentrations and hepatic Cu,Zn-superoxide dismutase activities were significantly (P<0.05) depressed. The size of the Cu-deficient hearts was greatly enlarged as estimated from the absolute heart weight and the ratio of heart weight to body weight. The abundances of mRNAs for atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin in left ventricles were all significantly increased in the Cu- deficient hearts. Furthermore, Cu deficiency activated the expression of the c-myc oncogene in the left ventricle. This study thus demonstrated that a molecular program of alterations in embryonic genes, similar to that shown in the work-overloaded heart, was activated in the hypertrophied heart induced by Cu deficiency.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Ceruloplasmin; Copper; Diet; Disease Models, Animal; Gene Expression; Liver; Mice; Myocardium; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Proto-Oncogene Proteins c-myc; Superoxide Dismutase

TGF-beta stimulation of cardiac myocytes induces a hypertrophic responsiveness to beta-adrenoceptor stimulation. This study investigates whether this beta-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC).. Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-beta or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-beta 1.. ODC activity and expression increased within 2 h in TGF-beta 1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (alpha-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-beta no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-beta 1, but not in ventricles from their nontransgenic counterparts.. This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to beta-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-beta. It is concluded that TGF-beta induces a hypertrophic responsiveness to beta-adrenoceptor stimulation that is characterized by ODC induction.

Topics: Adrenergic beta-Agonists; Analysis of Variance; Animals; Animals, Genetically Modified; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Enzyme Activation; Isoproterenol; Male; Myocardium; Ornithine Decarboxylase; Perfusion; Phenylalanine; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Stimulation, Chemical; Transforming Growth Factor beta

We examined the mechanism of atrial natriuretic factor (ANF) transcription by isoproterenol (ISO), an agonist for the beta-adrenergic receptor (betaAR), in cardiac myocytes. ISO only modestly activated members of the mitogen-activated protein kinase family. ISO-induced ANF transcription was not affected by inhibition of mitogen-activated protein kinases, whereas it was significantly inhibited by KN93, an inhibitor of Ca(2+)/calmodulin-dependent kinase (CaM kinase II). Production of 3'-phosphorylated phosphatidylinositides (3 phosphoinositides) was also required for ISO-induced ANF transcription. ISO caused phosphorylation (Ser-473) and activation of Akt through CaM kinase II- and 3 phosphoinositides-dependent mechanisms. Constitutively active Akt increased myocyte surface area, total protein content, and ANF expression, whereas dominant negative Akt blocked ISO-stimulated ANF transcription. ISO caused Ser-9 phosphorylation and decreased activities of GSK3beta. Overexpression of GSK3beta inhibited ANF transcription, which was reversed by ISO. ISO failed to reverse the inhibitory effect of GSK3beta(S9A), an Akt-insensitive mutant. Kinase-inactive GSK3beta increased ANF transcription. Cyclosporin A partially inhibited ISO-stimulated ANF transcription, indicating that calcineurin only partially mediates ANF transcription. These results suggest that both CaM kinase II and 3 phosphoinositides mediate betaAR-induced Akt activation and ANF transcription in cardiac myocytes. Furthermore, betaAR-stimulated ANF transcription is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of GSK3beta.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Gene Expression Regulation; Glycogen Synthase Kinase 3; MAP Kinase Signaling System; Myocardium; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphorylation; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Retroviridae Proteins, Oncogenic; Transcription, Genetic

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Humans; Luciferases; MEF2 Transcription Factors; Mice; Mice, Transgenic; Myocardium; Myogenic Regulatory Factors; Myosin Heavy Chains; NFATC Transcription Factors; Nuclear Proteins; Promoter Regions, Genetic; Rats; Signal Transduction; Transcription Factors

Dietary copper restriction causes heart hypertrophy in animal models. Several studies have indicated that this cardiomyopathy is mediated by oxidative stress. Metallothionein (MT), a low molecular weight and cysteine-rich protein, functions in protecting the heart from oxidative injury. We therefore used a cardiac-specific MT-overexpressing transgenic mouse model to test the hypothesis that MT inhibits copper deficiency-induced heart hypertrophy. Dams of both transgenic pups and non-transgenic littermates were fed a copper-adequate or copper-deficient diet, starting on the fourth day post-delivery, and the weanling mice were continued on the dams' diets until they were killed. Heart hypertrophy developed in copper-deficient pups by the fourth week of the combined pre- and post-weaning feeding and aggressively progressed until the end of the experiment (6 weeks). MT overexpression did not prevent the occurrence of heart hypertrophy, but inhibited the progression of this cardiomyopathy, which correlated with its suppression of cardiac lipid peroxidation. Corresponding to the progression of heart hypertrophy, myocardial apoptosis and atrial natriuretic peptide (ANP) production in the left ventricle were detected in non-transgenic copper-deficient mice; these effects were significantly suppressed in transgenic copper-deficient mice. Measurement of apoptosis by TUNEL assay and Annexin V-FITC confocal microscopy in primary cultures of cardiomyocytes revealed that ANP was largely responsible for the myocyte apoptosis and that MT inhibited ANP-induced apoptosis. The data clearly demonstrate that elevation of MT in the heart inhibits oxidative injury and suppresses the progression of heart hypertrophy in copper deficiency, although it does not block its initiation. The results suggest that MT inhibits the transition from heart hypertrophy to failure by suppressing apoptosis through inhibition of both cardiac ANP production and its apoptotic effect.

Topics: Animals; Antioxidants; Apoptosis; Atrial Natriuretic Factor; Cardiomegaly; Copper; In Situ Nick-End Labeling; Metallothionein; Mice; Mice, Transgenic; Myocardium; RNA, Messenger

The signal transduction mechanisms mediating hypertrophic responses in myocardial cells (MCs) remain uncertain. We investigated the role of the extracellular signal-regulated kinase (ERK) cascade in myocardial cell hypertrophy by the strategy of using the adenovirus-mediated overexpression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which is the upstream activator of ERK. We generated recombinant adenoviruses expressing constitutively active MEK1 (MEK1 EE) and dominant negative MEK1 (MEK1 DN). Overexpression of MEK1 EE in MCs activated ERK1/2 and subsequently induced atrial natriuretic peptide (ANP) mRNA expression. In addition, MEK1 EE overexpression resulted in an increase in cell size and sarcomeric reorganization. In contrast, overexpression of MEK1 DN in MCs inhibited endothelin-1 (ET-1)-, phenylephrine (PE)-, leukemia inhibitory factor (LIF)-, isoproterenol (ISP)-, and mechanical stretch-induced ERK activation and ANP mRNA expression. MEK1 DN overexpression inhibited ET-1-, PE-, LIF-, and ISP-induced increases in cell size and sarcomeric reorganization. Consistent with the observed effects on cellular morphology, overexpression of MEK1 EE resulted in an increase in amino acid incorporation, while overexpression of MEK1 DN inhibited ET-1-, PE-, LIF-, ISP-, and mechanical stretch-induced increases in amino acid incorporation. These results indicate that the ERK cascade plays an important role in the signaling pathway leading to the development of myocardial cell hypertrophy.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Enzyme Activation; Genetic Vectors; Hypertrophy; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardium; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sarcomeres

The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophic response, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present study utilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin-1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with the farnesyltransferase inhibitor BMS-191563 (25 microM) increased the level of unfarnesylated ras in the cytosolic fraction, and caused a concomitant 42 +/- 2% decrease in immunodetectable farnesylated ras in the particulate fraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3H-leucine uptake (80 +/- 10% decrease: n = 6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46 +/- 6% decrease: n = 6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50 +/- 10% decrease (n = 8; P<0.05) of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistent with the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulated extracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependent stimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, the pretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANP and -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression of the natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present study has shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate the hypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the small GTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathways are also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneity in their recruitment.

Topics: Alkyl and Aryl Transferases; Animals; Atrial Natriuretic Factor; Calmodulin; Cardiomegaly; Cells, Cultured; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Farnesyltranstransferase; Gene Expression; Heart; Mitogen-Activated Protein Kinases; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Norepinephrine; Protein Precursors; ras Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sulfonamides

Previous studies from our laboratory and others indicate that contraction-induced mechanical loading of cultured neonatal rat ventricular myocytes produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo, and that these changes occur via the activation of serine-threonine protein kinases. These may include the extracellular regulated protein kinases (ERK1 and ERK2), the c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and one or more isoenzymes of protein kinase C. In this study, we assessed whether one or more of these kinases are activated by stimulated contraction, and whether activation was isoenzyme-specific. Low-density, quiescent cultures of neonatal rat ventricular myocytes were maintained in serum-free medium, or electrically stimulated to contract (3 Hz) for up to 48 h. ERK and JNK activation was assessed by Western blotting with polyclonal antibodies specific for the phosphorylated forms of both kinases. PKC activation was analysed by subcellular fractionation, detergent extraction, and Western blotting using isoenzyme-specific monoclonal antibodies. Stimulated contractile activity produced myocyte hypertrophy, as indicated by increased cell size, a 15+/-5% increase in total protein/DNA ratio, and induction of ANF and beta MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by;two-fold. Subcellular fractionation revealed a time-dependent increase in PKC delta, and to a much lesser extent PKC xi, in a Triton X-100-soluble membrane fraction within 5 min of the onset of stimulated contraction. PKC alpha was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Western; Cardiomegaly; Cells, Cultured; Culture Media, Serum-Free; Electric Stimulation; Electrophoresis, Polyacrylamide Gel; Electrophysiology; Enzyme Activation; Heart Ventricles; Isoenzymes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Myosin Heavy Chains; Promoter Regions, Genetic; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Subcellular Fractions; Tetradecanoylphorbol Acetate; Time Factors; Transfection

Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feed-forward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.

Topics: Age Factors; Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cardiomegaly; Gene Expression Regulation; Mice; Mice, Transgenic; Promoter Regions, Genetic; Ventricular Function

Cardiac hypertrophy and function were studied 6 wk after constriction of the thoracic aorta (TAC) in transgenic (TG) mice expressing constitutively active mutant alpha(1B)-adrenergic receptors (ARs) in the heart. Hearts from sham-operated TG animals and nontransgenic littermates (WT) were similar in size, but hearts from TAC/TG mice were larger than those from TAC/WT mice, and atrial natriuretic peptide mRNA expression was also higher. Lung weight was markedly increased in TAC/TG animals, and the incidence of left atrial thrombus formation was significantly higher. Ventricular contractility in anesthetized animals, although it was increased in TAC/WT hearts, was unchanged in TAC/TG hearts, implying cardiac decompensation and progression to failure in TG mice. There was no increase in alpha(1A)-AR mRNA expression in TAC/WT hearts, and expression was significantly reduced in TAC/TG hearts. These findings show that cardiac expression of constitutively actively mutant alpha(1B)-ARs is detrimental in terms of hypertrophy and cardiac function after pressure overload and that increased alpha(1A)-AR mRNA expression is not a feature of the hypertrophic response in this murine model.

Topics: Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Binding, Competitive; Blood Pressure; Cardiac Myosins; Cardiomegaly; Constriction, Pathologic; Down-Regulation; Heart; Lung; Mice; Mice, Inbred Strains; Mice, Transgenic; Myocardium; Myosin Light Chains; Organ Size; Pressure; Promoter Regions, Genetic; Radioligand Assay; Receptors, Adrenergic, alpha-1; RNA, Messenger; Thrombosis

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and beta(1D)-integrin (beta1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that beta1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) beta1D protein expression was upregulated perinatally; 2) alpha(1)-adrenergic stimulation of cardiac myocytes increased beta1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of beta1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free beta1D cytoplasmic domains inhibited alpha(1)-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of beta1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by alpha(1)-adrenergic stimulation; and 4) blunting of the alpha(1)-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that beta1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Antibodies; Atrial Natriuretic Factor; Cardiomegaly; Cardiotonic Agents; Cell Size; Cytoplasm; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Genes, Reporter; Heart Ventricles; Integrin beta1; Molecular Sequence Data; Muscle Fibers, Skeletal; Muscle, Skeletal; Myocardium; Phenylephrine; Phosphorylation; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction

Activation of the malate/aspartate and alpha -glycerophosphate shuttles (the NADH shuttles) has been identified in glycolytically active newborn myocardium. The goal of this study was to determine if the NADH shuttles and their regulatory genes are activated in hypertrophied myocardium as substrate utilization shifts away from fatty acids and toward glucose and lactate. Capacity of the shuttles was determined in cardiac mitochondria isolated one week, one month, and three months following aortic banding or sham operation. Myocardial steady-state mRNA and protein levels of regulatory enzymes were also measured. Despite a significant increase in left ventricular mass and activation of the atrial natriuretic peptide gene, no change in malate/aspartate nor alpha -glycerophosphate shuttle capacity was found at any of the three time points studied. Reactivation of the genes encoding the regulatory inner mitochondrial membrane proteins was not found in the hypertrophied myocardium, though these genes were down regulated one week following aortic-banding. These results suggest that sufficient malate/aspartate and alpha -glycerophosphate shuttle capacity exists in cardiac mitochondria to accommodate increased shuttle flux as hypertrophied myocardium becomes more glycolytically active.

Topics: Adenosine Triphosphate; Animals; Aorta; Aspartic Acid; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Fatty Acids; Glucose; Glycerophosphates; Immunoblotting; Lactic Acid; Malate Dehydrogenase; Malates; Male; Mitochondria; Models, Biological; Myocardium; NAD; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

RGS family members are GTPase-activating proteins for heterotrimeric Gq and Gi proteins. RGS genes are expressed in heart tissue and in cultured cardiomyocytes. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure.. We investigated the ability of RGS proteins to block G-protein signaling in vivo by using a cultured cardiomyocyte transfection system. Endothelin-1, angiotensin II, and phenylephrine signal through Gq or Gi family members and promote the hypertrophy of cardiomyocytes. We found that phenylephrine-mediated and endothelin-1-mediated induction of the atrial natriuretic factor and myosin light chain-2 genes was inhibited in cells that were transfected with RGS4. Phenylephrine-mediated gene induction was not inhibited in cells that were transfected with N128A-RGS4, a point mutant form that lacks GTPase-activating protein activity. Phenylephrine-mediated myofilament organization and cell growth were also blocked in cells by RGS4.. These results demonstrate that RGS protein can inhibit G-protein-mediated signaling in vivo and suggest that increased expression of RGS protein may be a counterregulatory mechanism to inhibit G protein signaling.

Topics: Actin Cytoskeleton; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Endothelin-1; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Genes, Reporter; GTP-Binding Proteins; Luciferases; Muscle Fibers, Skeletal; Myocardium; Phenylephrine; Point Mutation; Proteins; Rats; Rats, Sprague-Dawley; RGS Proteins; Signal Transduction; Subcellular Fractions; Sympathomimetics; Transcriptional Activation; Transfection

To evaluate the role of angiotensin II (AII) on diastolic function during post-myocardial infarction (MI) ventricular remodeling, coronary ligation or sham operation was performed in male Sprague-Dawley rats. Experimental animals were maintained on either irbesartan, a selective AT1-receptor antagonist, or no treatment. Measurement of cardiac hypertrophy, diastolic function, and sarcoendoplasmic reticulum adenosine triphosphatase (ATPase; SERCA) and phospholamban (PLB) gene expression was assessed at 6 weeks after MI. Myocardial infarction caused a significant increase in myocardial mass and left ventricular (LV) filling pressure, whereas LV systolic pressure and +dP/dt were reduced. The time constant of isovolumic relaxation (tau) was markedly prolonged after MI. Post-MI hypertrophy was associated with substantial increases in the messenger RNA (mRNA) expression of atrial natriuretic peptide (ANP), but no significant changes in SERCA or PLB levels. Although irbesartan treatment did not significantly alter post-MI LV systolic or filling pressures, it nevertheless effectively decreased ventricular hypertrophy, improved tau, and normalized ANP expression. These results demonstrate that AT1-receptor antagonism has important effects on myocardial hypertrophy and ANP gene expression, which are independent of ventricular loading conditions. In addition, the improvement in diastolic function was not related to changes in SERCA and PLB gene expression, suggesting that enhanced myocardial relaxation was related to the blockade of AII effects on myocyte function or through a reduction of ventricular hypertrophy itself or both.

Topics: Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Biphenyl Compounds; Body Weight; Calcium-Binding Proteins; Calcium-Transporting ATPases; Cardiomegaly; Diastole; Gene Expression; Heart Ventricles; Hemodynamics; Hypertrophy; Irbesartan; Male; Myocardial Infarction; Organ Size; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; RNA, Messenger; Tetrazoles

Angiotensin-converting enzyme (ACE) inhibitors have proven an effective means to control hypertension and manage cardiac hypertrophy. It is presently unknown if newer specific angiotensin II subtype 1 receptor (AT1R) antagonists are as effective or more effective in treating these conditions compared with ACE inhibitors. There is evidence that these classes of drugs may affect cardiac hypertrophy by different mechanisms. This study compared the effect of irbesartan, an AT1R antagonist, with that of captopril, an ACE inhibitor, on expression of early genetic markers of cardiac hypertrophy in lean male SHHF/Mcc-fa(cp) rats. SHHF/Mcc-fa(cp) rats (n = 10/group) were given captopril (100 mg/kg/day), irbesartan (50 mg/kg/day), or placebo for 16 weeks. Irbesartan and captopril significantly reduced systolic pressure and produced similar rightward shifts in the angiotensin I dose-response curve. Renal renin gene expression was increased 8.6-fold by irbesartan and 17.7-fold by captopril. The only effect on echocardiographic findings was a similar decrease in aortic peak velocity, an index of systolic function, by both treatments. Early markers of cardiac hypertrophy were significantly attenuated by both drugs. Both drugs produced marked and equivalent reductions in left ventricular atrial natriuretic peptide (ANP) messenger RNA (mRNA) levels compared with controls. This decrease in ANP gene expression was accompanied by a decrease in plasma ANP concentration in the treatment groups. The shift from V1 to V3 myosin isozymes was similarly decreased in both treatment groups, compared with controls. These data suggest that captopril and irbesartan are similarly effective in controlling expression of genes associated with ventricular hypertrophy in heart failure-prone SHHF/Mcc-fa(cp) rat.

Topics: Angiotensin I; Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Biphenyl Compounds; Blood Pressure; Body Weight; Captopril; Cardiomegaly; Dose-Response Relationship, Drug; Echocardiography; Gene Expression; Heart Failure; Irbesartan; Isoenzymes; Male; Myosin Heavy Chains; Organ Size; Rats; Rats, Inbred Strains; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Renin; RNA, Messenger; Systole; Tetrazoles

To clarify the mechanism of cardiac hypertrophy in carnitine-deficient JVS mice, we studied the possible role of catecholamine metabolism. Cardiac hypertrophy occurs 2 weeks after birth. The turnover of norepinephrine in the ventricles of JVS mice at 2 weeks was 3 times that of control, but it was not different from control at 5 days when the heart weight was not changed. To evaluate the accelerated norepinephrine turnover, we examined the effects of catecholamine metabolism inhibitors (alpha-methyltyrosine and 6-hydroxydopamine) and catecholamine receptor blockades (propranolol, prazosin and yohimbine) on the ratio of heart weight to body weight (HW/BW) and on the augmented expression of atrial natriuretic peptide (ANP) and the down-regulated carnitine deficiency-associated gene expressed in ventricle (CDV-1). The HW/BW ratio in JVS mice treated with catecholamine metabolism inhibitors and receptor blockades was significantly lower than in JVS mice without treatment, but still higher than in controls treated with each drug and in JVS mice treated with carnitine. The HW/BW ratio of JVS mice with propranolol was not significantly different from that of JVS mice treated with catecholamine metabolism inhibitors and was significantly lower than that of JVS mice treated with prazosin and yohimbine. Northern blot analysis showed that the altered expression of ANP and CDV-1 was not corrected in the ventricles of JVS mice treated with any of the drugs except carnitine. These results suggest that the catecholamine metabolism accelerated in JVS mice ventricles at 2 weeks is not the major cause of cardiac hypertrophy, but probably promotes cardiac hypertrophy mainly through the beta-adrenergic signaling pathway. The aberrant gene expression of ANP and CDV-1 found in JVS mice seems to be independent of catecholamine metabolism, and mediated primarily by the systemic carnitine deficiency.

Topics: Adrenergic Antagonists; Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Carnitine; Cyclosporine; Disease Models, Animal; Mice; Microtubule-Associated Proteins; Muscle Proteins; Norepinephrine; RNA, Messenger

This study analyzed the regulation and the role of the cardiac steroidogenic system in myocardial infarction (MI).. Seven days after MI, rats were randomized to untreated infarcted group or spironolactone- (20 and 80 mg x kg-1 x d-1), losartan- (8 mg x kg-1 x d-1), spironolactone plus losartan-, and L-NAME- (5 mg x kg-1 x d-1) treated infarcted groups for 25 days. Sham-operated rats served as controls. In the noninfarcted myocardium of the left ventricle (LV), MI raised aldosterone synthase mRNA (the terminal enzyme of aldosterone synthesis) by 2. 0-fold and the aldosterone level by 3.7-fold. Conversely, MI decreased 11beta-hydroxylase mRNA (the terminal enzyme of corticosterone synthesis) by 2.4-fold and the corticosterone level by 1.9-fold. MI also induced a 1.9-fold increase in cardiac angiotensin II level. Such cardiac regulations were completely prevented by treatment of the infarcted heart with losartan. The MI-induced collagen deposition in noninfarcted LV myocardium was prevented by 1.6-fold by both low and high doses of spironolactone and by 2.5-fold by losartan. In addition, norepinephrine level was unchanged in infarcted heart but was attenuated by both losartan and spironolactone treatments.. MI is associated with tissue-specific activation of myocardial aldosterone synthesis. This increase is mediated primarily by cardiac angiotensin II via AT1-subtype receptor and may be involved in post-MI ventricular fibrosis and in control of tissue norepinephrine concentration.

Topics: Aldosterone; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Fibrosis; Gene Expression; Heart; Heart Ventricles; Male; Myocardial Infarction; Myocardium; Norepinephrine; Rats; Rats, Wistar; Steroids

Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Crosses, Genetic; Echocardiography; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Heart; Heterozygote; Humans; Mice; Mice, Transgenic; Myocardium; Receptor Protein-Tyrosine Kinases; Receptors, Adrenergic, beta-2; Signal Transduction; Ventricular Function, Left

Transgenesis has become a useful tool in effecting a complete or partial remodeling of the cardiac contractile apparatus. Although gene dosage effects were initially a concern, recent data showed that the heart is able to accommodate varying levels of transgenic over-expression without detectable ill effects. The present study was designed to test the limits of the transgenic paradigm in terms of the production of a cardiac phenotype due simply to the over-expression of a contractile protein. To this end, eight lines of mice which express an isoform of the essential myosin light chain 1 that is normally found in the adult ventricle (ELC1v) were generated. Overt phenotype was correlated both with the level of expression/protein replacement and copy number of the transgene. Two of the lines showed essentially complete replacement of the atrial isoform (ELC1a) with ELC1v. However, the phenotypes of the two lines differed dramatically. The line with the lower copy number (37 copies), and moderate over-expression (16 fold) showed no overt pathology while a line with very high copy number (94 copies) and extremely high levels of over-expression (27-50 fold) developed a significant atrial hypertrophy, dilation and cardiomyopathy. These data indicate that very high expression levels of a contractile protein can cause a cardiac pathology that is unrelated to its degree of replacement in the sarcomere and the unique role(s) it may assume in motor protein function.

Topics: Actins; Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Base Sequence; Biomarkers; Calcium-Transporting ATPases; Cardiomegaly; Connectin; Gene Dosage; Gene Expression; Heart Atria; Heart Defects, Congenital; Heart Ventricles; Mice; Mice, Transgenic; Molecular Sequence Data; Muscle Proteins; Myocardial Contraction; Myocardium; Myosin Light Chains; Protein Biosynthesis; Protein Isoforms; Protein Kinases; RNA, Messenger; Sequence Homology, Amino Acid; Transcription, Genetic; Ventricular Dysfunction, Left

Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload-induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Blood Pressure; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Enzyme Activation; Gene Expression; Gene Transfer Techniques; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation; Myocardium; Protein Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction

Atrial natriuretic factor (ANF) inhibits proliferation in non-myocardial cells and is thought to be anti-hypertrophic in cardiomyocytes. We investigated the possibility that the anti-hypertrophic actions of ANF involved the mitogen-activated protein kinase signal transduction cascade. Cultured neonatal rat ventricular myocytes treated for 48 h with the alpha(1)-adrenergic agonist phenylephrine (PE) had an 80% increase in cross-sectional area (CSA). ANF alone had no effect but inhibited PE-induced increases in CSA by approximately 50%. The mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD098059 minimally inhibited PE-induced increases in CSA, but it completely abolished ANF-induced inhibition of PE-induced increases. ANF-induced extracellular signal-regulated protein kinase (ERK) nuclear translocation was also eliminated by PD098059. ANF treatment caused MEK phosphorylation and activation but failed to activate any of the Raf isoforms. ANF induced a rapid increase in ERK phosphorylation and in vitro kinase activity. PE also increased ERK activity, and the combined effect of ANF and PE appeared to be additive. ANF-induced ERK phosphorylation was eliminated by PD098059. ANF induced minimal phosphorylation of JNK or p38, indicating that its effect on ERK was specific. ANF-induced activation of ERK was mimicked by cGMP analogs, suggesting that ANF-induced ERK activation involves the guanylyl cyclase activity of the ANF receptor. These data suggest that there is an important linkage between cGMP signaling and the mitogen-activated protein kinase cascade and that selective ANF activation of ERK is required for the anti-hypertrophic action of ANF. Thus, ANF expression might function as the natural defense of the heart against maladaptive hypertrophy through its ability to activate ERK.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Cyclic GMP; Enzyme Activation; Fluorescent Antibody Technique; Mitogen-Activated Protein Kinase Kinases; Myocardium; Phenylephrine; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley

Adrenoreceptor agonists induce a hypertrophic phenotype in vitro and in vivo. To investigate the molecular remodeling in chronic cardiac hypertrophy we infused adult male mice with vehicle. isoproterenol, phenylephrine or both agonists for 3, 7 or 14 days. All drugs increased cardiac mass. After minipump removal cardiac mass regressed to control levels within 7 days after PE and ISO treatment whereas ISO + PE treated hearts were incompletely regressed. ANF and beta-MHC, but not alpha-MHC, expression were increased by agonists at all time points. GATA-4, Nkx-2.5, Egr-1, c-jun and c-fos expression were increased after 3, 7 and 14 days of treatment. Expression was greatest after ISO+PE> >ISO>PE>vehicle infusion suggesting a synergistic effect of adrenoreceptor stimulation and indicating a greater effect of beta- than alpha-adrenergic action in vivo. After PE or ISO drug withdrawal the HW/BW was normal and Egr-1, c-jun, c-fos and GATA-4, but not Nkx2.5, expression dropped to control levels. HW/BW regression was incomplete after ISO+PE and elevated levels of Egr-1, c-jun and Nkx2.5 expression remained. A hydralazine-mediated reduction in blood pressure had no effect on the agonist-induced cardiac hypertrophy or gene expression. In conclusion, we found that continued agonist stimulation, and not blood pressure. is responsible for the maintained increase in gene expression. Further, we found the decrease in gene expression in the regression after drug withdrawal was gene specific.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Cardiomegaly; DNA-Binding Proteins; GATA4 Transcription Factor; Gene Expression Regulation; Genes, Immediate-Early; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Isoproterenol; Male; Mice; Mice, Inbred C57BL; Myosin Heavy Chains; Phenylephrine; Receptors, Adrenergic; Transcription Factors; Xenopus Proteins

Chronic alcohol consumption produces alcoholic heart muscle disease (AHMD), a prevalent form of congestive heart failure. Several hypotheses have been proposed to explain the damaging effects of alcohol on the heart, but neither the mechanism nor the ultimate toxin has been established. In this study, we use transgenic overexpression of alcohol dehydrogenase to elevate cardiac exposure to acetaldehyde, the major and most reactive metabolite of alcohol. Overexpression of alcohol dehydrogenase by 40-fold produced no detectable deleterious effects to the heart in the absence of alcohol. In the presence of alcohol, transgenic hearts contained 4-fold higher acetaldehyde than control hearts. Chronic alcohol exposure produced many changes similar to AHMD in transgenic hearts. Compared with control hearts, these pathological changes occurred more rapidly and to a greater extent: alcohol-exposed transgenic hearts were almost twice as large as control hearts. They demonstrated ultrastructural damage consistent with AHMD and had much lower contractility than alcohol-exposed control hearts. In addition, the transgenic hearts showed greater changes in mRNA expression for alpha-skeletal actin and atrial natriuretic factor than alcohol-exposed control hearts. Alterations in NAD(+)/NADH levels were insufficient to account for such severe damage in cardiomyopathic hearts. The increased damage produced in transgenic hearts suggests an important role for acetaldehyde in AHMD.

Topics: Acetaldehyde; Actins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Alcoholic; Disease Models, Animal; Ethanol; L-Lactate Dehydrogenase; Mice; Mice, Transgenic; Microscopy, Electron; Myocardial Contraction; NAD; RNA, Messenger; Time Factors

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) upregulation are genetic markers for the chronic hypertrophic phenotype but also have important acute physiologic effects on salt and water balance and blood pressure control. The presence of a dual NP-system led us to hypothesize a differential expression of ANP and BNP in response to an acute hemodynamic stress of volume overload in the left ventricle (LV) and right ventricle (RV). Accordingly, we examined the temporal relationship between the RV and LV expression of ANP and BNP mRNA and NP receptor mRNA levels on days 1, 2, 3, and 7 after induction of aortocaval fistula in the rat. LV end-diastolic pressure was increased 1.5-fold by day 3 and 2.0-fold by day 7 compared to control (P<0.05). LV weight increased by day 7 compared to control (2.34+/-0.04 vs 3.07+/-0.10 mg/g, P<0.05) while RV weight did not change over the 7 days. There was a 7-fold increase of ANP mRNA in LV at day 1, which was sustained through day 7, while LV BNP mRNA levels did not differ from controls over the 7 days. In contrast, RV mRNA transcript levels for ANP and BNP were increased >2-fold by day 2 and this increase was sustained throughout 7 days. NP clearance receptor was decreased by 75% by day 7 in the LV but did not change in the RV. Thus, LV ANP mRNA levels increased before the onset of LV hypertrophy and RV BNP mRNA levels increased in the absence of RV hypertrophy. The disparate response of BNP and the NP clearance receptor transcript levels in the LV and RV may be related to differences in load and/or differential expression of the NP system in the LV and RV in response to acute haemodynamic stress.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Gene Expression Regulation; Hemodynamics; Male; Myocardium; Natriuretic Peptide, Brain; Organ Size; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Transcription, Genetic

Neuregulins are a family of growth-promoting peptides known to be important in neural and mesenchymal tissue development. Targeted disruption of neuregulin (NRG)-1 or one of two of its cognate receptors, ErbB2 or ErbB4, results in embryonic lethality because of failure of the heart to develop. Although expression of NRGs and their receptors declines after midembryogenesis, both ErbB2 and ErbB4 are present in cardiac myocytes, and NRG-1 expression remains inducible in primary cultures of coronary microvascular endothelial cells from adult rat ventricular muscle. In neonatal rat ventricular myocytes, a soluble NRG-1, recombinant human glial growth factor-2, increased [(3)H]phenylalanine uptake and induced expression of atrial natriuretic factor (ANF) and sarcomeric F-actin polymerization. The effect of NRG-1 on [(3)H]phenylalanine uptake and sarcomeric F-actin polymerization was maximal at 20 ng/ml but declined at higher concentrations. NRG-1 activated p42/p44 mitogen-activated protein kinase (MAPK) [extracellular signal-regulated kinase (ERK)-2/ERK1] and ribosomal S6 kinase (RSK)-2 (90-kDa ribosomal S6 kinase), both of which could be inhibited by the MAPK/ERK kinase-1 antagonist PD-098059. NRG-1 also activated 70-kDa ribosomal S6 kinase, which was inhibited by either rapamycin or wortmannin. Activation of these pathways exhibited the same "biphasic" response to increasing NRG-1 concentrations. Wortmannin and LY-294002 blocked sarcomeric F-actin polymerization but not [(3)H]phenylalanine uptake or ANF expression, whereas PD-098059 consistently blocked both [(3)H]phenylalanine uptake and ANF expression but not actin polymerization. In contrast, rapamycin inhibited [(3)H]phenylalanine uptake and F-actin polymerization but not ANF expression. Thus NRG-ErbB signaling triggers multiple nonredundant pathways in postnatal ventricular myocytes.

Topics: Actins; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Heart Ventricles; Humans; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardium; Neuregulin-1; Phosphatidylinositol 3-Kinases; Protein Precursors; Rats; Recombinant Proteins; Ribosomal Protein S6 Kinases; Sarcomeres

This study was designed to examine the effects of interleukin-1 beta (IL-1 beta) on myocyte (MC) hypertrophy and the production of A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in rat ventricular cardiocyte culture, and to investigate the role of nonmyocyte (NMC) in this process. We examined the effects of IL-1 beta on the production of ANP and BNP in comparison with the effects of endothelin-1 (ET-1) by using two types of neonatal rat cardiocyte culture; MC-enriched culture and MC-NMC coculture. In the MC-enriched culture, the increase in secretion of ANP and BNP was small in treatment with IL-1 beta (1000 pg/ml), while ET-1 (10 nM) markedly augmented the secretion of ANP and BNP. In the MC-NMC coculture, IL-1 beta and ET-1 each significantly augmented the secretion of ANP and BNP. The degree of the increase of ANP and BNP was equivalent between IL-1 beta and ET-1. As for the morphological changes of MCs, IL-1 beta induced the star-shaped MC hypertrophy characterized by elongation and pointed edges only in the MC-NMC coculture, while ET-1 induced the MC hypertrophy characterized by shapes of squares, triangles or circles in both cultures. This study shows that IL-1 beta induces unique cardiac hypertrophy and the marked secretion of ANP and BNP, and that NMC is indispensable when treated with IL-1 beta.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin-1; Gene Expression Regulation; Heart Ventricles; Interleukin-1; Kinetics; Myocardium; Natriuretic Peptide, Brain; Rats; Rats, Wistar

Cardiac hypertrophy is a fundamental adaptive response to hemodynamic overload; how mechanical load induces cardiac hypertrophy, however, remains elusive. It was recently reported that activation of a calcium-dependent phosphatase, calcineurin, induces cardiac hypertrophy. In the present study, we examined whether calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy.. Pressure overload produced by constriction of the abdominal aorta increased the activity of calcineurin in the rat heart and induced cardiac hypertrophy, including reprogramming of gene expression. Treatment of rats with a calcineurin inhibitor, FK506, inhibited the activation of calcineurin and prevented the pressure overload-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Load-induced expression of immediate-early-response genes and fetal genes was also suppressed by the FK506 treatment.. The present results suggest that the calcineurin signaling pathway plays a pivotal role in load-induced cardiac hypertrophy and may pave the way for a novel pharmacological approach to prevent cardiac hypertrophy.

Topics: Animals; Aorta, Abdominal; Atrial Natriuretic Factor; Blood Volume; Body Weight; Calcineurin; Calcineurin Inhibitors; Cardiomegaly; Constriction, Pathologic; Disease Models, Animal; Echocardiography; Fibrosis; Gene Expression; Genes, Immediate-Early; Heart Rate; Immunosuppressive Agents; Male; Myocardium; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Signal Transduction; Tacrolimus

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Male; Mice; Mice, Transgenic; Monosaccharide Transport Proteins; Muscle Proteins; Myocardial Contraction; Myocardium; Natriuretic Peptide, Brain; Organ Size

To study the effects of long-term treatment with the type 1 angiotensin (AT1) receptor antagonist losartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril, on cardiac adrenomedullin (ADM), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression.. Spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were given losartan (15 mg/kg per day) or enalapril (4 mg/kg per day) orally for 10 weeks. The effects of drugs on systolic blood pressure, cardiac hypertrophy, ANP, BNP and ADM mRNA and immunoreactive-ANP (IR)-ANP, IR-BNP and IR-ADM levels in the left ventricle and atria were compared.. Losartan and enalapril treatments completely inhibited the increase of systolic blood pressure occurring with ageing in SHR. The ratio of heart to body weight was reduced in both losartan- and enalapril-treated SHR and WKY rats. Treatment with losartan or enalapril reduced left ventricular ANP mRNA and IR-ANP in both strains, and ventricular BNP mRNA levels in SHR rats. Inhibition of ACE, AT1 receptor antagonism, changes in blood pressure or cardiac mass had no effect on left ventricular ADM gene expression in SHR and WKY rats. In addition, atrial IR-ANP and IR-ADM levels increased in SHR whereas IR-BNP levels decreased in WKY and SHR rats in response to drug treatments.. Our results show that ventricular ADM synthesis is an insensitive marker of changes in haemodynamic load or cardiac hypertrophy. Furthermore, the expression of ADM, ANP and BNP genes is differently regulated both in the left ventricle and atria in response to AT1 receptor antagonism and ACE inhibition.

Topics: Adrenomedullin; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Enalapril; Gene Expression; Heart; Hypertension; Losartan; Male; Natriuretic Peptide, Brain; Peptides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Reference Values

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase, calcineurin, as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in cardiac hypertrophy. The aim of the present study was to test whether calcineurin is involved in the signal transduction of angiotensin II (AngII)-induced cardiac myocyte hypertrophy and fibroblast hyperplasia. Firstly, we observed that calcineurin activity was significantly increased in AngII-stimulated cardiac myocytes as well as fibroblasts, but was markedly inhibited by Losartan (50 micromol/l), H7 (50 micromol/l), and Fura-2/AM (5 micromol/l). It is indicated that AngII-induced activation of calcineurin is through an ATI receptor, may be dependent on the sustained increases of [Ca2+]i, and be regulated by protein kinase C. In a second experiment, we found that cyclosporin (0.1-10micromol/l), a specific inhibitor of calcineurin, decreased the protein synthesis rate in AngII-stimulated cardiomyocytes and the DNA synthesis rate in AngII-treated fibroblasts in a dose-dependent manner. In the latter experiment, calcineurin inhibition reduced the mRNA level of the atrial natriuretic factor gene. These results indicate that calcineurin is involved in the signal transduction of AngII-induced cardiomyocyte hypertrophy and fibroblast hyperplasia.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcineurin; Calcineurin Inhibitors; Calcium; Cardiomegaly; Cell Division; Cells, Cultured; Cyclosporine; DNA; Fibroblasts; Fura-2; Hyperplasia; Losartan; Myocardium; Protein Kinase C; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; RNA, Messenger; Signal Transduction

Transgenic mice with a dysfunctional guanylyl cyclase A gene (GCA -/-) are unable to transduce the signals from atrial naturetic peptide and develop hypertension and cardiac hypertrophy. Magnetic resonance imaging (MRI) was performed to assess cardiac hypertrophy in these animals, using wild-type siblings as controls. Anesthetized mice were studied by gated multislice, multiphase cine MRI at 1.5 T. Simpson's rule was used to estimate left ventricle (LV) mass and volumes from short-axis images. Correlation between LV mass evaluated by MRI and at necropsy was excellent, with LVnecropsy = 1.04 x LVMRI + 4.69 mg (r2 = 0.95). By MRI, GCA -/- LV mass was significantly different when compared with isogenic controls [GCA -/-, 226 +/- 43 mg (n = 14) vs. controls, 156 +/- 14 mg (n = 10); P < 0.0001]. LV volumes and ejection fraction in the two groups were not significantly different. MRI provides an accurate means for the noninvasive assessment of murine cardiac phenotype and may be useful in following the effects of genetic modification.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; Guanylate Cyclase; Heart Ventricles; Hypertension; Magnetic Resonance Imaging; Mice; Mice, Transgenic; Mutation; Signal Transduction

The hypertrophic response is characterized by increased myofibril/sarcomere organization, induction of the cardiac specific atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2v) genes, and an increase in total cell volume. The alpha1-adrenergic agonist phenylephrine induces both the morphological and biochemical markers of hypertrophy in cultured neonatal rat ventricular cardiomyocytes. Previous studies have suggested a functional requirement for the heterotrimeric G-protein, Galphaq, for a subset of the hypertrophic phenotypes. The small GTPases Ras and Rho have also been implicated in phenylephrine-induced hypertrophy. To further delineate the role of Galphaq in hypertrophy, a constitutively active mutant of Galphaq was transiently transfected in primary rat ventricular cardiomyocytes. This molecule was sufficient to induce ANF-, AP1-, and MLC-2-driven gene expression. Co-transfection of Galphaq and dominant negative Ras or dominant negative Raf resulted in dose-dependent inhibition of ANF-driven expression. Both dominant negative Rho, and the Rho inhibitor C3-transferase, also attenuated Galphaq- and Ras-induced ANF-driven gene expression. Cells transfected with active Galphaq did not show a detectable increase in activation of the mitogen activated protein kinases ERK or SAPK. However, activity of the MAP-kinases appears to be important for Galphaq-induced gene expression since the MAP-kinase phosphatase Clone 100 and catalytically inactive SAPK strongly inhibited Galphaq-induced ANF expression. Thus, our studies indicate Galphaq-induced hypertrophic gene expression requires the small G-proteins Ras and Rho. The data also indicates that Galphaq mediated gene expression is dependent on functional MAP-kinases and that multiple signaling pathways contribute to Galphaq-mediated cardiac cell hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiac Myosins; Cardiomegaly; Cells, Cultured; Enzyme Inhibitors; Flavonoids; Gene Expression; GTP-Binding Proteins; Myocardium; Myosin Light Chains; Promoter Regions, Genetic; ras Proteins; Rats; Rho Factor; Signal Transduction; Transcription Factor AP-1; Transfection

Cardiac remodeling secondary to myocardial infarction is associated with hypertrophy of surviving myocardium and altered cardiac gene expression. The present study examined the spatiotemporal expression of cardiac contractile protein and peptide hormone mRNA following left ventricular myocardial infarction (LVMI) in the rat heart.. LVMI was produced in Wistar rats by ligation of the left anterior descending coronary artery and mRNA levels of cardiac alpha-action (sACT), ventricular myosin light chain-2(MLC-2v), beta-myosin heavy chain (beta-MHC) and pre-proatrial natriuretic peptide (ppANP) were examined at 24 h, 1 and 4 weeks) post-LVMI by in situ hybridization histochemistry with 35S-labeled oligonucleotide probes.. Infarct size, determined at 1 week post-LVMI, was 44.5 +/- 2.7% of the combined left ventricular epi- and endocardial surface area. Myocyte fiber width, reflecting cellular hypertrophy, was increased in left ventricular, mid-septal and mid-right ventricular muscle fibers by 11-20% at 1 week post-LVMI (P < 0.05) and by 24-29% at 4 weeks (P < 0.05). At 24 h, 1 and 4 weeks post-LVMI, heart- and lung/body weight ratios were significantly elevated compared to sham-operated rats (1.3-1.8-fold, P < 0.01 and 1.6-2.9-fold, P < 0.005, respectively). PpANP mRNA levels in the left ventricle were increased 3.8- and 3.3-fold at 1 and 4 weeks (P < 0.05), with highest levels in the epicardium, papillary muscle, infundibulum and apex of the chamber. Septal and right ventricular ppANP mRNA levels were highest at 24 h post-LVMI (2.1- and 2.6-fold increase, P < 0.05) and remained elevated at 4 weeks, with maximum levels at the left endocardial surface of the septum and apex of the chambers. Atrial levels of cACT mRNA were increased 1.9-fold at 1 week post-LVMI (P < 0.05) and remained elevated at 4 weeks. Skeletal ACT mRNA, not normally expressed in the adult rat heart, was induced as early as 24 h post-LVMI in both atria, the septum and right ventricle, with discrete hybridization signal detected at the apex of the chambers and in the right ventricular free-wall, and later (1 week) in the left ventricular epicardium. MLC-2v mRNA levels were unaltered post-LVMI, except for a transitory loss of expression at 24 h in the left atria, ventricle and apical septum. In contrast, ventricular beta-MHC mRNA was markedly induced in regions containing increased ppANP mRNA, with a maximal 3.0- and 4.0-fold induction (P < 0.05) seen at 1 and 4 weeks in the left ventricle and a 3.7-fold induction at 4 weeks in the septum and right ventricle (P < 0.05).. The regional increases in induced cardiac hormone and contractile protein mRNA in similar subchamber regions of the rat heart post-LVMI implies mutual activation by mechanical and/or neuroendocrine stimuli in the transcriptional response to myocardial overload.

Topics: Actins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; In Situ Hybridization; Myocardial Infarction; Myocardium; Myosin Heavy Chains; Myosin Light Chains; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger

Increased ventricular expression of several genes, including atrial natriuretic factor (ANF), has been documented in experimental models of cardiac hypertrophy. It remains to be clarified whether altered expression of these genes is a consistent marker of the hypertrophy itself or a marker of some parallel pathogenetic process. Using a transgenic mouse model of hypertrophic cardiomyopathy as a tool, we assessed the relationship between the amount of ventricular ANF gene expression and the degree of hypertrophy as well as the relationship between the cells expressing ANF and tissue pathology. We determined that hypertrophy is not always associated with increased ventricular expression of ANF and that cells expressing ANF are found in regions of tissue pathology. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Cytoplasmic Granules; Disease Models, Animal; Female; Fibrosis; Gene Expression; Heart Ventricles; Mice; Mice, Transgenic; Models, Genetic

In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. We show that cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 interacts with the cardiac zinc finger transcription factor GATA4, resulting in synergistic activation of cardiac transcription. Transgenic mice that express activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure that mimic human heart disease. Pharmacologic inhibition of calcineurin activity blocks hypertrophy in vivo and in vitro. These results define a novel hypertrophic signaling pathway and suggest pharmacologic approaches to prevent cardiac hypertrophy and heart failure.

Topics: Angiotensin II; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Cell Nucleus; DNA-Binding Proteins; GATA4 Transcription Factor; Immunosuppressive Agents; Mice; Mice, Transgenic; Myocardium; Natriuretic Peptide, Brain; NFATC Transcription Factors; Nuclear Proteins; Phenylephrine; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Signal Transduction; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Zinc Fingers

c-Jun N-terminal protein kinase (JNK) and p38, two distinct members of the mitogen-activated protein (MAP) kinase family, regulate gene expression in response to various extracellular stimuli, yet their physiological functions are not completely understood. In this report we show that JNK and p38 exerted opposing effects on the development of myocyte hypertrophy, which is an adaptive physiological process characterized by expression of embryonic genes and unique morphological changes. In rat neonatal ventricular myocytes, both JNK and p38 were stimulated by hypertrophic agonists like endothelin-1, phenylephrine, and leukemia inhibitory factor. Expression of MAP kinase kinase 6b (EE), a constitutive activator of p38, stimulated the expression of atrial natriuretic factor (ANF), which is a genetic marker of in vivo cardiac hypertrophy. Activation of p38 was required for ANF expression induced by the hypertrophic agonists. Furthermore, a specific p38 inhibitor, SB202190, significantly changed hypertrophic morphology induced by the agonists. Surprisingly, activation of JNK led to inhibition of ANF expression induced by MEK kinase 1 (MEKK1) and the hypertrophic agonists. MEKK1-induced ANF expression was also negatively regulated by expression of c-Jun. Our results demonstrate that p38 mediates, but JNK suppresses, the development of myocyte hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cardiotonic Agents; Endothelin-1; Enzyme Inhibitors; Growth Inhibitors; Heart; Imidazoles; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Leukemia Inhibitory Factor; Lymphokines; Mitogen-Activated Protein Kinases; Myocardium; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Rats

Treatment of spontaneously hypertensive rats (SHR) with captopril (100 mg . kg-1 . day-1) throughout development and during the first 16 wk of life leads to a reduction in blood pressure and left ventricular hypertrophy. Blood pressures and hypertrophy are reduced in these animals (vs. untreated SHR) for up to 24 wk after discontinuation of the drug. We used conventional blot hybridization and Western analysis to examine hypertrophy-dependent gene expression during this period. Ventricular expression of the atrial natriuretic peptide gene was reduced by >90% at 16 wk of age in the captopril-treated SHR. Expression increased in the 24 wk after discontinuation of treatment, but remained well below that of the untreated SHR. A similar reduction in ventricular c-myc gene expression was seen with captopril treatment. Neither renal expression of the atrial natriuretic peptide gene nor ventricular expression of the c-fos gene was affected by captopril. This study demonstrates that captopril treatment during a critical period of development in the SHR leads to a sustained reduction in hypertrophy-dependent myocardial gene expression, which does not revert to levels seen in the untreated SHR after discontinuation of the drug.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Captopril; Cardiomegaly; Gene Expression; Genes, fos; Genes, myc; Heart; Hypertension; Kidney; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors; Ventricular Function

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.

Topics: Actins; Atrial Natriuretic Factor; Calcium; Calmodulin; Cardiomegaly; Cell Division; Cells, Cultured; Flavonoids; Gene Expression Regulation; Genes, ras; Heart; Mitogen-Activated Protein Kinase 1; Ouabain; Protein Kinase C; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Tetradecanoylphorbol Acetate

Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that beta1 integrin function is involved in the alpha1-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti-beta1 integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of beta1 integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed beta1A integrin or the cardiac/skeletal muscle-specific beta1 isoform (beta1D) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free beta1 integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.

Topics: Adenoviridae; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Extracellular Matrix; Gene Expression Regulation, Viral; Heart Ventricles; Integrin beta1; Isomerism; Myocardium; Phenylephrine; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Signal Transduction; Transfection

1. Adrenomedullin is a recently discovered vasodilating and natriuretic peptide whose physiological and pathophysiological roles remain to be established. Like atrial natiuretic peptide adrenomedullin is expressed in the left ventricle. Ventricular expression of atrial natriuretic peptide is known to be markedly increased by volume or pressure overload. In this study we investigated whether ventricular expression of adrenomedullin is similarly stimulated under such conditions. 2. Ventricular adrenomedullin and atrial natriuretic peptide mRNA levels as well as those of a loading control mRNA (glyceraldehyde-3-phosphate dehydrogenase) were quantified by Northern blot analysis in (a) rats with severe post-infarction heart failure induced by left coronary ligation at 30 days post-surgery and (b) in rats with pressure-related cardiac hypertrophy induced by aortic banding at several time points (0.5, 1 and 4 h, and 1, 4, 7 and 28 days) after surgery. Levels were compared with those in matched sham-operated controls. 3. The mRNA level of atrial natriuretic peptide was markedly increased (8-10-fold) in the left ventricle of animals with post-infarction heart failure. In contrast, there was only a modest (40%) increase in the level of adrenomedullin mRNA. In rats with pressure-induced cardiac hypertrophy the ventricular level of atrial natriuretic peptide mRNA was again markedly increased (maximum 10-fold). The increase was first noticeable at 24 h post-banding and persisted until 28 days. In contrast, there was no change in adrenomedullin mRNA level compared with sham-operated rats at any time point. 4. Despite having similar systemic effects, the expression of adrenomedullin and atrial natriuretic peptide in the left ventricle is differently regulated. The findings imply distinct roles for the two peptides. The results do not support an important role for ventricular adrenomedullin expression in the remodelling process that occurs during the development of cardiac hypertrophy but suggest that ventricular adrenomedullin participates in the local and/or systemic response to heart failure.

Topics: Adrenomedullin; Animals; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Gene Expression Regulation; Heart Failure; Heart Ventricles; Male; Myocardium; Peptides; Rats; Rats, Inbred WKY; Rats, Wistar; RNA, Messenger

To investigate potential therapeutic effects of atrial natriuretic peptide (ANP) gene delivery on renal and cardiac disorders, adenovirus harboring the human ANP gene (Ad.RSV-cANP) was delivered into Dahl salt-sensitive (DSS) rats on a high-salt diet. A single intravenous injection of the ANP gene caused a significant delay of blood pressure increase 3 days post-injection and the effect lasted for more than 5 weeks. A maximal blood pressure reduction of 32.8 mmHg was observed after ANP gene delivery, as compared with that of control rats injected with Ad.CMV-LacZ. Immunoreactive human ANP can be detected in the heart, lung, and kidney of rats after gene delivery. ANP gene delivery caused significant increases in renal blood flow, glomerular filtration rate, sodium output, urine excretion, and urinary cGMP levels. These beneficial effects were reflected morphologically by a reduction in cardiomyocyte size, attenuation of the glomerular-sclerotic lesions, tubular injury and arterial thickening. This study demonstrated the usefulness of somatic gene transfer as a new tool for ANP gene delivery in studying salt-related hypertension and renal and cardiovascular diseases. In addition, the findings also suggest that ANP gene delivery may have potential in therapeutic applications.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Cardiomegaly; Genetic Therapy; Genetic Vectors; Glomerular Filtration Rate; Hypertension; Kidney; Male; Rats; Rats, Inbred Strains; Regional Blood Flow; Sodium, Dietary

Cardiac hypertrophic and contractile responses were studied in mice administered growth hormone (GH) and insulin-like growth factor (IGF-I) (8 mg . kg-1 . day-1), alone or in combination (IGF-I/GH), for 2 wk. Also, changes in expression of selected left ventricular (LV) genes in response to IGF-I/GH were compared with those in other forms of cardiac hypertrophy. GH or IGF-I alone at three to four times the usual dose in rats failed to produce increases in heart and LV weights and hemodynamic effects; however, IGF-I/GH was synergistic, increasing body weight and LV weights by 39 and 35%, respectively. A measure of myocardial contractility (maximal first derivative of LV pressure, catheter-tip micromanometry) was increased by 34% in the IGF/GH group, related in part to a force-frequency effect, since the heart rate increased by 21%. Other mice were treated surgically to produce pressure overload (transverse aortic constriction) or volume overload (arteriovenous fistula) for 2 wk; LV weights were then matched to those in the IGF-I/GH group, and mRNA levels of selected markers were assessed. In contrast to the increased mRNA levels of atrial natriuretic factor, alpha-skeletal actin, and collagen III generally observed in overloaded hearts, changes in IGF-I/GH-treated mice were not significant. Thus high-dose IGF-I/GH produce cardiac hypertrophy and a positive inotropic effect without causing significant changes in expression of fetal and other selected myocardial genes, suggesting that this hypertrophy may be of a more physiological type than that due to mechanical overload.

Topics: Actins; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Collagen; Female; Gene Expression Regulation; Heart; Heart Rate; Heart Ventricles; Hemodynamics; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Mice; Mice, Inbred C57BL; Myocardium; Rats; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Ventricular Function, Left

Adult rat cardiomyocytes in long-term culture reexpress several fetal cardiac proteins which also reappear during overload heart hypertrophy in vivo. IGF-I decreases reexpression of some of these proteins and stimulates myofibrillogenesis. IGF-I might therefore contribute to enhancing readaptation of the heart to overload. In order to test this hypothesis, hypertension was induced in male Wistar Kyoto rats by constriction of the left renal artery, and an infusion of 500 microg/day of recombinant human IGF-I (rhIGF-I) or vehicle was started after the operation via intraabdominally implanted osmotic minipumps. In the vehicle-treated hypertensive animals body weight gain was reduced after 3, 7 and 14 days, whereas rhIGF-I-treated hypertensive animals continued to gain weight like sham-operated animals. Left ventricular weight and the left, but not the right ventricle/body weight ratio increased more in rhIGF-I- than in vehicle-infused rats. Left ventricular IGF-I mRNA levels remained unchanged after renal clipping in both vehicle- and rhIGF-I-treated rats. However, beta-myosin heavy chain (MHC) mRNA in the left ventricle was 6- to 10-fold increased in clipped controls during the whole postoperative period, and rhIGF-I reduced this increase by more than 50% on days 7 and 14. On the first postoperative day, rhIGF-I prevented the decrease (50%) of alpha-MHC mRNA and the increase (2.5-fold of atrial natriuretic factor mRNA in the left ventricle. Renal clipping did not alter cardiac alpha-actin, but enhanced skeletal alpha-actin mRNA expression in the left ventricle up to 2.5-fold. However, both mRNAs were unaffected by rhIGF-I treatment. Restoration of body weight gain and stimulation of left ventricular cardiac weight by rhIGF-I as well as partial reversion of hypertension-induced changes in cardiac protein expression may reflect beneficial effects contributing to enhance readaptation of the heart to overload.

Topics: Actins; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Growth; Heart; Heart Rate; Humans; Insulin-Like Growth Factor I; Male; Myocardium; Myosin Heavy Chains; Rats; Rats, Inbred WKY; Recombinant Proteins; RNA, Messenger

gp130, a signal transducer of the IL-6-related cytokines, is expressed ubiquitously, including in the heart. The activation of gp130 in cardiac myocytes was reported to induce myocardial hypertrophy. The downstream side of gp130 consists of two distinct pathways in cardiac myocytes, one a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, the other a mitogen-activated protein kinase (MAPK) pathway. In the present study, we examined whether the JAK/STAT pathway, especially the STAT3-mediated pathway, plays a critical role in gp130-dependent myocardial hypertrophy by transfecting wild-type and mutated-type STAT3 cDNA to cardiac myocytes.. We constructed three kinds of replication-defective adenovirus vectors carrying wild-type (AD/WT) or mutated-type (AD/DN) STAT3 cDNA or adenovirus vector itself (AD). Cultured murine cardiac myocytes infected with adenovirus were stimulated with leukemia inhibitory factor (LIF), and the expression of c-fos and atrial natriuretic factor (ANF) mRNAs and [3H]leucine incorporation were examined. There were no significant differences in MAPK activity among the three groups. Compared with AD-transfected cardiac myocytes, induction of c-fos and ANF mRNAs and protein synthesis after LIF stimulation were significantly augmented in AD/WT-transfected cells. In contrast, induction of c-fos and ANF mRNA expression and protein synthesis were attenuated after LIF stimulation in cardiac myocytes transfected with AD/DN.. These results suggest that the STAT3-dependent signaling pathway downstream of gp 130 promotes cardiac myocyte hypertrophy under stimulation with LIF.

Topics: Adenoviridae; Animals; Antigens, CD; Atrial Natriuretic Factor; Cardiomegaly; Cytokine Receptor gp130; DNA-Binding Proteins; Genetic Vectors; Growth Inhibitors; Interleukin-6; Leucine; Leukemia Inhibitory Factor; Lymphokines; Membrane Glycoproteins; Mice; Mutation; Myocardium; Phosphorylation; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transfection; Tyrosine

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.

Topics: Adenoviridae; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cell Size; Cells, Cultured; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Transfer Techniques; GTP Phosphohydrolases; GTP-Binding Proteins; Myocardium; Phenylephrine; Protein Biosynthesis; Proto-Oncogene Proteins c-raf; rac GTP-Binding Proteins; Rats; Rats, Sprague-Dawley; Sarcomeres; Signal Transduction

Chronic, complete AV block (CAVB) in the dog leads to ventricular hypertrophy, which has been described as an independent risk factor for arrhythmias. In this model, we examined (1) whether the short- and long-term electrical adaptations predispose to acquired torsade de pointes arrhythmias (TdP) and (2) the nature of the structural and functional adaptations involved.. We determined (1) endocardial right (RV) and left (LV) ventricular APD, DeltaAPD (LV APD-RV APD), presence of EADs at 0 weeks (acute: AAVB), and CAVB (6 weeks) and inducibility of TdP by pacing and d-sotalol (n=10); (2) steady-state and dynamic LV hemodynamics at 0 and 6 weeks (n=6); (3) plasma neurohumoral levels in time (n=7); (4) structural parameters of the LV and RV of CAVB dogs (n=6) compared with sinus rhythm (SR) dogs (n=6); and (5) expression of ventricular mRNA atrial natriuretic factor (ANF) in CAVB (n=4) and SR (n=4) dogs. Compared with AAVB, CAVB led to nonhomogeneous prolongation of LV and RV APD and different sensitivity for d-sotalol, leading to EADs (4 of 14 versus 9 of 18, P<0.05), increased DeltaAPD (45+/-30 versus 125+/-60 ms, P<0.05), and induction of TdP in most dogs (0% versus 60%, P<0.05). CAVB led to biventricular hypertrophy, whereas LV function was similar in AAVB and CAVB. The neurohumoral levels were transiently elevated. The LV and RV collagen and the capillary/fiber ratio remained normal, whereas ventricular ANF mRNA was not detectable.. The electrical remodeling occurring after CAVB predisposes the heart to acquired TdP, whereas the structural changes (hypertrophy) are successfully aimed at maintaining cardiac function.

Topics: Action Potentials; Adaptation, Physiological; Animals; Atrial Natriuretic Factor; Cardiomegaly; Chronic Disease; Coronary Vessels; Disease Models, Animal; Dogs; Electrocardiography; Electrophysiology; Female; Fibrosis; Gene Expression; Heart Block; Heart Ventricles; Hemodynamics; Male; Norepinephrine; Organ Size; RNA, Messenger; Torsades de Pointes

Our objectives were (i) to evaluate the expression of several genes involved in the remodelling of cardiac extracellular matrix (ECM), with a special interest on SPARC (secreted protein acidic and rich in cysteine) a glycoprotein with anti-adhesive properties, and (ii) to characterise structural changes in the left (LV) and right (RV) ventricles of rats subjected to continuous beta-adrenergic stimulation. The rats were infused for 3 or 7 days with isoproterenol (ISO, 4 mg/kg/day) or vehicle. Hybridisation analysis was done for SPARC, atrial natriuretic peptide (ANP),alpha2 (I) [COL-I] and alpha1 (III) [COL-III] procollagens, TGF-beta1 and TGF-beta3 mRNA content. Interstitial and perivascular collagen deposition in both ventricles was measured after specific staining. The mean cross-sectional area of LV cardiomyocytes was evaluated by quantitative histomorphometry. ISO provoked an increase of LV mass, and a progressive enlargement of cardiomyocytes: their cross-sectional area raised from 205+/-8 micrometer2 in vehicle-treated animals to 247+/-4 and 296+/-9 micrometer2 after 3 or 7 days of ISO infusion, respectively (P<0.001). SPARC messenger abundance increased by more than 50% in LV and RV, a first evidence of its expression in the myocardium of adult rats. Transcripts of ANP, COL-III, TGF-beta1 and TGF-beta3 increased in both ventricles. COL-I transcript increased in LV (75 and 116% on days 3 and 7), but not in RV. In LV, collagen accumulated in the interstitium (2.69+/-0.20v 9. 23+/-0.50% of tissue area for vehicle and ISO 7 days groups, P<0.05) and around coronary arteries (1.04+/-0.11v 4.47+/-0.48% of lumen area for vehicle and ISO 7 days,P<0.05). Cardiac fibrosis was less marked in RV. In conclusion, early expression of SPARC, an anti-adhesive protein, and preferential expression of COL-III, a distensible form of collagen, should increase ECM plasticity and facilitate ventricular remodelling.

Topics: Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; Collagen; Extracellular Matrix; Fibrosis; Gene Expression Regulation; Heart; Heart Ventricles; Isoproterenol; Male; Myocardium; Osteonectin; Rats; Rats, Wistar; Transforming Growth Factor beta; Up-Regulation

Prolonged stimulation of cardiac alpha1-adrenergic receptors causes myocyte hypertrophy, although the receptor subtypes involved remain controversial. We have used a potent and selective alpha1A agonist, A-61603, to test whether activation of the alpha1A-adrenergic receptor subtype is sufficient to mediate the morphological, biochemical and molecular alterations associated with cardiomyocyte hypertrophy. In neonatal rat cardiomyocyte cultures, 48 h incubation with 50 nm A-61603 led to a marked increase in myocardial cell size that was associated with a significant elevation in the rate of protein synthesis. The increased rate of incorporation of radiolabelled amino acids into protein stimulated by A-61603 was totally abolished by the selective alpha1A antagonist KMD-3213. A-61603 increased ANF secretion three-fold, and ANF mRNA 12-fold above control levels in cardiomyocyte cultures. RNase protection analysis demonstrated a A-61603-mediated two to three-fold increase in alpha1A-adrenergic receptor mRNA with a concomitant 50% decrease in alpha1B mRNA levels by 48 h. Identical responses of differential regulation of alpha1A- and alpha1B-adrenergic receptor mRNA were observed with phenylephrine. Both the stimulation of alpha1A- and repression alpha1B-adrenergic receptor mRNA caused by A-61603 could be abolished by 10-20 nm KMD-3213. The present data provide evidence that selective activation of alpha 1A-adrenergic receptors on cardiomyocytes is sufficient to mediate the phenotypic changes associated with cardiac hypertrophy. In addition, the differential regulation of alpha1A and alpha1B mRNA in response to selective alpha1A-adrenergic receptor stimulation suggests that cross-talk between receptor subtypes may be involved in regulating receptor populations during chronic agonist exposure.

Topics: Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Heart; Imidazoles; Indoles; Myocardium; Propranolol; Proteins; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-1; RNA, Messenger; Tetrahydronaphthalenes

In previous studies we demonstrated that in normotensive rats, but not in spontaneously hypertensive rats (SHR), atrial natriuretic peptide (ANP) enhances bradycardic reflexes through an action on cardiac vagal afferent pathways. The present study aimed to determine whether cardiac hypertrophy, hypertension, or a nonreversible genetic factor accounted for the insensitivity of SHR to ANP action on cardiac reflex pathways. SHR were treated with the angiotensin-converting enzyme (ACE) inhibitor perindopril (3 mg/kg per day) for 6 weeks from 4 to 9 weeks of age (SHR-S, n=10) or for 9 weeks from 4 to 12 weeks of age (SHR-L, n=10) or were untreated (SHR, n=10) to produce differential effects on blood pressure and left ventricle/body weight ratio (LV/BW). Untreated normotensive Wistar-Kyoto rats (WKY, n=10) were also studied. At 13 weeks of age, all rats were instrumented with aortic and jugular catheters, and at 14 weeks we measured heart rate reflexes to rapid intravenous infusions of methoxamine (100 microg/kg, cardiac baroreflex) and serotonin (5 to 60 microg/kg, von Bezold-Jarisch cardiac chemosensitive reflex), with either alpha-rat ANP (150 ng/kg per minute IV) or saline vehicle (270 microL/h IV) infusion. Perindopril treatment for 6-week (SHR-S) and 9-week (SHR-L) durations maintained blood pressure at normotensive levels in both groups. SHR-S exhibited a small degree of cardiac hypertrophy (LV/BW was 8% higher than in WKY but 11% less than in untreated SHR), but LV/BW was normalized in SHR-L (to within 1% of WKY LV/BW). In WKY, ANP significantly (P<0.05) enhanced bradycardic responses to both the cardiac baroreflex (by 42+/-10%) and von Bezold-Jarisch chemosensitive reflex (by 17+/-5%) activation but had no effect in SHR. The cardiac reflex action of ANP was restored in SHR-L (ANP enhanced reflex bradycardia by 28+/-12% and 36+/-8%, baroreflex and von Bezold-Jarisch reflex, respectively; P<0.05), but SHR-S, which developed some cardiac hypertrophy, remained unresponsive to ANP. Our results suggest that the inability of ANP to sensitize cardiac vagal (nonarterial) afferents in SHR was not due to an inherited irreversible component, or the hypertension per se, but was associated with the presence of cardiac hypertrophy. A functional consequence of hypertension-induced cardiac hypertrophy may be the inhibition of the cardioprotective action of ANP through cardiac vagal reflexes.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Baroreflex; Blood Pressure; Bradycardia; Cardiomegaly; Heart Rate; Hypertension; Indoles; Male; Perindopril; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Species Specificity; Weight Gain

The aim of this study was to investigate the relationships between levels of natriuretic peptides and adrenomedullin and 24 h blood pressure levels in elderly hypertensives.. We performed both 24 h ambulatory blood pressure monitoring and measurement of plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and adrenomedullin in 118 asymptomatic hypertensive elderly (> 60 years old) patients. We classified the subjects into groups with isolated clinic hypertension (n = 40) and sustained hypertension (n = 78). We also measured the levels of these peptides in 37 elderly normotensive subjects.. Plasma ANP and BNP levels were slightly increased in patients with isolated clinic hypertension compared with elderly normotensives. Among the hypertensives, plasma ANP and BNP levels were more closely related to 24 h blood pressure levels than to office blood pressure levels. Sustained hypertensives showed significantly increased plasma levels of ANP and BNP compared with isolated clinic hypertensives, while adrenomedullin levels were similar in the two groups. Elderly hypertensives with left ventricular hypertrophy detected by electrocardiography had significantly higher levels of ANP and BNP, and higher BNP/ANP ratios than those without left ventricular hypertrophy, while there was no significant difference in adrenomedullin levels between the two groups.. Our results suggest that measurements of ANP and BNP may be useful in detecting left ventricular hypertrophy and in differentiating isolated clinic hypertension from sustained hypertension in elderly hypertensive patients.

Topics: Adrenomedullin; Aged; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Circadian Rhythm; Echocardiography; Female; Humans; Hypertension; Male; Middle Aged; Natriuretic Peptide, Brain; Peptides

Candoxatril is an inhibitor of neutral endopeptidase, a membrane-bound enzyme that degrades atrial natriuretic peptide. The effects of candoxatril on hemodynamic parameters and cardiovascular hypertrophy were evaluated in the rat model of myocardial infarction. Myocardial infarction was induced by left coronary artery ligation in rats and they were treated either with candoxatril (10mg/kg per day) or a vehicle for up to 4 weeks. Systolic blood pressure and body weight did not change for up to 4 weeks between the 2 groups. At the end of treatment, hemodynamic parameters were measured, and then plasma, heart, lungs and kidneys were collected. Kidney neutral endopeptidase, as measured by the quantitative autoradiographic method, was significantly inhibited in candoxatril-treated rats compared with that in controls (66.6+/-3.2% of control, p<0.001). On the contrary, there were no significant differences in right atrial pressure, left ventricular end-diastolic pressure, systemic pressure, and plasma level of atrial natriuretic peptide between the 2 groups. There were also no significant differences in cardiac weight and lung weight. These data indicate that inhibition of neutral endopeptidase by candoxatril at a dose of 10 mg/kg per day did not oppose cardiac hypertrophy in the rat model of myocardial infarction in spite of significant neutral endopeptidase inhibition.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Cardiomegaly; Drug Evaluation, Preclinical; Hemodynamics; Indans; Kidney; Male; Myocardial Infarction; Myocardium; Neprilysin; Propionates; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Ventricular Remodeling

We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.

Topics: Actins; Animals; Atrial Natriuretic Factor; Calcium-Binding Proteins; Cardiomegaly; Cells, Cultured; Echocardiography; Gene Expression Regulation; Heart Ventricles; Humans; Mice; Mice, Transgenic; Myocardial Infarction; Myocardium; Myosin Heavy Chains; Nerve Growth Factors; Norepinephrine; Receptors, Adrenergic, alpha-1; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Tissue Distribution

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2alpha (PGF2alpha), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2alpha- and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2alpha treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2alpha increased the beat rate to 3.3 times that of control. After exposure to the beta-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2alpha' 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2alpha by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2alpha. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal alpha-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal alpha-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Topics: Actins; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Cells, Cultured; Dinoprost; Endothelins; Growth Inhibitors; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Myocardium; Phenylephrine; Rats; RNA, Messenger

Activation of the atrial natriuretic peptide (ANP) gene is regarded as one of the earliest and most reliable markers of hypertrophy in the ventricular cardiac myocyte. We have examined the role of the nonreceptor tyrosine kinases in the signaling mechanism(s) leading to hypertrophy using human ANP gene promoter activity as a marker. Endothelin (ET), a well known hypertrophic agonist, increased activity of c-Src, c-Yes, and Fyn within minutes and promoted a selective redistribution of each of these kinases within the cell. Overexpression of c-Src effected a significant increase in activity of a cotransfected human ANP promoter-driven chloramphenicol acetyl transferase reporter, while expression of either c-Yes or Fyn was considerably less effective in this regard. ET-dependent stimulation of the human ANP gene promoter was partially inhibited by co-transfection with dominant negative Ras or dominant negative Src or Csk or by treatment with the potent Src family-selective tyrosine kinase inhibitor PP1, suggesting that the Src family kinases are involved in signaling ET-dependent activation of this promoter. Both ET- and Src-dependent activation of the ANP promoter required the presence of a CArG motif in a serum response element-like structure between -422 and -413 but did not appear to require assembly of a ternary complex for full activity. These findings support a role for Src in the activation of ANP gene expression and suggest that this kinase may contribute in an important way to the signaling mechanisms that activate hypertrophy in the cardiac myocyte.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Biological Transport; Cardiomegaly; Cell Compartmentation; Cells, Cultured; CSK Tyrosine-Protein Kinase; Endothelins; Humans; Myocardium; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Proto-Oncogene Proteins c-yes; Proto-Oncogene Proteins pp60(c-src); ras Proteins; Rats; Receptors, Endothelin; Signal Transduction; src-Family Kinases

Topics: Atrial Natriuretic Factor; Cardiomegaly; Female; Heart Atria; Humans; Middle Aged; Mitral Valve Stenosis; Oliguria; Postoperative Complications; Tricuspid Valve Insufficiency

Partial inhibition of cardiac Na/K-ATPase by digitalis drugs such as ouabain is the initial event leading to positive inotropy in the heart. We showed recently that exposure of rat cardiac myocytes to ouabain concentrations that produce positive inotropy, but no overt toxicity, caused inductions of some early response genes and hypertrophy of these myocytes. The aim of this work was to determine if ouabain also affects the expressions of certain late response genes that are regulated by other hypertrophic stimuli. Non-toxic concentrations of ouabain (5-100 microM) increased mRNAs of skeletal alpha-actin, atrial natriuretic factor, myosin light chain 2, and transforming growth factor beta: indicating that ouabain's effects on these marker genes are similar to those of hypertrophic stimuli that mimic the effects of pressure overload. Expression of skeletal alpha-actin was more sensitive to ouabain than that of atrial natriuretic factor, suggesting significant differences in the ouabain-specific pathways of the induction of these fetal genes. The effects of ouabain on skeletal alpha-actin gene were transcriptional, and required an increase in net influx of extracellular Ca2+. Protein kinase C and Ca(2+)-calmodulin kinases, but not protein kinase A, were involved in the signal pathways leading to the induction of skeletal alpha-actin gene. These data and our prior findings indicate that an increase in net influx of Ca2+ through partial inhibition of Na/K-ATPase initiates protein kinase-dependent pathways resulting in alterations in cardiac growth and expressions of both early and late response genes.

Topics: Actins; Animals; Atrial Natriuretic Factor; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Dactinomycin; DNA; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Genes, fos; Heart; Myocardium; Myosin Light Chains; Ouabain; Protein Biosynthesis; Protein Kinase C; Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

The induction of the atrial natriuretic factor (ANF) gene during alpha 1-adrenergic stimulation of neonatal rat ventricular myocytes has served as a model for gene expression during cardiac muscle cell hypertrophy. This study describes and identifies a single regulatory element that mediates expression of the ANF gene. Deletional mutations were generated in a 639-bp fragment of the ANF promoter that confers alpha 1-adrenergic inducibility to a luciferase reporter gene in transient transfection assays in ventricular myocytes. The results of gel mobility shift and diethylpyrocarbonate (DEPC) interference studies with nuclear cardiac cell extracts identified the nucleotide contract points for a novel A/T-rich element (ANF-AT) at positions -582/-575 that partially mediates alpha 1-adrenergic inducibility. Mutations in the ANF-AT element reduced alpha-adrenergic inducibility of an ANF-TK-luciferase fusion gene in cardiac cells by 35% but had no effect on expression in other muscle and non-muscle cells tested. Gel mobility supershift assays with antibodies directed against the MEF-2 protein, the homeobox protein MHox, or the zinc finger protein HF-1b, document that these factors are not major components of the endogenous ANF-AT binding activity in cardiac muscle cells. The current study provides evidence for a role for a novel A/T-rich element in the regulation of ANF gene expression in cardiac ventricular myocytes.

Topics: Animals; Atrial Natriuretic Factor; Binding Sites; Binding, Competitive; Cardiomegaly; Cells, Cultured; DNA-Binding Proteins; Gene Expression Regulation; Luciferases; MEF2 Transcription Factors; Myocardium; Myogenic Regulatory Factors; Rats; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sp4 Transcription Factor; Transcription Factors; Transcription, Genetic

To characterize cardiac hypertrophy in juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency, we investigated how the hypertrophy develops and whether it is associated with altered expression of any specific genes, especially atrial natriuretic peptide (ANP) and contractile protein genes, in the hypertrophied ventricle. Cardiac hypertrophy in JVS mice became apparent at 10 days after birth and progressed during development. The hypertrophy was observed in the ventricles but not in the atria. ANP mRNA was more intensively expressed in JVS ventricles than in control even at 5 days. Carnitine administration ameliorated the cardiac hypertrophy and suppressed the augmentation of ANP mRNA in the ventricles. Isoform change of expression of alpha-actin genes from cardiac to skeletal was seen in the ventricles of JVS mice at 2 weeks. There was no difference in the ratio of beta-myosin heavy chain mRNA to alpha-myosin heavy chain mRNA between control and JVS mice at 5 days, but at 2 weeks the ratio was significantly lower in JVS mice than in control. These results suggest that the molecular characteristics of cardiac hypertrophy caused by carnitine deficiency are different from those of cardiac hypertrophy caused by aortic constriction.

Topics: Actins; Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Carnitine; Disease Models, Animal; Gene Expression Regulation, Developmental; Heart Ventricles; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Myocardial Contraction; Myosin Heavy Chains; Organ Size; RNA, Messenger; Time Factors

Post-natal growth of cardiac muscle cells occurs by hypertrophy rather than division and is associated with changes in gene expression and muscle fiber morphology. We show here that the protein kinase MEKK1 can induce reporter gene expression from the atrial natriuretic factor (ANF) promoter, a genetic marker that is activated during in vivo hypertrophy. MEKK1 induced both stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) activity; however, while the SAPK cascade stimulated ANF expression, activation of the ERK cascade inhibited expression. C3 transferase, a specific inhibitor of the small GTPase Rho, also inhibited both MEKK- and phenylephrine-induced ANF expression, indicating an additional requirement for Rho-dependent signals. Microinjection or transfection of C3 transferase into the same cells did not disrupt actin muscle fiber morphology, indicating that Rho-dependent pathways do not regulate actin morphology in cardiac muscle cells. While active MEKK1 was a potent activator of hypertrophic gene expression, this kinase did not induce actin organization and prevented phenylephrine-induced organization. These data suggest that multiple signals control hypertrophic phenotypes. Positive and negative signals mediated by parallel MAP kinase cascades interact with Rho-dependent pathways to regulate hypertrophic gene expression while other signals induce muscle fiber morphology in cardiac muscle cells.

Topics: 3T3 Cells; Actins; ADP Ribose Transferases; Animals; Atrial Natriuretic Factor; Botulinum Toxins; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cell Cycle Proteins; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; GTP Phosphohydrolases; Immediate-Early Proteins; Luminescent Proteins; MAP Kinase Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase 1; Muscle Fibers, Skeletal; Myocardium; Phenylephrine; Phosphoprotein Phosphatases; Protein Kinases; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rho Factor; Signal Transduction; Transcription Factor AP-1

Chronic pressure overload is known to increase cardiac mass and expression levels of both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNAs. Although mechanical stretching of cardiac myocytes could cause these changes, humoral factor(s) secondary to pressure overload may also be involved. To dissociate humoral effects from the effects of mechanical loading on cardiac hypertrophic responses, we examined expression of ANP and BNP at both mRNA and protein levels and proportions of myosin isoforms in transplanted cervical hearts that were mechanically unloaded under conditions with or without hypertension by aortic coarctation. Seven days after transplantation, cardiac atrophy that usually occurs in transplanted hearts without hypertension by coarctation was prevented in the transplanted hearts with hypertension by coarctation. The levels of expression of ANP and BNP mRNAs were increased in the transplanted hearts with relative to those without hypertension by coarctation. The plasma level of angiotensin II was higher in rats with than without hypertension by coarctation. Plasma endothelin-1 levels were not significantly different between the two groups. In addition, levels of expression of ANP and BNP mRNAs were increased in the transplanted hearts without hypertension relative to those in the in situ hearts. The proportion of the V3 myosin isoform was also increased in the transplanted hearts without hypertension relative to the in situ hearts. These results indicate that humoral factor(s) secondary to the pressure overload produced by aortic coarctation enhanced the cardiac hypertrophic response and elevated the levels of mRNAs encoding these embryonic markers. Moreover, our findings regarding ANP and BNP expression in the transplanted hearts provide additional evidence that the fetal genes are reexpressed during the process of cardiac atrophy as well as in cardiac hypertrophy.

Topics: Angiotensin II; Animals; Aortic Coarctation; Atrial Natriuretic Factor; Atrophy; Blood Pressure; Body Weight; Cardiomegaly; Endothelin-1; Heart Rate; Heart Transplantation; Hypertension; Male; Myosins; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats; Rats, Inbred Lew; RNA, Messenger; Transcription, Genetic; Transplantation, Heterotopic; Transplantation, Isogeneic

Left ventricular assist devices (LVAD) provide lifesaving circulatory support to patients awaiting heart transplantation. To date, the extent to which sustained mechanical unloading alters the phenotype of pathologic myocardial hypertrophy in dilated cardiomyopathy is unknown.. We examined left ventricular size, myocyte and myocardial immunoreactivity for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in eight patients with advanced dilated cardiomyopathy before and after LVAD support. The mean duration of congestive heart failure was 18 +/- 5 months, and LVAD support averaged 42 +/- 4 days before heart transplantation.. Echocardiographically determined left ventricular mass decreased from 505 +/- 83 to 297 +/- 52 gm (p < 0.05) during LVAD support, whereas minimum myocyte diameter decreased from 28.1 +/- 0.9 to 21.7 +/- 0.6 microns (p < 0.01) in transmural myocardial tissue specimens. Overall left ventricular ANP immunopositivity decreased from 48% at LVAD placement to 12% at transplantation (p < 0.05), whereas BNP immunopositivity decreased from 28% to 4% after LVAD support. Moreover, a gradient of ANP and BNP immunostaining from subendocardium to epicardium observed before mechanical unloading diminished after LVAD support. Analysis of the relationship between left ventricular mass and ANP immunopositivity revealed a close and highly significant correlation between these variables.. These studies demonstrate remarkable left ventricular plasticity even in the presence of advanced cardiomyopathy. Parallel reductions in myocardial mass and myocyte size with reductions in ventricular ANP and BNP immunostaining indicate a novel regression of the phenotype of pathologic hypertrophy within the human myocardium after LVAD support.

Topics: Adolescent; Adult; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Dilated; Echocardiography, Doppler; Endocardium; Female; Heart; Heart-Assist Devices; Humans; Immunoenzyme Techniques; Male; Middle Aged; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Ventricular Function, Left

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; DNA-Binding Proteins; Dogs; Gene Expression Regulation; Genes, Regulator; Promoter Regions, Genetic; Receptors, Atrial Natriuretic Factor; Transcription Factor AP-1; Transcriptional Activation

We have reported previously that calcitonin gene-related peptide (CGRP) exerts hypertrophic effects, defined in the broadest sense as increased mass of protein per cell, in adult rat ventricular in vitro. The aim of the present investigation was to determine whether the peptide also increases the cell surface area of, and induces expression of ANP and skeletal alpha-actin mRNA in hypertrophying neonatal rat ventricular cardiomyocytes. Cells cultured in the presence of CGRP were invisibly hypertrophied after 48 h compared to cells cultured in serum-free MEM for the same period. CGRP, 100 pM and 1 nM, increased cell surface area significantly and to values 1.82- and 2.15-fold greater, respectively, than in the absence of peptide (659.64 +/-23.48 microns 2, n = 10). The selective antagonist at CGRP1, receptors, CGRP8-37(200nM), significantly attenuated the effects of CGRP (100 pM and 1 nM). CGRP caused a marked up-regulation of the expression of mRNA encoding skeletal alpha-actin and ANP, respectively, maximally after 12 h and at a concentration of 100 pM, to values approximately 3.6- and 2.5-fold greater than in the absence of peptide. These effects of the peptide were completely abolished in the presence of CGRP8-37(100 nM). In conclusion, CGRP increases cell surface area and induces expression of ANP and skeletal alpha-actin mRNA in hypertrophying cardiomyocytes via the CGRP1, receptor subtype.

Topics: Actins; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Northern; Calcitonin Gene-Related Peptide; Cardiomegaly; Cell Size; Cells, Cultured; Culture Media, Serum-Free; Heart Ventricles; Humans; Miotics; Myocardium; Peptide Fragments; Rats; Rats, Wistar; Receptors, Calcitonin Gene-Related Peptide; RNA, Messenger; Up-Regulation

Pericardial fluid (PF) may contain myocardial growth factors that exert paracrine actions on cardiac myocytes. The aims of this study were (1) to investigate the effects of human PF and serum, collected from patients undergoing cardiac surgery, on the growth of cultured adult rat cardiac myocytes and (2) to relate the growth activity of both fluids to the adaptive changes in overloaded human hearts. Both PF and serum increased the rate of protein synthesis, measured by [14C]phenylalanine incorporation in adult rat cardiomyocytes (PF, +71.9 +/- 8.2% [n = 17]; serum, +14.9 +/- 6.5% [n = 13]; both P < .01 versus control medium). The effects of both PF and serum on cardiomyocyte growth correlated positively with the respective left ventricular (LV) mass. However, the magnitude of change with PF was 3-fold greater than with serum (P < .01). These trophic effects of PF were mimicked by exogenous basic fibroblast growth factor (FGF2) and inhibited by anti-FGF2 antibodies and transforming growth factor-beta (TGF-beta), suggesting a relationship to FGF2. In addition, FGF2 concentration in PF was 20 times greater than in serum. On the other hand, the LV mass-dependent trophic effect, present in both fluids, was independent of FGF2 concentration or other factors, such as angiotensin II, atrial natriuretic factor, and TGF-beta. These data suggest that FGF2 in human PF is a major determining factor in normal myocyte growth, whereas unidentified LV mass-dependent factor(s), present in both PF and serum, participates in the development of ventricular hypertrophy.

Topics: Adult; Aged; Aged, 80 and over; Angiotensin II; Animals; Atrial Natriuretic Factor; Body Fluids; Cardiomegaly; Female; Fibroblast Growth Factor 2; Heart; Heart Ventricles; Humans; Male; Middle Aged; Myocardium; Pericardium; Rats; Rats, Wistar; Transforming Growth Factor beta

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system.. The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123.. These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cattle; Coculture Techniques; Culture Media, Conditioned; Endothelin Receptor Antagonists; Endothelin-1; Hypertrophy; Lipoproteins, LDL; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Paracrine Communication; Rats; Rats, Wistar; Receptors, Angiotensin; Receptors, Endothelin; RNA, Messenger; Transforming Growth Factor beta; Ventricular Function

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

Topics: Aging; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Animals; Atrial Natriuretic Factor; Captopril; Cardiomegaly; Gene Expression Regulation; Heart; Heart Failure; Hypertension; Male; Myosin Heavy Chains; Polymerase Chain Reaction; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Renin; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

Topics: Atrial Natriuretic Factor; Calcium-Transporting ATPases; Cardiomegaly; Gene Expression Regulation; Heart Failure; Humans; Hypertension, Pulmonary; Myocardium; Myosin Heavy Chains; Receptors, Adrenergic, beta; RNA, Messenger; Tissue Distribution

In cardiac hypertrophy or ventricular remodeling, not only the enlargement of myocytes but also interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study, we examined the mechanism of cyclic mechanical stretch-induced myocyte hypertrophy, highlighting the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MC) and cardiac nonmyocytes (NMC) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. Cyclic mechanical stretch was applied to the cultures with a Flexercell Stress Unit. In addition to cell size, we examined atrial natriuretic peptide/brain natriuretic peptide (ANP/BNP) production as the most sensitive biological markers for MC hypertrophy. Cyclic stretch did not induce hypertrophic responses in MC when they were cultured without NMC. In contrast, when MC were co-cultured with NMC, cyclic stretch induced further increase in ANP/BNP production (2.2-fold and 2.1-fold increases versus non-stretch group, after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II type 1 receptor antagonist. Moreover, ANP/BNP production in the co-culture was significantly suppressed by BQ-123, an endothelin A receptor antagonist, whether cyclic stretch was applied or not. This study raised the possibility that NMC mediate the hypertrophic effect of mechanical stress on MC by increasing endothelin production. It was also suggested that, in this process, angiotensin II is involved in the crosstalk between MC and NMC.

Topics: Angiotensin Receptor Antagonists; Animals; Atrial Natriuretic Factor; Benzimidazoles; Biphenyl Compounds; Cardiomegaly; Cells, Cultured; Coculture Techniques; Endothelin Receptor Antagonists; Fibroblasts; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Peptides, Cyclic; Rats; Stress, Mechanical; Tetrazoles

Whereas numerous studies have examined the cardiac tissue content and secretion of atrial natriuretic peptide (ANP), the response of brain natriuretic peptide (BNP) in states of experimental cardiac overload is less well documented. Our recent partial cloning of the ovine BNP gene has enabled us to study changes in cardiac tissue concentration, together with tissue and circulating molecular forms of ANP and BNP, in response to cardiac overload induced by rapid ventricular pacing (n = 7) and aortic coarctation (n = 6). In normal sheep, although highest levels of BNP were found in atrial tissue (15-fold those of the ventricle), the BNP/ANP concentration ratio in the ventricles was 10- to 20-fold higher than the ratio calculated for atrial tissue. Compared with normal sheep, significant depletion of both ANP and BNP concentrations within the left ventricle occurred after rapid ventricular pacing. Size exclusion and reverse phase HPLC analysis of atrial and ventricular tissue extracts from normal and overloaded sheep showed a single peak of high molecular weight BNP consistent with the proBNP hormone. In contrast, immunoreactive BNP extracted from plasma drawn from the coronary sinus was all low molecular weight material. Further analysis of plasma BNP using ion exchange HPLC disclosed at least 3 distinct immunoreactive peaks consistent with ovine BNP forms 26-29 amino acid residues in length. These findings show that BNP is stored as the prohormone in sheep cardiac tissues and that complete processing to mature forms occurs at the time of secretion. The capacity to process the prohormone at secretion is not impaired by chronic heart failure.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Pacing, Artificial; Cardiomegaly; Chromatography, High Pressure Liquid; Disease Models, Animal; Heart Failure; Molecular Weight; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Radioimmunoassay; Sheep

Topics: Aged; Aortic Valve; Atrial Natriuretic Factor; Cardiomegaly; Endothelium, Vascular; Exercise; Female; Forearm; Heart Failure; Heart Valve Prosthesis; Humans; Male; Middle Aged; Mitral Valve; Oxygen; Regional Blood Flow; Vasodilation

This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca2+]i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca2+]i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: > 90% of ET receptors in cardiomyocytes are of the ETA subtype, whereas fibroblasts had a nearly equal proportion of the ETA and ETB subtypes. ET-1 receptor densities, affinities, and ET-1-induced [Ca2+]i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca2+]i responses induced by Ang II at concentrations > 10(-8) mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT1) on fibroblasts was significantly reduced (42,970 +/- 3330 versus 73,870 +/- 7940 sites per cell for control cells, P < .01), but AT1 receptor affinity was unchanged after volume overload. The maximum increase in [Ca2+]i evoked by 10(-6) to 10(-4) mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ETA subtype and fibroblasts possessing both ETA and ETB receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT1 receptors on fibroblasts, whereas total binding and [Ca2+]i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Calcium; Cardiomegaly; Cells, Cultured; Endothelins; Male; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptors, Angiotensin; Receptors, Endothelin

1,25(OH)2 Vitamin D3 (VD3) and retinoic acid (RA) function as ligands for nuclear receptors which regulate transcription. Though the cardiovascular system is not thought to represent a classical target for these ligands, it is clear that both cardiac myocytes and vascular smooth muscle cells respond to these agents with changes in growth characteristics and gene expression. In this study we demonstrate that each of these ligands suppresses many of the phenotypic correlates of endothelin-induced hypertrophy in a cultured neonatal rat cardiac ventriculocyte model. Each of these agents reduced endothelin-stimulated ANP secretion in a dose-dependent fashion and the two in combination proved to be more effective than either agent used alone (VD3: 49%; RA:52%; VD3 + RA:80% inhibition). RA, at concentrations known to activate the retinoid X receptor, and, to a lesser extent, VD3 effected a reduction in atrial natriuretic peptide, brain natriuretic peptide, and alpha-skeletal actin mRNA levels. Similar inhibition (VD3:30%; RA:33%; VD3 + RA:59% inhibition) was demonstrated when cells transfected with reporter constructs harboring the relevant promoter sequences were treated with VD3 and/or RA for 48 h. These effects were not accompanied by alterations in endothelin-induced c-fos, c-jun, or c-myc gene expression, suggesting either that the inhibitory locus responsible for the reduction in the mRNA levels lies distal to the activation of the immediate early gene response or that the two are not mechanistically coupled. Both VD3 and RA also reduced [3H]leucine incorporation (VD3:30%; RA:33%; VD3 + RA:45% inhibition) in endothelin-stimulated ventriculocytes and, once again, the combination of the two was more effective than either agent used in isolation. Finally, 1,25(OH)2 vitamin D3 abrogated the increase in cell size seen after endothelin treatment. These findings suggest that the liganded vitamin D and retinoid receptors are capable of modulating the hypertrophic process in vitro and that agents acting through these or similar signaling pathways may be of value in probing the molecular mechanisms underlying hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Base Sequence; Calcitriol; Cardiomegaly; Chloramphenicol O-Acetyltransferase; DNA Primers; Endothelins; Gene Expression; Genes, Reporter; Heart; Humans; In Vitro Techniques; Molecular Sequence Data; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transfection; Tretinoin

We used the human atrial natriuretic peptide (hANP) gene as a model to investigate the causal relationship between immediate early gene expression and the subsequent activation of the embryonic gene repertoire in cardiac hypertrophy. Using transient transfection analysis, we found that overexpression of individual Jun family members, alone or in combination, displayed unique activity that varied as a function of the promoter and the nature of the transfected myocyte populations under examination. In neonatal cardiac ventriculocytes, both c-jun and to a lesser extent, JunB stimulated hANP promoter activity (approximately 7- and 3- fold, respectively). When cotransfected together, a synergistic activation was observed (approximately 16-fold activation), a finding that stands in contrast to the behavior of JunB (i.e. neutral or inhibitory) with other 12-O- tetradeconoylphorbol 13-acetate response element-dependent promoters. In atriocytes, on the other hand, JunB did not itself activate the hANP promoter, and it antagonized c-jun- mediated transcription. JunD, a third member of this gene family, was devoid of activity in these transfected cultures. These findings suggest that the hANP gene promoter exhibits a broad range of responses to the individual products of the jun gene family. The response in any single situation is a function of the relative concentrations and subunit composition of the prevailing activator protein-1 complexes.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression Regulation; Gene Transfer Techniques; Genes, fos; Genes, jun; Humans; Myocardium; Rats; Transcription Factor AP-1

In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.

Topics: Actin Cytoskeleton; Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Cardiomegaly; Endothelins; Enzyme Activation; Flavonoids; Gene Expression; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocardium; Myosin-Light-Chain Kinase; Phenylephrine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Rats; Rats, Sprague-Dawley; Receptors, Cell Surface

An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Down-Regulation; Molecular Sequence Data; Oligonucleotides, Antisense; Phenylephrine; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Topics: Adrenergic Agonists; Animals; Atrial Natriuretic Factor; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Glycogen Synthase Kinase 3; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardium; Phosphatidylinositols; Protein Kinase C; Protein Kinases; Protein-Tyrosine Kinases; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Tetradecanoylphorbol Acetate

1. The effects of prolonged chlorothiazide treatment of left ventricular failure on cardiac hypertrophy, circulating vasoactive hormones and exchangeable body sodium were examined in rats with chronic myocardial infarction induced by left coronary artery ligation. Chlorothiazide therapy commenced either immediately or 2 weeks after infarction. For 4 weeks, the rats were given either chlorothiazide (50 mg day-1 kg-1) in their drinking water or drinking water alone. 2. Cardiac weight increased in untreated rats with infarction in comparison with sham-operated controls, indicating the presence of chronic left ventricular dysfunction, although exchangeable body sodium, plasma renin activity, plasma vasopressin and plasma osmolality remained unchanged. 3. Chlorothiazide raised haematocrit and plasma renin activity equally in rats with and without infarction, although exchangeable body sodium, plasma vasopressin and plasma osmolality were not changed by the treatment. Plasma atrial natriuretic peptide was 2-fold higher in rats with infarction and this response was not affected by chlorothiazide treatment. Chlorothiazide therapy did not prevent or reverse cardiac hypertrophy. 4. Chronic diuretic therapy in this experimental model of heart failure did not reduce extracellular sodium, plasma vasopressin or the extent of ventricular hypertrophy, possibly because the condition was associated with activation of the renin-angiotensin system.

Topics: Animals; Arginine Vasopressin; Atrial Natriuretic Factor; Cardiomegaly; Chlorothiazide; Chronic Disease; Diuretics; Female; Myocardial Infarction; Myocardium; Organ Size; Rats; Rats, Wistar; Sulfonamides; Time Factors; Ventricular Dysfunction, Left

The transgenic rat TGR(mRen2)27, carrying the mouse Ren-2 gene, is a new model to elucidate the role of the local renin-angiotensin system in vivo. However, the role of the local renin-angiotensin system in the heart remains to be determined in TGR(mRen2)27.. TGR(mRen2)27 were treated with various antihypertensive drugs for 6 weeks to examine the effects on cardiac hypertrophy and gene expression. Cardiac mRNAs were examined by Northern blot analysis. In TGR(mRen2)27, left ventricular hypertrophy was associated with a decrease in alpha-myosin heavy chain expression of 31% and an increase in skeletal alpha-actin and atrial natriuretic polypeptide expression by 2.6- and 21-fold, respectively (P < .05), thereby showing the shift of myocardium to a fetal phenotype. Furthermore, cardiac collagen and laminin expressions were increased in TGR(mRen2)27 (P < .05), suggesting the occurrence of cardiac remodeling. Although treatment of TGR(mRen2)27 with a high dose of TCV-116 (angiotensin AT1 receptor antagonist) or manidipine (calcium antagonist) combined with atenolol (beta 1-adrenergic receptor blocker) completely normalized blood pressure, TCV-116 regressed cardiac hypertrophy and suppressed the changes in cardiac mRNA levels of TGR(mRen2)27 much more potently than manidipine with atenolol. Furthermore, the inhibitory effects of a low dose of TCV-116 on cardiac hypertrophy and altered gene expressions of TGR(mRen2)27 were greater than those of doxazosin (alpha 1-adrenergic receptor blocker) combined with atenolol, despite their similar hypotensive effects.. Our present observations provide evidence that the cardiac renin-angiotensin system in TGR(mRen2)27 is responsible for cardiac hypertrophy, phenotypic modulation, and remodeling.

Topics: Actins; Analysis of Variance; Animals; Animals, Genetically Modified; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Collagen; Heart; Heart Rate; Laminin; Male; Mice; Muscle, Skeletal; Myocardium; Organ Size; Phenotype; Rats; Rats, Sprague-Dawley; Renin; Renin-Angiotensin System; RNA, Messenger; Transcription, Genetic

1. We assessed the changes of atrial natriuretic peptide and brain natriuretic peptide gene expression associated with progression and regression of cardiac hypertrophy in renovascular hypertensive rats (RHR). 2. Two-kidney, one-clip hypertensive rats (6-week-old male Wistar) were made and studied 6 (RHR-1) and 10 weeks (RHR-2) after the procedure. Regression of cardiac hypertrophy was induced by nephrectomy at 6 weeks after constriction, and the nephrectomized rats were maintained further for 4 weeks (nephrectomized rat: NEP). Sham operation was performed, and the rats were studied after 6 (Sham-1) and 10 weeks (Sham-2). Atrial natriuretic peptide and brain natriuretic peptide gene expression in the left ventricle was analysed by Northern blotting. 3. Plasma atrial natriuretic peptide and brain natriuretic peptide were significantly higher in RHR-1 and RHR-2 than in Sham-1, Sham-2 and NEP. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in RHR-1 were approximately 7.2-fold and 1.8-fold higher than those in Sham-1, respectively, and the corresponding levels in RHR-2 were 13.0-fold and 2.4-fold higher than those in Sham-2, respectively. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels of NEP were normalized. Levels of atrial natriuretic peptide and brain natriuretic peptide mRNA were well correlated positively with left ventricular weight/body weight ratios. There was a significant positive correlation between the levels of atrial natriuretic peptide and brain natriuretic peptide mRNA (r = 0.86, P < 0.01). 4. We conclude that the expression of atrial natriuretic peptide and brain natriuretic peptide genes is regulated in accordance with the degree of myocardial hypertrophy and that the augmented expression of these two natriuretic peptides may play an important role in the maintenance of cardiovascular haemodynamics in renovascular hypertension.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Blotting, Northern; Cardiomegaly; DNA Primers; Gene Expression Regulation; Hypertension, Renal; Male; Molecular Sequence Data; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats; Rats, Inbred SHR; Rats, Wistar; RNA, Messenger

Myocardial infarction was induced by rats by ligation of the left coronary artery. Treatment with TM1, a prodrug of SQ 28,603, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11), was started 18-20 hours after ligation and was continued for 4 weeks (100 mg/kg, orally, twice daily). Morphological and biochemical parameters were assessed at the endo of therapy. The treatment resulted in a significant reduction of heart hypertrophy, which was restricted to the parts of myocardium hemodynamically upstream of the infarcted left ventricle. The weights of the right ventricle and atria were reduced by 15-20%, whereas the treatment had no effect on the left ventricle and septum weights. Treatment led to an almost complete inhibition of plasma NEP activity and to a slight decrease (-14%, p < 0.05) in plasma ACE activity. Plasma ANF level increased 3.8-fold after ligation, and treatment resulted in a slight ( + 29%) and nonsignificant additional increase in the ANF level. The amount of hydroxyproline in the right ventricle was enhanced by + 207% in control ligated rats and by +140% (NS) in treated rats. These data indicated that prolonged NEP inhibition exerts a favorable effect in heart failure by reducing the development of right ventricular and atrial hypertrophy. These effects may result from an improvement in hemodynamic conditions, leading to a reduction in cardiac preload.

Topics: Alanine; Animals; Atrial Natriuretic Factor; Cardiomegaly; Enzyme Inhibitors; Ligation; Male; Myocardial Infarction; Neprilysin; Rats; Rats, Wistar

Several prostaglandins [prostaglandin (PG) A2, -B2, -D2, -E2, -F2 alpha, and -I2 and carbaprostacyclin] and the thromboxane analogue U-46619 were analyzed for the ability to induce hypertrophy of rat neonatal cardiac ventricular myocytes. Myocyte hypertrophy was induced specifically by PGF2 alpha. Myocytes exposed to this prostanoid in culture increased in size and protein content. The contractile fibrils within the cells became organized into parallel arrays, and the cells tended to cluster and beat spontaneously. PGF2 alpha also induced the expression of c-fos, atrial natriuretic factor (ANF), and alpha-skeletal actin in these cells. The effects of PGF2 alpha were compared with several known cardiac myocyte hypertrophy factors (phenylephrine, endothelin-1, leukemia inhibitory factor, cardiotrophin-1, and angiotensin II). PGF2 alpha was found to be intermediate in potency among the factors but induced a level of ANF production that was approximately 10-fold higher than any of the other effectors. Responsiveness to PGF2 alpha was not limited to neonatal cardiocytes. Ventricular myocytes isolated from adult rats also responded specifically to PGF2 alpha with a morphological change similar to that observed with phenylephrine and by producing ANF. In rats, chronic administration of fluprostenol, a potent agonist analogue of PGF2 alpha, resulted in a dose-dependent increase in heart weight- and ventricular weight-to-body weight ratios. The amount of PGF2 alpha extractable from the hearts of rats with cardiac hypertrophy induced by myocardial infarction was also found to be greater than that in sham-operated control rats. These results indicate that PGF2 alpha may play an important role in inducing cardiac hypertrophy.

Topics: Aging; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Dinoprost; Heart; Male; Myocardial Infarction; Myocardium; Phenylephrine; Prostaglandins; Prostaglandins F, Synthetic; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

To evaluate the effect of 7-day angiotensin II antagonism with losartan, an AT1-receptor antagonist, on systolic blood pressure, renal sodium and water excretion and on the atrial natriuretic factor system in one-kidney, one clip hypertensive rats.. The one-kidney, one clip hypertensive rats were separated into four groups: untreated (group 1), low-sodium diet (group 2), losartan (20 mg/kg orally, group 3) and low-sodium diet with losartan (group 4). All of the rats were kept in metabolic cages with urinary volume, urinary sodium level and water intake being evaluated daily. Body weight and blood pressure were assessed before treatment and at the end of the observation period. Renal glomerular and papillary atrial natriuretic factor receptors were assessed by radioligand binding experiments.. No differences were observed either in body weight or in blood pressure between groups at the outset After 1 week, blood pressure was 184+/-4, 184+/-7, 170+/-5 and 78+/-8 mmHg, in groups 1, 2, 3 and 4, respectively. Group 3 rats failed to gain weight and had high urinary volume. In contrast, group 4 rats lost 15% of their original body weight. Both of the losartan-treated groups presented an apparently reduced cardiac hypertrophy but it was only clear in the low-sodium diet group. Both of the losartan-treated groups had high plasma renin activity. All of the three treated groups showed upregulation of glomerular and no changes in papillary atrial natriuretic factor receptors. Overall, mortality was 18, 27, 0 and 36% in groups 1, 2, 3 and 4, respectively.. Losartan administration reduces blood pressure in one-kidney, one clip rats only when combined with a low-sodium diet. Both low-sodium diet and angiotensin II antagonism upregulate renal glomerular but not papillary atrial natriuretic factor receptors, suggesting a divergent regulatory mechanism.

Topics: Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Body Water; Cardiomegaly; Cardiovascular Diseases; Disease Models, Animal; Drinking; Drug Antagonism; Heart; Hypertension; Kidney; Losartan; Male; Natriuresis; Organ Size; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Renin; Time Factors

This study investigates basal and angiotensin II (Ang II)-induced [Ca2+]i concentrations in cells from hearts of rats that have undergone cardiac hypertrophy due to volume overload. [Ca2+]i measurements assessed by digital imaging using fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from adult rat hearts with a 4-week aortocaval shunt. Long-term aortocaval shunt induced a significant increase in atrial (72%) and ventricular (41%) weights and a large elevation in plasma atrial natriuretic peptide-(1-98) concentration (160%). For adult cardiomyocytes [Ca2+]i measurements are reported as diastolic (average of the lowest points) and systolic intracellular Ca2+ values (average of the maximum points corresponding to the diastolic points) over a 30-second time interval. Basal diastolic [Ca2+]i (99 +/- 4.1 nmol/L for experimental cells versus 90 +/- 4.8 for control cells) was not altered, whereas basal systolic [Ca2+]i was significantly greater in ventricular cardiomyocytes from overload hearts (155 +/- 2.3 versus 129 +/- 4.4 nmol/L for control cells, P < .05). Ang II increased intracellular Ca2+ spike frequency in a concentration-dependent manner in cardiomyocytes from control and overload myocardium. Basal and Ang II-induced intracellular Ca2+ spike frequencies were not modified in cardiomyocytes from hypertrophied hearts. Basal [Ca2+]i in ventricular fibroblasts from overload myocardium was significantly increased (128 +/- 5.1 nmol/L for fibroblasts from hypertrophied hearts versus 104 +/- 3.5 for control cells, P < .05). Ang II-induced [Ca2+]i was lower in fibroblasts from overload myocardium (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Angiotensin II; Animals; Atrial Natriuretic Factor; Calcium; Cardiac Volume; Cardiomegaly; Cells, Cultured; Culture Media; Cytosol; Fibroblasts; Male; Myocardium; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Vasoconstrictor Agents

Adult mammalian ventricular cardiomyocytes are terminally differentiated cells that enlarge adaptively by hypertrophy. In this situation, genes normally expressed in the fetal ventricular cardiomyocyte (e.g. atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM) alpha-actin) are re-expressed, and there is transient expression of immediate early genes (e.g. c-fos). Using appropriate reporter plasmids, we studied the effects of transfection of the constitutively active or dominant negative mitogen-activated protein kinase kinase MEK1 on ANF, beta-MHC, and SkM alpha-actin promoter activities in cultured ventricular cardiomyocytes. ANF expression was stimulated (maximally 75-fold) by the hypertrophic agonist phenylephrine in a dose-dependent manner (EC50, 10 microM), and this stimulation was inhibited by dominant negative MEK1. Cotransfection of dominant negative MEK1 with a dominant negative mitogen-activated protein kinase (extracellular signal-regulated protein kinase (ERK2)) increased this inhibition. Transfection with constitutively active MEK1 constructs doubled ANF promoter activity. The additional cotransfection of wild-type ERK2 stimulated ANF promoter activity by about 5-fold. Expression of beta-MHC and SkM alpha-actin was also stimulated. Promoter activity regulated by activator protein-1 or c-fos serum response element consensus sequences was also increased. We conclude that the MEK1/ERK2 cascade may play a role in regulating gene expression during hypertrophy.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Chickens; Gene Expression; Heart Ventricles; Humans; Luciferases; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Phenotype; Point Mutation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rabbits; Rats; Rats, Sprague-Dawley; Serine; Transfection

We examined the relationship between cardiac hypertrophy, myosin heavy chain (MHC) isoform expression, and production of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) before and after the development of DOCA-salt hypertension. DOCA-salt rats exhibited significant left ventricular hypertrophy at the prehypertensive stage (1 week of treatment), without MHC isoform switch or change in natriuretic peptide gene expression. In the hypertensive stage (5 weeks of treatment), pronounced left ventricular hypertrophy was observed, and this was characterized by an increase in beta-MHC protein, resulting in a switch from 90% alpha-MHC to 51% alpha-MHC and 49% beta-MHC. ANF and BNP mRNA levels and peptide content were significantly increased at this stage. Unexpectedly, the MHC isoform switch was evident in the non-hypertrophied right ventricle to the same degree as in the left ventricle. Natriuretic peptide production was also increased in the right ventricle at 5 weeks of treatment, but to a lesser degree than in the left ventricle. In contrast, in the hypertrophied left atrium there was no MHC isoform switch, while ANF and BNP mRNA levels were augmented. Plasma ANF was significantly increased in the prehypertensive stage; this was accompanied by a partial depletion of atrial ANF stores. Plasma BNP was increased only in the hypertensive stage, reflecting an increase in ventricular BNP synthesis and secretion. These results suggest that 1) cardiac hypertrophy, MHC isoform expression, and stimulation of natriuretic peptide production are processes that may be dissociated from each other; 2) increases in plasma ANF without a concomitant increase in plasma BNP reflect atrial hemodynamic overload, while increases in both ANF and BNP in plasma are associated with ventricular hypertrophy; and 3) there exist differences in the storage, secretion, and processing patterns of ANF and BNP in the atria.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Blotting, Northern; Body Weight; Cardiomegaly; Centrifugation, Density Gradient; Chromatography, High Pressure Liquid; Desoxycorticosterone; Hypertension; Isomerism; Male; Myocardium; Myosin Subfragments; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Organ Size; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA; Sodium Chloride

Atrial natriuretic peptide (ANP), a cardiac-specific hormone, is stored in the atria and released in response to atrial stretch. During cardiac hypertrophy, ANP gene expression is markedly upregulated in the left ventricle (LV). Because the hearts of normotensive senescent rats exhibit left atrial (LA) and left ventricular (LV) hypertrophy and dilatation, we examined ANP mRNA levels by Northern blot analysis and ANP peptide concentrations by radioimmunoassay in atria, LVs, and plasma of rats at 2, 6, 18, and 22-24 mo of age. Compared with LVs of 6-mo-old rats, the LV-to-body weight ratio was elevated 30% by 18 mo of age, whereas levels of ANP mRNA were elevated twofold (not significant) and sevenfold (P < 0.05) in the LV of 18- and 22- to 24-mo-old rats, respectively. The concentration of immunoreactive ANP (ir-ANP) exhibited a four- to fivefold increase in LVs of 18- and 22- to 24-mo-old rats compared with values for 6-mo-old rats (43 +/- 4 pmol/g wet wt; means +/- SE). Among 18-and 22- to 24-mo-old rats a significant correlation was observed between ANP peptide concentration and LV hypertrophy (r 2 = 0.64). Levels of ANP mRNA and ir-ANP in the atria exhibited only modest changes with aging.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression; Heart; Heart Atria; Heart Ventricles; Male; Rats; Rats, Wistar; RNA, Messenger

The aim was to compare activities of the type A and type B natriuretic factor genes during development of cardiac hypertrophy by use of a non-radioactive method designed for assessment of stable atrial and brain natriuretic factor (ANF, BNF) transcript levels in biopsy sized tissue samples.. At 1 and 7 days after aortocaval shunt or sham surgery in rats, quantitative reverse transcriptase mediated polymerase chain reaction (Q-RT-PCR) was used to determine mRNA levels in cardiac tissues. Phosphoglycerate kinase-1 (PGK-1) mRNA levels served as an external standard for Q-RT-CR.. The shunt increased left ventricular end diastolic pressure at days 1 and 7, and cardiac weight was increased by day 7. By day 1, left ventricular BNF mRNA levels were twice those of controls, whereas ANF mRNA levels were not changed. By day 7, left ventricular BNF mRNA levels were increased 15-fold, and those for ANF were increased fivefold; the BNF mRNAs were also increased in right atria and right ventricle, about fivefold in both cases.. Both natriuretic factor genes were activated by cardiac volume overload, and the increase in the level of left ventricular BNF transcripts-observed for the first time-was in fact more rapid and exceeded that of ANF. The Q-RT-PCR assay will be of value to investigate the response to increased work load of cardiac muscle in vivo.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Cardiomegaly; DNA Primers; Gene Expression Regulation; Male; Molecular Sequence Data; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Polymerase Chain Reaction; Rats; Rats, Wistar

Utilizing an in vitro model system of cardiac muscle cell hypertrophy, we have identified a retinoic acid (RA)-mediated pathway that suppresses the acquisition of specific features of the hypertrophic phenotype after exposure to the alpha-adrenergic receptor agonist phenylephrine. RA at physiological concentrations suppresses the increase in cell size and induction of a genetic marker for hypertrophy, the atrial natriuretic factor (ANF) gene. RA also suppresses endothelin 1 pathways for cardiac muscle cell hypertrophy, but it does not affect the increase in cell size and ANF expression induced by serum stimulation. A trans-activation analysis using a transient transfection assay reveals that neonatal rat ventricular myocardial cells express functional RA receptors of both the retinoic acid receptor and retinoid X receptor (RAR and RXR) subtypes. Using synthetic agonists of RA, which selectively bind to RXR or RAR, our data indicate that RAR/RXR heterodimers mediate suppression of alpha-adrenergic receptor-dependent hypertrophy. These results suggest the possibility that a pathway for suppression of hypertrophy may exist in vivo, which may have potential therapeutic value.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cell Size; Endothelins; Genetic Markers; Heart; Humans; In Vitro Techniques; Myocardium; Phenylephrine; Rats; Receptors, Adrenergic, alpha; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. To assess the pathophysiological role of BNP in ventricular overload, we have examined the gene expression of BNP, In comparison with that of ANP, in a model of cardiac hypertrophy using cultured neonatal rat ventricular cardiocytes. During cardiocyte hypertrophy evoked by endothelin-1, Phenylephrine, or PMA, the steady state level of BNP mRNA increased as rapidly as the "immediate-early" induction of the c-fos gene expression, and reached a maximal level within 1 h. Actinomycin D, a transcriptional inhibitor, completely diminished the response, while the translational blocked with cycloheximide did not inhibit it. In contrast, ANP mRNA began to increase 3 h after the stimulation, and accumulated during cardiocyte hypertrophy. The BNP secretion from ventricular cardiocytes was also stimulated, more rapidly than the ANP secretion. Furthermore, the turnover of BNP mRNA was significantly faster than that of ANP mRNA, being consistent with the existence of AUUUA motif in the 3'-untranslated region of BNP mRNA. These results demonstrate that the gene expression of BNP is distinctly regulated from that of ANP at transcriptional and posttranscriptional levels, and indicate that the characteristics of the BNP gene expression are suitable for its possible role as an " emergency" cardiac hormone against ventricular overload.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Base Sequence; Cardiomegaly; Cells, Cultured; Chromatography, Gel; Chromatography, High Pressure Liquid; DNA Primers; Endothelins; Enzyme Activation; Gene Expression Regulation; Heart Ventricles; Kinetics; Molecular Sequence Data; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Phenylephrine; Protein Kinase C; Radioimmunoassay; Rats; Rats, Wistar; Restriction Mapping; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic

To determine whether similar or divergent pathways mediate atrial natriuretic factor (ANF) induction in neonatal and hypertrophied adult ventricular myocardium, and to assess whether studies using an in vitro model system of hypertrophy have fidelity to the in vivo context during pressure overload hypertrophy, we generated transgenic mice which harbor either 638 or 3,003 bp of the rat ANF 5' flanking region ligated upstream from a luciferase reporter. Luciferase activity in the ventricles of day 1 transgenic neonates was 8-24-fold higher than the levels expressed in the ventricles of adult mice. Adult mice expressed the luciferase reporter in an appropriate tissue-specific manner. Transverse aortic constriction of adult mice harboring ANF reporter transgenes demonstrated no significant increase in reporter activity in the ventricle. These findings demonstrate that distinct regions of the ANF 5'-flanking region are required for inducible expression of the ANF gene in the hypertrophic adult ventricle compared with those required for atrial-specific and developmentally appropriate expression in the intact neonatal heart. Furthermore, the cis regulatory elements necessary for induction of ANF expression in endothelin-1 or alpha 1-adrenergically stimulated cultured neonatal ventricular myocytes are not sufficient for induction in the in vivo context of pressure overload hypertrophy.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Chloramphenicol O-Acetyltransferase; Female; Gene Expression; Heart; Heart Ventricles; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Myocardium; Polymerase Chain Reaction

Long-term, serum supplemented cultures of rat adult ventriculocytes were utilized to study the tropic effects of the alpha-agonist phenylephrine and of the carnitine palmitoyltransferase I inhibitor etomoxir. Cell protein and the rate of incorporation of phenylalanine were measured, corrected for cellular DNA content and utilized as an index for hypertrophy and of anabolic activity of the cells, respectively. The mRNA level of ANF was utilized as an index for the pathological phenotypic change (i.e., switch to fetal gene program), and that of the Na-channel--a constantly expressed gene in normal and hypertrophic cardiomyocytes--served as an internal control. Both mRNAs were quantified at various stages in culture by competitive reverse transcriptase PCR. The size of control myocytes steadily increased for over 3 weeks. The cells were completely redifferentiated and reached a maximum of anabolic activity 2 weeks after plating. Secretion and mRNA levels of ANF were increased severalfold after 7-8 days. Addition of 10 microM phenylephrine considerably speeded up cell growth. Maximum anabolic activity and complete redifferentiation were reached already after 1 week. Levels of mRNA and of ANF release increased 30-40 fold. Interestingly, induction of ANF gene transcription lagged behind the redifferentiation of the cells. Ten microM etomoxir inhibited the oxidation of palmitic acid and stimulated that of exogenous glucose by adult cardiomyocytes. In spite of its clear effect on fuel utilization, etomoxir had no direct hypertrophic effect on the myocytes in culture and did not inhibit the stimulatory action of alpha-agonists. Reactivation of the fetal gene program, as visualized by ANF production, was not reversed by etomoxir.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Cardiomegaly; Cell Division; Cells, Cultured; DNA; Epoxy Compounds; Gene Expression; Glucose; Molecular Sequence Data; Myocardium; Oxidation-Reduction; Palmitic Acid; Palmitic Acids; Phenylalanine; Phenylephrine; Protein Biosynthesis; Rats; Sodium Channels

Cardiac phenotypic modulation and remodeling appear to be involved in the pathophysiology of cardiac hypertrophy and heart failure. We undertook this study to examine whether angiotensin II (Ang II) in vivo, independent of blood pressure, contributes to cardiac phenotypic modulation and remodeling. A low dose (200 ng/kg per minute) of Ang II was continuously infused into rats by osmotic minipump for 24 hours or 3 or 7 days to examine the effects on the expression of cardiac phenotype-related or fibrosis-related genes. This Ang II dose caused a small and gradual increase in blood pressure over 7 days. Left ventricular mRNAs for skeletal alpha-actin, beta-myosin heavy chain, atrial natriuretic polypeptide, and fibronectin were already increased by 6.9-, 1.8-, 4.8-, and 1.5-fold, respectively, after 24 hours of Ang II infusion and by 6.9-, 3.3-, 7.5-, and 2.5-fold, respectively, after 3 days, whereas ventricular alpha-myosin heavy chain and smooth muscle alpha-actin mRNAs were not significantly altered by Ang II infusion. Ventricular transforming growth factor-beta 1 and types I and III collagen mRNA levels did not increase at 24 hours and began to increase by 1.4-, 2.8-, and 2.1-fold, respectively, at 3 days. An increase in left ventricular weight occurred 3 days after Ang II infusion. Treatment with TCV-116 (3 mg/kg per day), a nonpeptide selective angiotensin type 1 receptor antagonist, completely inhibited the above-mentioned Ang II-induced increases in ventricular gene expressions and weight. Hydralazine (10 mg/kg per day), which completely normalized blood pressure, did not block cardiac hypertrophy or increased cardiac gene expressions by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Angiotensin II; Animals; Atrial Natriuretic Factor; Base Sequence; Body Weight; Cardiomegaly; Collagen; Gene Expression; Heart; Male; Molecular Sequence Data; Phenotype; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

We studied plasma concentration, content, and mRNA for atrial natriuretic peptide (ANP-mRNA) in heart chambers of monocrotaline-treated rats. Three distinct groups emerged: group 1, with moderate congestive heart failure (CHF; pleural effusion < 1 ml; no peritoneal effusion); group 2, with severe CHF (pleural and peritoneal effusion > 1 ml); and group 3, with right hypertrophy and no CHF. Group 1 and 2 rats had right atrial and ventricular hypertrophy, raised plasma ANP (from 16.31 +/- 11.32 to 98.50 +/- 22.50 and 124.09 +/- 57.29 pg/ml, respectively; P < 0.001), and depletion of right atrial ANP (from 143.23 +/- 29.79 to 21.70 +/- 17.70 and 18.12 +/- 14.64 nmol/g, respectively; P < 0.001). Ventricular ANP concentration was unchanged. ANP-mRNA rose in the right atrium [10.6 (P < 0.02) and 7.9 (P < 0.01) times] and right ventricle (53.0 and 46.6 times; P < 0.01). In left unhypertrophied chambers it also increased, although to a smaller extent. Group 3 rats had isolated right ventricular hypertrophy, normal ANP levels in plasma and tissues, and no activation of synthesis. These data suggest that 1) plasma concentration and ANP synthesis are increased only in animals with CHF, 2) activation of ANP synthesis is maximal in early stages of CHF and is not related to the degree of hypertrophy, and 3) ANP-mRNA is also expressed in unhypertrophied heart chambers of rats with CHF but is not expressed in hypertrophied chambers of animals without CHF.

Topics: Animals; Ascitic Fluid; Atrial Natriuretic Factor; Cardiomegaly; Female; Gene Expression; Heart Failure; Hypertrophy, Right Ventricular; Monocrotaline; Myocardium; Organ Specificity; Pleural Effusion; Rats; Rats, Sprague-Dawley; RNA, Messenger

Left ventricular hypertrophy in response to pressure overload may be modified by neurohumoral activation. To investigate the contribution of the renin-angiotensin system, we studied rats after banding of the ascending aorta that developed severe left ventricular hypertrophy associated with normal plasma renin but elevated cardiac angiotensin-converting enzyme (ACE) levels. Rats were treated with vehicle, ACE inhibitor (ramipril), angiotensin II type 1 receptor antagonist (losartan), or vasodilator (hydralazine) during weeks 7 through 12 after aortic banding. A significant regression of left ventricular mass index as determined by serial echocardiography was observed in ramipril- and losartan-treated groups during weeks 9 through 12 after banding, whereas hypertrophy further increased in vehicle- and hydralazine-treated groups. Twelve weeks after banding, relative left ventricular weights and myocyte widths were markedly increased in vehicle- and hydralazine-treated groups, whereas ramipril and losartan significantly reduced these parameters. In addition, molecular adaptations in left ventricular hypertrophy, such as upregulation of left ventricular atrial natriuretic peptide and downregulation of sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels, were blunted by ramipril or losartan treatment. Hypertrophic regression was associated with reduced mortality in rats treated with ramipril (11%) and losartan (13%) versus hydralazine (20%) and vehicle (31%). Thus, the renin-angiotensin system may be involved in the maintenance of chronic left ventricular hypertrophy. Blockade of the system may result in regression of the hypertrophic phenotype and improve survival in rats despite persistent pressure overload.

Topics: Animals; Atrial Natriuretic Factor; Calcium-Transporting ATPases; Cardiomegaly; Echocardiography; Hemodynamics; Hypertension; Male; Myocardium; Rats; Rats, Wistar; Renin-Angiotensin System; RNA, Messenger; Sarcoplasmic Reticulum; Survival Analysis

Alterations in left atrial (LA) and left ventricular (LV) compliance and arterial and coronary sinus atrial natriuretic factor (ANF) concentrations at baseline and in response to both volume depletion and expansion were investigated in 15 conscious dogs with aortic banding-induced LV hypertrophy (LVH) (LV/body wt increased by 64%), which also induced LAH (LA/body wt increased by 61%). With volume expansion coronary sinus ANF increased more (P < 0.05) in dogs with LVH (+427 +/- 88 pg/ml) compared with control dogs (+146 +/- 45 pg/ml). Arterial ANF levels also rose more with volume expansion in LVH. In dogs with LVH, the LV end-diastolic pressure-diameter relationship was shifted to the left with a steeper slope with volume expansion, such that at any given diastolic dimension, diastolic pressure was higher. In contrast, the pressure-dimension relationship for the LA appendage was shifted in the opposite direction during both atrial systolic and diastolic phases, with a more shallow slope in hypertrophy compared with control dogs, resulting in an augmented pressure-dimension product during volume loading in LAH. In conclusion, in dogs with LVH and LAH, enhanced ANF was revealed in the coronary sinus and systemic circulation during volume expansion, which could be due, in part, to a more compliant, but hypertrophied, LA, which responded to equivalent volume loading with an augmented pressure-dimension product.

Topics: Animals; Aorta; Atrial Function, Left; Atrial Natriuretic Factor; Blood Pressure; Blood Volume; Cardiomegaly; Compliance; Constriction; Dogs; Hemodynamics; Osmolar Concentration; Plasma Substitutes; Ventricular Function, Left

Antihypertensive treatments were given to young and adult SHRs, to prevent and reverse hypertension, respectively. Cardiac hypertrophy and the steady state level of the "fetal" genes, ANP, alpha-skeletal actin (alpha-skA), and beta myosin heavy chain (beta-MHC) mRNAs were assessed. Our findings show that the reduction of blood pressure does not consistently result in a similar regression of the "fetal gene program".

Topics: Actins; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Gene Expression; Hypertension; Myosins; Rats; Rats, Inbred SHR

The cardiovascular consequences of endothelin (ET) blockade with the ETA-receptor antagonist FR 139317 were evaluated by determining the long-term effects of the drug on hemodynamic, hormonal, renal and structural parameters in stroke-prone spontaneously hypertensive rats (SHR-SP). Young SHR-SP on a high-sodium diet develop malignant hypertension accompanied by renovascular and cerebrovascular lesions. In control SHR-SP the systolic blood pressure increased from 196 +/- 3 to 260 +/- 4 mm Hg, whereas in animals treated with FR 139317 (20 mg/kg intraperitoneally, twice daily) it increased only from 196 +/- 4 to 212 +/- 3 mm Hg during a treatment period of 6 weeks. There was also an increase in heart weight. At the end of the experiment the plasma levels of atrial natriuretic peptide and brain natriuretic peptide were significantly lower in the group treated with FR 139317 than in the controls. The endothelin plasma levels were significantly higher and the plasma renin activity was lower in the group treated with the endothelin receptor antagonist. These data indicate that endothelin is involved in the maintenance of high blood pressure and cardiac hypertrophy in malignant hypertension, as exemplified by SHR-SP.

Topics: Animals; Atrial Natriuretic Factor; Azepines; Cardiomegaly; Endothelin Receptor Antagonists; Endothelins; Hypertension; Indoles; Rats; Rats, Inbred SHR; Renin

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression Regulation; Genes, ras; Genes, Reporter; Genes, src; Hypertrophy; Luciferases; Myocardium; Phenotype; Promoter Regions, Genetic; Rats

The cardiovascular consequences of endothelin (ET) blockade with the ETA receptor antagonist FR 139317 were evaluated by determining long-term effects of the drug on hemodynamic, hormonal, and structural parameters in stroke-prone spontaneously hypertensive rats (SHR-SP). Young SHR-SP on a high-sodium diet develop malignant hypertension accompanied by renovascular and cerebrovascular lesions. In control SHR-SPs the systolic blood pressure increased from 196 +/- 3 to 260 +/- 4 mm Hg, whereas in animals treated with FR 139317 (20 mg/kg, i.p., b.i.d.) blood pressure increased only from 196 +/- 4 to 212 +/- 3 mm Hg during a treatment period of 6 weeks. The increase in heart weight was also delayed. At the end of the experiment, the plasma levels of ANP and BNP were significantly lower in the group treated with FR 139317 than in the controls. The plasma ET levels were significantly higher and the plasma renin activity was lower in the group treated with the ET receptor antagonist. These data indicate that ET is involved in the maintenance of high blood pressure and cardiac hypertrophy in malignant hypertension, as exemplified by an SHR-SP rat model.

Topics: Animals; Atrial Natriuretic Factor; Azepines; Cardiomegaly; Endothelin Receptor Antagonists; Endothelins; Hypertension; Indoles; Rats; Rats, Inbred SHR; Receptor, Endothelin A

1. We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. The production and secretion of BNP and ANP in the hypertrophied ventricles were markedly augmented, serving as a compensation mechanism against ventricular overload by their natriuretic, diuretic and vasodilatory actions. 2. In the present study, we prepared an in vitro model of cardiocyte hypertrophy using cultured neonatal rat ventricular cardiocytes and alpha1-adrenergic stimulation, and examined the gene expressions of BNP and ANP during the process of cardiocyte hypertrophy. 3. The treatment of cultured ventricular cardiocytes with phenylephrine evoked cardiocyte hypertrophy around 24 h after the treatment, which was characterized by augmented expression of the myosin light chain-2 gene and increase in cell size. 4. In this model of cardiocyte hypertrophy, the steady-state level of BNP mRNA rapidly increased to the maximal level within 1 h after the treatment. In contrast, ANP mRNA began to increase at 3 h, and accumulated during the course of cardiocyte hypertrophy. The secretion of BNP from ventricular cardiocytes was also stimulated more rapidly than the ANP secretion. 5. These results indicate that the gene expression of BNP is distinctly regulated from that of ANP in cardiocyte hypertrophy, and suggest a discrete pathophysiological role of BNP as an 'emergency' cardiac hormone against ventricular overload.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Cells, Cultured; Gene Expression Regulation; Heart Ventricles; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats; Rats, Wistar

Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli.

Topics: Animals; Animals, Newborn; Aorta, Abdominal; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Constriction, Pathologic; Culture Media, Conditioned; Fibroblasts; Heart Ventricles; Male; Myocardium; Norepinephrine; Prazosin; Propranolol; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Transgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Diglycerides; Gene Expression; Heart Ventricles; Humans; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Myocardium; Myosins; Organ Size; Point Mutation; Promoter Regions, Genetic; Radioligand Assay; Receptors, Adrenergic, alpha-1; Reference Values; RNA, Messenger; Type C Phospholipases

Around the time of birth, cardiac muscle cells lose the capacity to divide and, from this time on, growth of the heart occurs by hypertrophy where each cells gets bigger. The hypertrophic response is characterized by changes in gene expression including expression of the atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2) genes. In cultured neonatal ventricular myocytes, hypertrophy also involves reorganization of contractile proteins into sarcomeric units. We have investigated the role of the Raf-1 kinase in this response. Activation of an estradiol-regulated Raf-1 protein kinase led to activation of mitogen-activated protein (MAP) kinase and activated expression from the ANF and MLC-2 promoters. Raf-1-induced activation of these genes was inhibited by a kinase deficient mutant of the 44-kDa MAP kinase, Erk1 indicating a requirement for MAP kinases in the Raf-1-induced response. However, activation of Raf-1 was not sufficient to induce the organization of actin into sarcomeric units. Transfection of dominant negative Raf-1 inhibited phenylephrine-induced activation of the ANF and MLC-2 promoters. Transactivation was rescued by the introduction of increased amounts of c-Raf suggesting a role for Raf-1 in the response to alpha-adrenergic agonists. These results suggest that activation of Raf-1 kinase is a critical component of the signal transduction pathway leading to changes in gene expression associated with hypertrophy but that Raf-1 is not sufficient for the regulation of actin organization during the hypertrophic response.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Gene Expression; Heart Ventricles; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Myocardium; Myosins; Phenylephrine; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Rats; Recombinant Proteins; Transcription, Genetic; Transfection

We studied the effects of physical endurance training on atrial natriuretic peptide (ANP) gene expression in beagle dogs, Wistar rats, and spontaneously hypertensive rats (SHR). The dogs underwent a gradually increased running training up to 40 km/day on a treadmill for 55 wk while the nontrained sibling control dogs were kept in their cages throughout the study. Endurance training caused a significant 13% (P < 0.05) increase in ventricular hypertrophy but did not change plasma immunoreactive (ir)-ANP levels at rest or ventricular ANP mRNA or irANP levels. When normotensive Wistar rats ran up to 2,200 m/day for 8 wk, no significant change was seen in ventricular hypertrophy or in plasma or ventricular irANP levels at rest compared with nontrained controls. However, endurance training caused a 2.2-fold increase in epicardial ANP mRNA levels (P < 0.05). In the SHR strain, running training up to 900 m/day for 31 wk increased ventricular hypertrophy of trained SHR by 7% (P < 0.01) and caused a concomitant 1.6- to 1.7-fold elevation in ventricular irANP and ANP mRNA levels (P < 0.01-0.001) compared with nontrained SHR. In contrast, changes in atrial ANP mRNA or irANP levels in response to training were small in all three protocols. This study shows that in the normal heart induction of ANP synthesis by endurance training is not associated with ventricular hypertrophy. Moreover, the common stimulus for ventricular ANP synthesis induced by both chronic pressure overload and physical training may be mechanical stretching of cardiac myocytes, because endurance training further stimulated ANP synthesis in hypertrophied ventricles in SHR.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Northern; Body Weight; Cardiomegaly; Dogs; Eating; Female; Lactates; Lactic Acid; Male; Myocardium; Organ Size; Physical Conditioning, Animal; Physical Endurance; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar; RNA, Messenger

The failing heart is characterized by impaired cardiac muscle function and increased interstitial fibrosis. Our purpose was to determine whether the functional impairment of the failing heart is associated with changes in levels of mRNA encoding proteins that modulate parameters of contraction and relaxation and whether the increased fibrosis observed in the failing heart is related to elevated expression of genes encoding extracellular matrix components. We studied hearts of 18- to 24-month-old spontaneously hypertensive rats with signs and symptoms of heart failure (SHR-F) or without evidence of failure (SHR-NF) and of age-matched normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, SHR-NF exhibited left ventricular (LV) hypertrophy (2.2-fold) and right ventricular (RV) hypertrophy (1.5-fold), whereas SHR-F were characterized by comparable LV hypertrophy (2.1-fold) and augmented RV hypertrophy (2.4-fold; all P < .01). Total RNA was isolated from ventricles and subjected to Northern blot analysis. In SHR-F hearts, the level of alpha-myosin heavy chain mRNA was decreased in both ventricles to 1/3 and 1/5 of the SHR-NF and WKY values, respectively (both P < .01). Levels of beta-myosin heavy chain, alpha-cardiac actin, and myosin light chain-2 mRNAs were not significantly altered in hearts of SHR-NF or SHR-F. Levels of alpha-skeletal actin were twofold greater in SHR-NF hearts compared with WKY hearts and were intermediate in SHR-F hearts. Levels of atrial natriuretic factor (ANF) mRNA were elevated threefold in the LV of SHR-NF (P < .05) but were not significantly increased in the RV of SHR-NF compared with WKY rats. During the transition to failure (SHR-F versus SHR-NF), ANF mRNA levels increased an additional 1.6-fold in the LV and were elevated 4.7-fold in the RV (both P < .05). Levels of sarcoplasmic reticulum Ca(2+)-ATPase (SRCA) mRNA were maintained in the LV of hypertensive and failing hearts at levels not significantly different from WKY values. In contrast, the level of RV SRCA mRNA was 24% less in SHR-NF compared with WKY rats, and during the transition to failure, this difference was not significantly exacerbated (29% less than the WKY value). The levels of fibronectin and pro-alpha 1(I) and pro-alpha 1(III) collagen mRNAs were not significantly elevated in either ventricle of the SHR-NF group but were fourfold to fivefold higher in both ventricles of SHR-F (all P < .05).(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Calcium-Transporting ATPases; Cardiac Output, Low; Cardiomegaly; Contractile Proteins; Extracellular Matrix Proteins; Gene Expression; Genes; Heart; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Sarcoplasmic Reticulum

There is increasing evidence that the renin-angiotensin system may play a important role in cardiac hypertrophy. To assess the role of angiotensin II in the induction of cardiac hypertrophy, three groups of adult mice were subjected to left ventricular pressure overload by transverse aortic constriction (TAC). For the next 7 days the groups received either the specific angiotensin II subtype 1 receptor (AT1) antagonist (losartan, 1.05 g/l; n = 17), an angiotensin enzyme inhibitor (captopril, 2 g/l; n = 17), or no treatment (n = 22) administered in the drinking water and compared with three similarly treated sham-operated groups (n = 7 each). TAC resulted in a significant increase in heart weight-to-body weight ratio (0.634 +/- 0.087 vs. 0.525 +/- 0.039, g/g x 100, P < 0.05), which was prevented by losartan (0.506 +/- 0.069, g/g x 100, P < 0.0001) despite similar hemodynamic load (proximal systolic pressure 146 +/- 31 vs. 136 +/- 32 mmHg, untreated vs. losartan, P = NS). Proximal systolic pressure was positively correlated with the development of ventricular hypertrophy. In the presence of AT1-receptor blockade, the increase in heart weight-to-body weight ratio at any given systolic pressure was significantly attenuated compared with untreated TAC mice. The increase in heart weight-to-body weight ratio was also significantly attenuated by captopril compared with untreated banded controls (0.542 +/- 0.091, g/g x 100, P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Aorta; Atrial Natriuretic Factor; Biphenyl Compounds; Captopril; Cardiomegaly; Constriction; Gene Expression; Heart Ventricles; Hemodynamics; Hypertension; Imidazoles; Losartan; Mice; Mice, Inbred C57BL; Tetrazoles

The aim of the study was to compare, in a rat model of congestive heart failure, the effect of captopril, a selective angiotensin-converting enzyme (ACE; EC 3.4.15.1) inhibitor, to that of alatriopril, a mixed inhibitor of ACE and atriopeptidase (EC 3.4.24.11), an enzyme implicated in the degradation of atrial natriuretic factor (ANF). Myocardial infarction was induced by ligation of the left coronary artery. Groups of rats received orally twice daily captopril (10 mg/kg), alatriopril (100 mg/kg) or vehicle. Treatments were started 18 to 20 h after ligation and continued for 4 weeks. Hypertrophic and hormonal changes reflecting congestive heart failure were assessed in rats with large infarcts by measuring the relative weight of cardiac tissues as well as by assaying ANF in heart and plasma and by measuring renin activity in plasma. Both treatments significantly reduced cardiac hypertrophy, but alatriopril showed a greater efficacy than captopril--the increase in relative heart weight reaching 38% with captopril and only 22% with alatriopril (P < .05). The hypertrophy of right ventricle was reduced by 47% with alatriopril and by 35% with captopril (N.S.), whereas the corresponding reductions for atria were 47% vs. 21% (P < .05). Both treatments prevented the ligation-induced increase of ANF level in the right ventricle. In contrast, plasma ANF level was significantly reduced after captopril but not after alatriopril treatment, a difference that probably reflects the protection of endogenous ANF in circulation resulting from atriopeptidase inhibition. Plasma renin was increased by 36-fold after captopril but only by 1.6-fold after alatriopril, a difference that presumably reflects the inhibition of renal renin secretion by endogenous ANF after alatriopril. These data suggest that enhancement of ANF levels in circulation via atriopeptidase inhibition magnifies the capacity of ACE inhibitors to prevent cardiac hypertrophy, and they show the potential therapeutic value of mixed ACE-atriopeptidase inhibitors in congestive heart failure.

Topics: Alanine; Amino Acid Sequence; Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Bradykinin; Captopril; Cardiomegaly; Dioxoles; Disease Models, Animal; Heart Failure; Hormones; Male; Molecular Sequence Data; Myocardial Infarction; Myocardium; Neprilysin; Peptidyl-Dipeptidase A; Rats; Rats, Wistar; Renin

The alpha-adrenergic agonist, phenylephrine, has been widely used to induce hypertrophy in cultured ventricular myocytes from neonatal rats. We have investigated the role of tyrosine phosphorylation in this signaling pathway using the tyrosine kinase inhibitor, genistein. We find that genistein treatment prevents phenylephrine-induced activation of three promoters (Fos, atrial natriuretic factor, ANF, and the myosin light chain 2, MLC-2), which are activated in the hypertrophic response. Genistein also inhibits phenylephrine-induced activation of the mitogen activated protein (MAP) kinases Erk1 and Erk2 and inhibits GTP loading of the Ras protein. These data demonstrate that a genistein-sensitive step is critical for the activation of the Ras-MAP kinase pathway by phenylephrine and suggest that this pathway is important in the regulation of the hypertrophic response.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Gene Expression; Genistein; Isoflavones; Mitogen-Activated Protein Kinase 1; Myocardium; Myosin-Light-Chain Kinase; Phenylephrine; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fos; Rats; Receptors, Adrenergic, alpha; Signal Transduction

Fibronectin expression was studied in the heart of rats given a continuous infusion of angiotensin II (Ang II). Northern blot analysis showed that left ventricular fibronectin steady-state mRNA increased fivefold to eightfold in response to pressor doses of Ang II after 24 hours. Accumulation of immunodetectable fibronectin in the ventricles occurred after the mRNA levels increased. The changes in fibronectin expression were reversible when Ang II treatment was withdrawn. The Ang II-induced increase in fibronectin mRNA accompanied similar increases for collagen type I, collagen type IV, and atrial natriuretic factor steady-state mRNA. Interstitial and perivascular fibrosis was identified in both ventricles of angiotensin-treated rats within 3 days. In situ hybridization identified cells associated with areas of fibrosis as the principal site of fibronectin mRNA accumulation in treated animals. By comparison, normal hearts showed fibronectin expression primarily within ventricular vascular tissue and the atrial endocardium. A dose-dependent reduction of fibronectin expression followed treatment with losartan, indicating an Ang II type 1 receptor-mediated effect. Normalization of blood pressure during Ang II infusion by either hydralazine or prazosin had different effects on the level of fibronectin steady-state mRNA, indicating that blood pressure elevation was not the principal factor responsible for fibronectin induction. Concurrent administration of angiotensin-converting enzyme inhibitors with Ang II attenuated the increased fibronectin expression. Our data indicate that Ang II induces an acute fibrotic response within the heart and suggests that Ang II stimulates fibronectin expression within nonmyocytic cardiac cells by a direct action.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Fibronectins; Gene Expression; Male; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

We investigated neurohumoral profiles and transmitter and neuroenzyme markers of cardiac autonomic innervation in control (unpaced) dogs and three groups of dogs with pacing-induced heart failure (paced, paced + beta-adrenergic blockade, and paced + cardiac denervation). Left ventricular ejection fraction decreased significantly and to a comparable extent in all paced groups. Pacing increased plasma norepinephrine (NE); increases in NE were not attenuated but instead tended to be exaggerated by treatment with propranolol or cardiac denervation. Atrial hypertrophy occurred in all paced groups compared with the control group. However, atrial and right ventricular hypertrophy were not as pronounced in the paced plus cardiac denervation group as in the paced and paced plus propranolol groups. Pacing also depleted neuropeptide Y and NE from all heart chambers; propranolol treatment did not modify these local tissue changes. Pacing caused selective depletion of neuroenzymes predominantly in the left ventricle; again, propranolol did little to modify these changes. In this study of paced animals with experimentally maintained cardiac dysfunction, failure to modify noradrenergic responses with intrapericardial cardiac denervation suggests that noncardiac sources contribute predominantly to high plasma NE. Failure to modify neurohumoral, neuropeptide, and neuroenzyme responses with beta-antagonist suggests this treatment has little practical direct influence on sympathetic vasomotor activity or neuronal function in heart failure.

Topics: Animals; Arginine Vasopressin; Atrial Natriuretic Factor; Bicarbonates; Biomarkers; Body Weight; Cardiac Pacing, Artificial; Cardiomegaly; Dogs; Electric Stimulation; Electrolytes; Heart; Heart Failure; Muscle Denervation; Neuropeptide Y; Neuropeptides; Norepinephrine; Oxygen; Partial Pressure; Propranolol; Reference Values; Respiration; Vagus Nerve; Ventricular Function, Left

The blood pressure was decreased after chronic treatment with enalapril, MK-954, and hydralazine in deoxycorticosterone acetate (DOCA)-salt-induced malignant hypertension of spontaneously hypertensive rats (SHR); however, ventricular weight and plasma brain natriuretic peptide (BNP) concentration were decreased after enalapril and MK-954 but not after hydralazine. The BNP secretory rates from the ventricle in enalapril- and MK-954-treated DOCA-salt SHR were decreased to approximately 50% of those in untreated DOCA-salt SHR. The BNP secretory rate from the ventricle was positively correlated with ventricular weight in untreated and treated DOCA-salt SHR. In contrast, acute administration of captopril or MK-954 did not decrease the BNP secretory rate from the heart. Results suggest that the decrease in plasma BNP after enalapril and MK-954 is attributed to a decline in the secretion from the ventricle but not from the atrium. The reduction in ventricular mass appeared to be related to this decline.

Topics: Animals; Atrial Natriuretic Factor; Biphenyl Compounds; Blood Pressure; Blood Urea Nitrogen; Cardiomegaly; Chromatography, High Pressure Liquid; Creatinine; Desoxycorticosterone; Enalapril; Heart; Hydralazine; Hypertension, Malignant; Imidazoles; Losartan; Male; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Rats; Rats, Inbred SHR; Sodium, Dietary; Tetrazoles

The role of angiotensin II via the angiotensin type 1 or type 2 receptor in the development of cardiac hypertrophy was determined in adult male Sprague-Dawley rats subjected to coarctation of the abdominal aorta. Five groups of animals were studied: coarctation, coarctation plus DuP 753, coarctation plus PD 123319, sham plus DuP 753, or sham operation. Type 1 receptor blockade was accomplished with DuP 753 given in the drinking water and type 2 blockade with PD 123319 delivered by osmotic minipumps beginning with the day of surgery until 72 hours after aortic coarctation. Mean carotid blood pressures and the carotid-femoral artery blood pressure gradients were not different among coarctation, coarctation plus DuP 753, and coarctation plus PD 123319 animals. However, ratios of heart weight to body weight were higher in coarctation (4.95 +/- 0.8) or coarctation plus PD 123319 (4.52 +/- 0.5) than in sham animals (3.6 +/- 0.4; P < .005 and .05, respectively). In coarctation plus DuP 753-treated animals heart weight-body weight ratios were not different from sham or sham plus DuP 753 animals (3.9 +/- 0.4 versus 3.6 +/- 0.4 or 3.3 +/- 0.08, respectively). Type 1 receptor mRNA levels were significantly increased in the coarctation group, with the highest levels in the coarctation plus DuP 753 and sham plus DuP 753 groups. To determine whether growth factors were involved in the hypertrophic process, we measured transforming growth factor-beta 1 mRNA levels. Northern analysis demonstrated a twofold increase in coarctation animals compared with sham or coarctation plus DuP 753-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angiotensin II; Animals; Aortic Coarctation; Atrial Natriuretic Factor; Biphenyl Compounds; Cardiomegaly; Gene Expression Regulation; Imidazoles; Losartan; Male; Rats; Rats, Sprague-Dawley; Receptors, Angiotensin; Tetrazoles; Transforming Growth Factor beta

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Heart Ventricles; Hydrolysis; Luciferases; Microinjections; Myocardium; Oncogene Protein p21(ras); Phenylephrine; Phosphatidylinositols; Rats; Receptors, Adrenergic, alpha-1

We have compared the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all peripheral tissues, the level of both natriuretic peptide transcripts was approximately 1-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than 1/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs suggests paracrine and/or autocrine function of these natriuretic peptides.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Brain; Cardiomegaly; DNA Primers; Female; Gene Expression; Humans; Liver; Lung; Male; Middle Aged; Molecular Sequence Data; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Organ Specificity; Ovarian Neoplasms; Ovary; Pancreas; Pituitary Gland; Polymerase Chain Reaction; Rats; Transcription, Genetic

The multiple K+ channels are crucial for repolarization and configuration of the action potential in the neuronal and cardiac cells. In this study, we report the regulatory mechanisms of rapidly inactivating Shaker Kv1.4 channel transcript in the rat heart. Quantitative PCR analysis showed that stimulation with high concentration of KCl, BAY-K 8644, or 12-O-tetradecanoyl phorbol-13-acetate resulted in an immediate and substantial increase (two- to threefold) of Kv1.4 mRNA levels in spontaneously beating myocytes prepared from neonatal rat ventricles. The Kv1.4 mRNA in the ventricle remains at a steady state level after birth and gradually declines with maturation. These results suggest that the Kv1.4 mRNA level is not static and undergoes dynamic modulation by multiple factors that activate intracellular signals. In addition, the expression patterns of Kv1.4 as well as the delayed rectifier Shaker K+ channel Kv1.5 mRNAs were examined in hypertrophied ventricles in which a plateau phase of action potential is remarkably prolonged. The Kv1.5 mRNA level was dramatically repressed while the Kv1.4 mRNA level was remarkably increased. This differential regulation was completely reversed by the normalization of hypertrophy, suggesting that the pathological alterations of K+ channel gene regulation may be involved in the occurrence of ventricular arrhythmias in hypertrophic hearts.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aging; Animals; Animals, Newborn; Atrial Natriuretic Factor; Base Sequence; Bucladesine; Cardiomegaly; Cells, Cultured; DNA Primers; Gene Expression Regulation; Heart; Heart Ventricles; Molecular Sequence Data; Myocardium; Nifedipine; Oligonucleotides, Antisense; Polymerase Chain Reaction; Potassium Channels; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA, Messenger

Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle in response to mechanical load. How muscle cells convert mechanical stimuli into growth signals has been a long-standing question. Using an in vitro model of load (stretch)-induced cardiac hypertrophy, we demonstrate that mechanical stretch causes release of angiotensin II (Ang II) from cardiac myocytes and that Ang II acts as an initial mediator of the stretch-induced hypertrophic response. The results not only provide direct evidence for the autocrine mechanism in load-induced growth of cardiac muscle cells, but also define the pathophysiological role of the local (cardiac) renin-angiotensin system.

Topics: Actins; Angiotensin I; Angiotensin II; Angiotensinogen; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Cytoplasmic Granules; Endothelins; Gene Expression Regulation; Genes, fos; Hypertrophy; In Vitro Techniques; Mechanoreceptors; Myocardium; Peptidyl-Dipeptidase A; Rats; Renin; RNA, Messenger; Stress, Mechanical

Plasma concentration of ANF and expression of left ventricular ANF-gene in rats with cardiac hypertrophy induced by abdominal aortic partial ligation were analyzed by RIA and Northern blot respectively. Results showed that plasma concentration of ANF and level of left ventricular ANF-mRNA in cardiac hypertrophic rats increased markedly, indicating that the cardiac load may induce transcription and expression of left ventricular ANF-gene. This effect could be potentiated by intracellular calcium modulator taurine and inhibited by vasodilator hydralazine.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Gene Expression; Hypertension; Male; Rats; Rats, Wistar; RNA, Messenger; Transcription, Genetic

alpha 1-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which alpha 1-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The alpha-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log Ki = -8.2) and (+)-niguldipine (log Ki = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies alpha-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alpha 1B selective antagonist chlorethylclonidine at a concentration of 10 microM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alpha 1A-adrenergic receptors mediate not only the early effects of alpha 1-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using alpha 1-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; Contractile Proteins; GTP-Binding Proteins; Hydrolysis; Luciferases; Myocardium; Oncogene Protein p21(ras); Phosphatidylinositols; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha; Recombinant Fusion Proteins

The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.

Topics: Alkaloids; Amino Acid Sequence; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Blotting, Northern; Cardiomegaly; Catechols; Cells, Cultured; Gene Expression; Heart Ventricles; Kinetics; Molecular Sequence Data; Myocardium; Nitriles; Oligopeptides; Phenanthridines; Platelet Aggregation Inhibitors; Polymerase Chain Reaction; Protease Inhibitors; Protein-Tyrosine Kinases; Radioimmunoassay; Rats; Receptors, Cell Surface; Receptors, Thrombin; RNA, Messenger; Thrombin; Tyrphostins

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic agonist.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Division; Cells, Cultured; Gene Expression; Genes, fos; Genes, ras; Genetic Markers; Heart Ventricles; Myocardium; Phenylephrine; Rats; Transfection

1 The effects of long-term angiotensin-converting enzyme inhibition with quinapril on arterial function were studied in spontaneously hypertensive rats, Wistar-Kyoto rats serving as normotensive controls. 2 Adult hypertensive animals were treated with quinapril (10 mg kg-1 day-1) for 15 weeks, which reduced their blood pressure and the concentrations of atrial natriuretic peptide in plasma and ventricular tissue to a level comparable with that in normotensive rats. 3 Responses of mesenteric arterial rings in vitro were examined at the end of the study. Compared with normotensive and untreated hypertensive rats, responses to noradrenaline were attenuated in hypertensive animals on quinapril, both force of contraction and sensitivity being reduced. Quinapril also attenuated maximal contractions but not sensitivity to potassium chloride. Nifedipine less effectively inhibited vascular contractions in normotensive and quinapril-treated than in untreated hypertensive rats. 4 Arterial relaxation responses by endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitrite, isoprenaline) mechanisms were similar in normotensive and quinapril-treated rats and more pronounced than in untreated hypertensive rats. 5 Cell membrane permeability to ions was evaluated by means of potassium-free solution-induced contractions of endothelium-denuded denervated arterial rings. These responses were comparable in normotensive and quinapril-treated rats and less marked than in untreated hypertensive rats. 6 Intracellular free calcium concentrations in platelets and lymphocytes, measured by the fluorescent indicator quin-2, were similar in normotensive and quinapril-treated rats and lower than in untreated hypertensive rats. 7 In conclusion, quinapril treatment improved relaxation responses and attenuated contractions in arterial smooth muscle of hypertensive rats. These changes may be explained by diminished cytosolic free calcium concentration, reduced cell membrane permeability, and alterations in dihydropyridine-sensitive calcium channels following long-term angiotensin-converting enzyme inhibition.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Calcium; Cardiomegaly; Hypertension; In Vitro Techniques; Isoquinolines; Male; Mesenteric Arteries; Muscle, Smooth; Muscle, Smooth, Vascular; Norepinephrine; Organ Size; Quinapril; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tetrahydroisoquinolines

SCH 34826, i.e., (S)-N-(N-(2,2[(2,2-dimethyl-1,3-dioxolan-4- yl)methoxy]-2-oxo-1-(phenyl-methyl)ethyl)-phenylalanyl)-beta-alanine, is a potent and selective inhibitor of neutral endopeptidase 24.11 (NEP), an enzyme that degrades the atrial natriuretic peptide (ANP). The effects of SCH 34826 on hypertension and left ventricular hypertrophy (LVH) in spontaneously hypertensive rats (SHRs) were evaluated following 1 month of treatment by measuring the blood pressure, cardiac weight, and left ventricular fibrosis. Adult SHRs were treated with SCH 34826 at 10, 30, or 100 mg/kg given orally twice daily or with vehicle. The systolic blood pressure (SBP) and heart rate (HR) were recorded weekly by the tail-cuff method. Cardiac structural damage was determined by morphometric analysis. Over the dose range examined, the drug produced no significant changes in either blood pressure or heart rate. Despite the lack of antihypertensive activity, SCH 34826 at 100 mg/kg reduced both the cardiac mass (-10%) and the amount of fibrotic tissue present in the left ventricle (-42%). These data indicate that chronic inhibition of NEP by SCH 34826 interacts with mechanisms underlying myocardial hypertrophy and cardiac remodeling.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Dioxolanes; Dipeptides; Hemodynamics; Hypertension; Male; Microscopy, Electron; Myocardium; Neprilysin; Rats; Rats, Inbred SHR; Ventricular Function, Left

To seek possible correlations between plasma atrial natriuretic factor (ANF) and left ventricular diastolic function (LVDF) in hypertensive patients.. Since LVDF abnormalities can be detected in patients with normal left ventricular mass, we studied a group of hypertensive patients without left ventricular hypertrophy.. Untreated hypertensive patients (n = 23) and normotensive control subjects (n = 19) were studied. LVDF indices were obtained by M-mode and pulsed Doppler echocardiography. Blood samples for plasma ANF were taken in the recumbent position from subjects on normal-sodium intake.. Plasma ANF levels were significantly higher in hypertensive patients than in normotensive subjects. All indices for systolic function were normal in both normotensive subjects and hypertensive patients. Left atrial diameter was significantly higher for hypertensive patients than for normotensive subjects. Considering LVDF, all indices for ventricular filling were found to be altered, on average, in hypertensive patients, the only exception being peak early velocity. In addition, significant correlations were found between plasma ANF and the pulsed Doppler parameters of left ventricular filling, peak atrial velocity and the peak early:peak atrial velocity ratio. Overall correlations between plasma ANF and left atrial diameter, and between left atrial diameter and left ventricular mass index were also observed.. The high levels of plasma ANF observed in our hypertensive patients and their correlation with the LVDF indices (which mainly reflect the atrial contribution to ventricular filling) could be the result of an increased atrial stretch due to diastolic ventricular dysfunction. This may exist in hypertensive patients before the development of ventricular hypertrophy.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Echocardiography, Doppler; Female; Humans; Hypertension; Male; Systole; Ventricular Function, Left

Left ventricular function (LVF) after reversal of left ventricular hypertrophy (LVH) with antihypertensive therapy is still controversial. The present study was undertaken in spontaneously hypertensive rats (SHR) to determine whether LVF of the regressed heart with lisinopril is normally maintained.. We compared cardiac function of SHR after reversal of LVH induced by lisinopril with that observed in control SHR and also with effects after a 4-week washout period.. Administration of lisinopril began at 15 weeks of age and continued for 20 weeks. Cardiac index, renal blood flow, leg muscle blood flow, plasma renin activity, atrial natriuretic peptide level, and norepinephrine concentration were determined.. Lisinopril decreased body weight, blood pressure and left ventricular weight and increased leg muscle blood flow; cardiac index and renal blood flow were unaltered. Although norepinephrine concentration was unchanged, plasma renin activity increased and atrial natriuretic peptide decreased in treated SHR. Peak left ventricular pumping ability during volume loading was comparable in the two groups. After a 4-week washout period, left ventricular mass and blood pressure increased but remained lower than controls; cardiac index at rest and during volume loading was similar in the two groups.. These data indicate that LVF of the regressed heart induced by lisinopril was well preserved at rest, during volume loading and also after spontaneous recurrence of hypertension in SHR.

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Enalapril; Hemodynamics; Hypertension; Lisinopril; Male; Norepinephrine; Rats; Rats, Inbred SHR; Renin; Ventricular Function, Left

We have developed murine models of viral myocarditis induced by encephalomyocarditis (EMC) virus in which severe myocarditis, congestive heart failure and dilated cardiomyopathy occur in high incidence. From these models, we have learned the natural history and pathogenesis and assessed new diagnostic methods and therapeutic and preventive interventions. Mural thrombi in the atria and ventricles, ventricular aneurysms, conduction disturbance and various arrhythmias were seen in these models. Anti-heart antibody were found in sera of mice and myosin isoenzyme were changed during the course of EMC virus myocarditis. Atrial natriuretic polypeptide was markedly increased in the ventricles in these mice. Successive infection with coxsackievirus and EMC virus developed lesions similar to chronic myocarditis. The myocardial uptake of antimyosin antibody was proved to be a useful method of diagnosis of myocarditis. Treatment with the nucleoside analogue, ribavirin and recombinant alpha interferon effectively inhibited myocardial virus replication and reduced myocardial damage. Passive immunization and virus-specific vaccine prevented development of myocarditis. The use of immunosuppressive therapy was associated with greater mortality when administered early in illness and beneficial effects were not seen by later administration. Angiotensin-converting enzyme inhibitor improved myocardial injury and congestive heart failure. A nonselective beta-adrenergic blocker with intrinsic sympathomimetic activity, carteolol, prevented the development of myocardial lesions similar to those in dilated cardiomyopathy after myocarditis in the chronic stage.

Topics: Adjuvants, Immunologic; Adrenergic beta-Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Autoantibodies; Cardiomegaly; Cardiomyopathy, Dilated; Encephalomyocarditis virus; Enterovirus Infections; Heart Aneurysm; Heart Failure; Immunization, Passive; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Myocarditis; Myocardium; Myosins; RNA, Viral

Sixteen patients with initial diastolic blood pressure greater than or equal to 120 mm Hg were treated for 1 year with extended-release nifedipine [nifedipine-GITS (gastrointestinal therapeutic system)]. Serial changes in left ventricular mass index and associated alterations in left ventricular systolic function, left ventricular filling, plasma renin activity, atrial natriuretic peptide, and catecholamines were evaluated. Blood pressure was significantly reduced from 200 +/- 8/122 +/- 3 mm Hg (mean +/- SEM) to 144 +/- 5/89 +/- 2 mm Hg (p less than 0.0001) at 1 year. Eleven patients (69%) required only nifedipine-GITS for blood pressure control and 5 (31%) required the addition of chlorthalidone. After 6 months, the left ventricular mass index was significantly reduced by 19% from 121 +/- 8 to 96 +/- 7 g/m2 and this reduction was sustained at 1 year. Septal and posterior wall thicknesses were reduced from 13.4 +/- 0.1 to 11.2 +/- 0.04 mm and from 12.8 +/- 0.1 to 10.0 +/- 0.03 mm (p less than 0.001), respectively. Prevalence of left ventricular hypertrophy decreased from 63 to 25%. Left ventricular fractional shortening increased from 34 to 42% (p less than 0.05) and the relationship between fractional shortening and end-systolic stress did not change. Over the year of sustained blood pressure reduction, the peak velocity of early filling increased from 58 to 63 cm/s (p = 0.07), the peak velocity of late filling did not change, and the ratio of late to early peak velocity of left ventricular filling significantly decreased (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Atrial Natriuretic Factor; Cardiomegaly; Chlorthalidone; Delayed-Action Preparations; Drug Therapy, Combination; Echocardiography, Doppler; Electrocardiography; Epinephrine; Hemodynamics; Humans; Hypertension; Middle Aged; Nifedipine; Norepinephrine; Renin

Aim of the study was to evaluate the effect of cardiopulmonary receptors activation and deactivation on antidiuretic hormone (ADH) and atrial natriuretic peptide (ANP) incretion in hypertensive and normotensive subjects. Twenty-one male subjects, 7 normotensives and 14 mild hypertensives, 7 without and 7 with left ventricular hypertrophy (LVH) were admitted to the study. Each subject underwent selective loading and unloading of cardiopulmonary receptors, by application of a positive (LBPP) or negative (LBNP) pressure to the lower body. Blood samples were taken for measurement of ANP, ADH, PRA, immunoreactive renin, aldosterone, noradrenaline and adrenaline. ADH plasma concentration increased during cardiopulmonary receptors inhibition, but this increase became statistically significant (p less than 0.05) at a step of LBNP (-40 mm Hg), in which an involvement of the sinoaortic receptors cannot be excluded. ANP plasma levels increased progressively during LBPP (p less than 0.05 at least). These changes were significantly reduced in hypertensive patients with LVH.

Topics: Adolescent; Adult; Atrial Natriuretic Factor; Cardiomegaly; Hemodynamics; Hormones; Humans; Hypertension; Lower Body Negative Pressure; Male; Middle Aged; Pressoreceptors; Pressure; Vasopressins

We investigated the role of atrial natriuretic factor (ANF) and the renin-angiotensin system as well as the effects of losartan in rats with aortocaval (AC) shunts. Right atrial and left ventricular end-diastolic pressures (LVEDP) were higher and mean arterial blood pressure (MAP) was lower in AC shunt animals than in their controls. AC shunt rats presented marked cardiac hypertrophy, decreased right atrial ANF concentration, and increased ventricular ANF content and concentration. Plasma ANF levels were elevated, and hematocrit was lower in AC shunt animals than in controls. Captopril or losartan treatment decreased MAP and returned LVEDP to sham-operated control values. A clear regression of cardiac hypertrophy was evident in both treated AC shunt groups, with plasma ANF levels tending to follow those in sham-operated rats. Plasma COOH-terminal ANF levels were decreased and urinary volume and hematocrit were increased in losartan-treated AC shunt animals. We conclude that chronic angiotensin converting enzyme inhibition and angiotension II receptor antagonism improved hemodynamic conditions, diminished water retention, reversed cardiac hypertrophy, and restored plasma and tissue ANF to more "normal" levels in rats with moderate high-output heart failure.

Topics: Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Biphenyl Compounds; Body Fluids; Captopril; Cardiac Output, High; Cardiomegaly; Hemodynamics; Homeostasis; Imidazoles; Losartan; Male; Myocardium; Organ Size; Rats; Rats, Sprague-Dawley; Renin; Tetrazoles; Time Factors

The physiology of atrial natriuretic peptide (ANP) secretion was studied in normotensive subjects and hypertensive patients both young and old. Basal plasma ANP concentration was least in young normotensives, intermediate in old normotensives and young hypertensives, and highest in old hypertensives. Nifedipine, a known stimulator of ANP secretion, acutely increased plasma ANP in young and old normotensive subjects but not in young hypertensive patients and half of the old hypertensive patients. Increase in serum ANP level in response to nifedipine did not augment its hypotensive effect. However, the increase of aldosterone in response to nifedipine-induced rise in plasma renin activity (PRA) seemed to be suppressed by elevated ANP.

Topics: Adult; Aged; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Electrocardiography; Female; Humans; Hypertension; Male; Middle Aged; Nifedipine; Pulse; Renin-Angiotensin System

Ventricular hypertrophy is characterized by augmentation of synthesis, storage, and release of atrial natriuretic peptide (ANP) from ventricular tissue, but the physiological stimulus for ANP release from ventricles is not known. We determined the effect of graded, passive myocardial stretch on ANP release in isolated, arrested, perfused heart preparations after removal of the atria in 13-20-month-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). By this age, ANP gene expression was increased in the hypertrophic ventricular cells of SHR, as reflected by elevated levels of immunoreactive ANP and ANP mRNA and the increased ANP secretion (SHR, 93 +/- 14 pg/ml, n = 22; WKY rats, 22 +/- 2 pg/ml, n = 20; p less than 0.001) from perfused ventricles after removal of the atria. The release of ANP from ventricles was examined at two levels of left ventricular pressure by increasing the volume of the intraventricular balloon for 10 minutes. Stretching of the ventricles produced a rapid but transient increase in ANP secretion. As left ventricular pressure rose from 0 to 14 and 26 mm Hg in WKY rats and from 0 to 13 and 27 mm Hg in SHR, increases in ANP release into the perfusate of 1.4 +/- 0.1-fold and 1.5 +/- 0.2-fold (p less than 0.05) in WKY rats and 1.1 +/- 0.1-fold and 1.6 +/- 0.2-fold (p less than 0.05) in SHR, respectively, were observed. There was a highly significant correlation between the left ventricular pressure level and the maximal concentration of ANP in the perfusate during stretching (p less than 0.001, r = 0.59, n = 42), as well as between the maximal ANP concentrations in perfusate during stretching and the ventricular weight/body weight ratios of the corresponding animals (r = 0.38, p less than 0.05, n = 42). High performance liquid chromatographic analysis revealed that the ventricles both before and during stretch primarily released the processed, active, 28-amino acid ANP-like peptide into the perfusate. These results indicate that stretching is a direct stimulus for ventricular ANP release and show that ANP is also a ventricular hormone.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Chromatography, High Pressure Liquid; Heart Ventricles; Hemodynamics; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Perfusion; Radioimmunoassay; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger

To analyze the determinants of left ventricular (LV) performance (myocardial afterload, chamber size, mass, and contractility) in Goldblatt hypertension, 19 anesthetized one-kidney, one-clip (1K1C) and 28 two-kidney, one-clip (2K1C) male Wistar rats were studied 58 to 62 days after clipping, together with 19 sham-operated and 13 normal rats (controls), by M-mode echocardiography using necropsy-validated methods of measurement. The LV fractional shortening was inversely related to end-systolic stress in all groups (r = -0.89 to -0.95, all P less than .00001): 7 2K1C (25%) and 9 1K1C (47%) had fractional shortening above the upper confidence limit in control animals. Both 1K1C and 2K1C with high LV performance had severe hypertension, inadequate LV hypertrophy, with resultant high wall stress (both P less than .005), increased LV chamber dimension (P less than .005 and P less than .05, respectively) and high afterload-corrected fractional shortening (both P less than .001); 2K1C also had high plasma renin activity and atrial natriuretic factor levels (both P less than .01). Rats with normal LV performance exhibited mild hypertension, adequate LV hypertrophy (normalizing wall stress), and normal LV chamber size and afterload-corrected fractional shortening. Thus, 8 1/2 weeks after clipping, adequate LV hypertrophy allows maintenance of normal LV function by normalizing myocardial afterload in a majority of rats with Goldblatt hypertension, whereas increased LV contractility (and possibly use of preload reserve in 1K1C) maintains normal LV function in the presence of inadequate LV hypertrophy and elevated wall stress, in a substantial minority of rats that developed more severe Goldblatt hypertension.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Heart Ventricles; Hypertension, Renovascular; Myocardial Contraction; Rats; Renin; Ventricular Function, Left

To investigate the ventricular expression of atrial natriuretic peptide (ANP) in human hypertrophic hearts, we conducted an immunohistochemical study of 130 endomyocardial biopsy specimens obtained from the right side of the ventricular septum (RVB), left ventricular free wall (LVB), or both from a total of 80 patients: 44 patients with hypertrophic cardiomyopathy (HCM), 14 with apical hypertrophic cardiomyopathy (APH), 13 with hypertensive hearts (HHD), and nine without hypertrophy (controls). No patients had apparent congestive heart failure. ANP was not seen in ventricular myocytes in controls but was identified in biopsy specimens of hypertrophic hearts, and its distribution was characteristic in each hypertrophic group: 15 RVB (37%) and two LVB (7%) of the HCM group, one RVB (7%) and two LVB (18%) of the APH group, and zero RVB (0%) and five LVB (46%) of the HHD group. Clinical data (including echocardiographic, hemodynamic, and angiographic data) were not directly related to ventricular ANP expression in HCM, APH, or HHD with one exception. In HHD patients, LVB specimens with ANP showed greater ventricular wall thickness than LVB specimens without ANP. According to histological data, however, the ANP-present RVB specimens of HCM or ANP-present LVB specimens of HHD had greater myocyte size than did the ANP-absent specimens. In addition, in HCM patients, the ANP-present RVB specimens showed more severe fibrosis and myofiber disarray than did the ANP-absent specimens. We conclude that a failing state and hemodynamic overload are not likely to be indispensable for ANP expression in human hypertrophic ventricles and that ventricular ANP expression occurs as a response to disease-specific changes: hemodynamic overload in HHD and histological changes such as myocardial fiber disarray, hypertrophy of myocytes, and fibrosis in HCM, which may reflect the characteristic distribution of intraventricular ANP.

Topics: Adult; Atrial Natriuretic Factor; Biopsy; Cardiomegaly; Cardiomyopathy, Hypertrophic; Female; Heart Ventricles; Humans; Hypertension; Immunoenzyme Techniques; Male; Myocardium

1. To determine the cellular mechanisms of atrial natriuretic peptide (ANP) release from ventricular cardiomyocytes, the secretory and the cardiac effects of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, were studied in isolated, perfused heart preparations from 2- and 21-month-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. TPA was added to the perfusion fluid for 30 min at a concentration of 46 nM after removal of atrial tissue. Additionally, atrial and ventricular levels of immunoreactive ANP (IR-ANP) and ANP mRNA, the distribution of ANP within ventricles as well as the relative contribution of atria and ventricles in the release of ANP were studied. 2. Ventricular hypertrophy that gradually developed in hypertensive rats resulted in remarkable augmentation of ANP gene expression, as reflected by elevated levels of immunoreactive ANP and ANP mRNA. The total amount of IR-ANP in the ventricles of the SHR rats increased 41 fold and ANP mRNA levels 12.9 fold from the age of 2 to 21 months. At the age of 21 months, levels of IR-ANP and ANP mRNA in the ventricles of SHR rats were 5.4 fold and 3.7 fold higher, respectively, than in the normotensive WKY rats. Immunohistochemical studies demonstrated ANP granules within the hypertrophic ventricles of the old SHR rats, but not within normal ventricular tissue. 3. In isolated perfused heart preparations, the severely hypertrophied ventricular tissue of SHR rats after atrialectomy secreted more ANP into the perfusate than did the control hearts. Interestingly, the ANP release from the hypertrophied ventricles of the old SHR rats increased considerably (from 413 + 30 to the maximum of 623 + 75 pgml-1, F = 10.8, P < 0.001, two-way analysis of variance), whereas only a small increase was seen in old WKY rats and no effect was observed in young animals of either strain. When intact rat hearts (without atrialectomy) were used, infusion of phorbol ester also increased the ANP secretion into the perfusate in young animals. 4. Our present results indicate that the phorbol ester TPA increases the release of ANP from the hypertrophied, but not from normal rat myocardium. Thus, hypertrophied rat ventricular myocytes appear to possess the cellular mechanisms necessary to secrete ANP by a regulated pathway. The results further suggest that protein kinase C activity may be involved in the the regulation of ANP secretion from ventricu

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression; Heart; Hemodynamics; In Vitro Techniques; Male; Perfusion; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Tetradecanoylphorbol Acetate

The purpose of the present study was to determine if ANF and the NH2-terminus of the ANF prohormone are secreted simultaneously in response to atrial distension in isolated perfused rat atria. The experiments were conducted in paced left atria perfused with a modified Krebs buffer. Atrial pressure was increased from a baseline level of 2 mmHg to 8-9 mmHg for 60 minutes (distension) and then returned to 2 mmHg for 60 minutes in one group (n = 9) of isolated atria. In a second group of atria (n = 6), atrial pressure was maintained at approximately 2 mmHg throughout the experimental period. ANF secretion averaged 100 pg/min during the three 10-minute periods immediately preceding the increased atrial pressure and increased to 600-800 pg/min (P less than 0.001) when atrial pressure was raised. Secretion of the proANF 31-67 peptide increased from a value of approximately 100 pg/min immediately prior to distension to a peak of over 200 pg/min during distension (P less than 0.01). Secretion of proANF 1-98 increased from an average of 1.25 ng/min during the three periods immediately prior to distension to a peak of 2.5 ng/min during distension (P less than 0.01). These data indicate that ANF and the NH2-terminus of the ANF prohormone appear to be simultaneously secreted by isolated paced atria.

Topics: Animals; Atrial Function; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Heart Atria; In Vitro Techniques; Male; Myocardium; Peptide Fragments; Protein Precursors; Rats

Plasma levels of atrial natriuretic peptide (ANP), renin activity (PRA), and endothelin (ET) are often elevated in uremic patients on hemodialysis treatment. The profile of these vasoactive hormones and their relationships with hemodynamic indices in patients on continuous ambulatory peritoneal dialysis (CAPD), however, are not clear. We therefore measured plasma concentrations of ANP, PRA, ET, together with parathyroid hormone (PTH) in 17 patients (mean age 38.5 years) on maintenance CAPD over a period of 12 weeks. Baseline ANP, PRA, and ET levels were significantly higher than those of healthy subjects, and no significant changes in these indices were observed over the 12 week period. There was a significant positive correlation between levels of plasma ANP and PRA [rank correlation coefficient, R(s) = 0.496, p less than 0.05] as has been reported in cardiac failure. Despite the absence of clinically overt heart failure, a significant proportion (approximately 50%) of our patients demonstrated evidence of myocardial dysfunction on echocardiography. Furthermore, a significant positive correlation was demonstrated between plasma ANP and left atrial size [R(s) = 0.61, p less than 0.01] and an inverse correlation existed between plasma ANP and the left ventricular ejection fraction [R(s) = 0.51, p less than 0.05]. Twelve patients (71%) had biochemical evidence of hyperparathyroidism with raised levels of serum PTH. Our study demonstrates increased levels of plasma ANP, PRA, and ET in uremic patients on long-term CAPD. A positive correlation exists between plasma ANP and PRA suggesting their myocardial function may be compromised and this was confirmed on echocardiography. The possibility that high circulating PTH concentrations contribute to impaired cardiac function in such patients, deserves further study.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Endothelins; Female; Humans; Male; Parathyroid Hormone; Peritoneal Dialysis, Continuous Ambulatory; Renin; Time Factors; Uremia

At least three cis-acting regulatory elements are required for expression of the rat atrial natriuretic factor (ANF) gene. One distal cis-acting regulatory element lies more than 640 base pairs from the transcription initiation site.. In this report, we identify two other proximal regulatory elements that lie within 609 base pairs of the transcription initiation site. One proximal regulatory element contains an activator protein-1 (AP-1)-like binding site and is recognized by the AP-1 protein, the c-fos/c-jun proto-oncogene heterodimer in vitro. The second regulatory element contains a cyclic AMP-responsive element (CRE)-like recognition site.. In vitro binding of the c-fos/c-jun heterodimer to ANF gene sequences suggests that the heterodimer may play a role in the regulation of gene transcription in vivo. This observation may also explain the correlation between c-fos/c-jun expression and ventricular ANF gene expression found in hypertrophic states. Nuclear extracts from normal cardiocytes contain proteins that bind these regulatory elements but do not appear to bind at the AP-1 site, suggesting that the levels of fos/jun heterodimer in nonhypertrophied cardiocytes are quite low.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Cardiomegaly; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation; Genes, Regulator; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Regulatory Sequences, Nucleic Acid; Trans-Activators; Transcription, Genetic; Transfection

To study the mechanisms that activate expression of the atrial natriuretic factor (ANF) gene during pressure-induced hypertrophy, we have developed and characterized an in vivo murine model of myocardial cell hypertrophy. We employed microsurgical techniques to produce a stable 35- to 45-mmHg pressure gradient across the thoracic aorta of the mouse that is associated with rapid and transient expression of an immediate-early gene program (c-fos/c-jun/junB/Egr-1/nur-77), an increase in heart weight/body weight ratio, and up-regulation of the endogenous ANF gene. These responses that are identical to those in cultured cell and other in vivo models of hypertrophy. To determine whether tissue-specific and inducible expression of the ANF gene can be segregated, we used a transgenic mouse line in which 500 base pairs of the human ANF promoter region directs atrial-specific expression of the simian virus 40 large tumor antigen (T antigen), with no detectable expression in the ventricles. Thoracic aortic banding of these mice led to a 20-fold increase in the endogenous ANF mRNA in the ventricle but no detectable expression of the T-antigen marker gene. This result provides evidence that atrial-specific and inducible expression of the ANF gene can be segregated, suggesting that a distinct set of regulatory cis sequences may mediate the up-regulation of the ANF gene during in vivo pressure overload hypertrophy. This murine model demonstrates the utility of microsurgical techniques to study in vivo cardiac physiology in transgenic mice and should allow the application of genetic approaches to identify the mechanisms that activate ventricular expression of the ANF gene during in vivo hypertrophy.

Topics: Animals; Antigens, Polyomavirus Transforming; Atrial Natriuretic Factor; Cardiomegaly; Disease Models, Animal; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression; Hemodynamics; Hypertension; Immediate-Early Proteins; Mice; Mice, Transgenic; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Transcription Factors

Inhibition of the metabolism of endogenous atrial natriuretic peptide (ANP), by continuous infusion of a specific inhibitor of neutral endopeptidase (membrane metalloendopeptidase E.C. 3.4.24.11), UK 73,967 (candoxatrilat), was undertaken in rats, in which chronic hypoxia was used as a stimulus to induce pulmonary hypertension and right ventricular hypertrophy. Inhibition of neutral endopeptidase 24.11 with low-dose and high-dose UK 73,967 (NEI) increased endogenous plasma ANP by greater than 155% during the development of pulmonary hypertension. NEI treatment reduced mean pulmonary arterial pressure in hypoxia as follows: vehicle 26.6 +/- 4.0 mm Hg; low-dose NEI 22.7 +/- 1.9 mm Hg, and high-dose NEI 22.6 +/- 2.5 mm Hg (both p less than 0.01 compared with hypoxic vehicle); however, it was without effect on pulmonary arterial pressure in normoxia (17.6 +/- 2.2 mm Hg) or on systemic blood pressure. The development of right ventricular hypertrophy was also reduced in both groups treated with NEI (right ventricular weight/left ventricular weight: 0.43 +/- 0.03 vehicle; 0.40 +/- 0.02 low-dose NEI and 0.40 +/- 0.02 high-dose NEI, both p less than 0.05 compared with vehicle). Remodelling of the pulmonary vasculature, characterized by extension of the muscle within the small pulmonary arteries toward the periphery of the lung, was reduced by NEI treatment (percentage of thick-walled peripheral vessels; 19.2 +/- 3.1% vehicle; 10.4 +/- 2.3% low-dose NEI and 8.1 +/- 1.8% high-dose NEI, both p less than 0.001 compared with vehicle). In the isolated blood perfused rat lung pulsed doses of NEI had no effect on pulmonary vascular tone in the absence of ANP. Specific inhibition of the enzyme neutral endopeptidase reduces vascular remodelling, the development of pulmonary hypertension, and right ventricular hypertrophy. Endogenous ANP modulates vascular remodelling in vivo. Retarding the metabolism of endogenous ANP through inhibition of neutral endopeptidase 24.11 represents a potential approach toward therapy. g

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cyclohexanecarboxylic Acids; Hypertension, Pulmonary; Hypoxia; Male; Neprilysin; Pulmonary Artery; Rats; Rats, Inbred Strains

We assessed the changes in atrial natriuretic peptide (ANP) and its messenger RNA (mRNA) levels in atria and ventricles in relation to hemodynamic factors during antihypertensive treatments in two-kidney, one-clip renovascular hypertensive rats (RHRs). Hypertension of 10-week duration caused a twofold increase in the left ventricular weight/body weight ratio, a significant increase in left ventricular end-diastolic pressure, and an eightfold increase in left ventricular ANP mRNA levels in RHRs, as compared with the levels in control rats. Uninephrectomy or 4 weeks of treatment with the converting enzyme inhibitor enalapril reduced the blood pressure to the control level, with the complete reversal of left ventricular hypertrophy, left ventricular end-diastolic pressure, and ANP mRNA levels. Four weeks of treatment with the arterial vasodilator hydralazine significantly, but not completely, reduced the high blood pressure, but it did not influence left ventricular hypertrophy, end-diastolic pressure, and ANP mRNA levels. The increased ANP synthesis observed in the right ventricles of RHRs also reverted to the control level by uninephrectomy or enalapril treatment, but not by hydralazine, with a time course similar to that of left ventricular ANP. In addition, uninephrectomy caused the left and right ventricular ANP and ANP mRNA levels of RHRs to fall to the levels of control rats as early as 1 week, despite persistent left ventricular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blotting, Northern; Cardiomegaly; Enalapril; Heart Ventricles; Hemodynamics; Hydralazine; Hypertension, Renovascular; Male; Nephrectomy; Rats; Rats, Inbred Strains; RNA, Messenger; Time Factors

The present study was designed to examine the effect of antihypertensive therapy on plasma and atrial concentration of atrial natriuretic peptides (ANP) in spontaneously hypertensive rats (SHR) by using alpha-methyldopa and hydralazine. Methyldopa and hydralazine treatment reduced blood pressure (P less than .05, P less than .05, respectively); however, ventricular weight was reduced by methyldopa (P less than .05) but not by hydralazine. Plasma ANP concentration in untreated SHR was higher than that observed in Wistar-Kyoto rats (WKY). Methyldopa treatment decreased plasma ANP concentration, but hydralazine treatment did not. Moreover, plasma ANP concentration and ventricular weight were positively correlated in untreated and treated SHR. The left atrial ANP concentration in untreated SHR was lower than that observed in WKY. Methyldopa treatment increased left atrial ANP concentration, but hydralazine treatment did not. These results suggest that the ANP release from the left atrium is chronically stimulated in adult SHR, and that a decrease in plasma ANP concentration by methyldopa treatment is, in part, associated with the decline of ANP release from the heart due to the reductions of blood pressure and cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Heart Atria; Heart Ventricles; Hydralazine; Hypertension; Male; Methyldopa; Organ Size; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors

1. Ventricular hypertrophy is characterized by stimulation of ventricular synthesis of atrial natriuretic peptide (ANP). To examine the role of ventricular ANP levels in the secretion of ANP into the circulation, atrial and ventricular levels of immunoreactive-ANP (IR-ANP) as well as ANP messenger RNA (mRNA), and the release of IR-ANP from isolated perfused hearts, both before and after atrialectomy, were measured simultaneously in control and minoxidil-treated Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. IR-ANP levels in the ventricles of untreated, 12 month-old SHR with severe ventricular hypertrophy were increased when compared to age-matched WKY rats. Minoxidil treatment for 8 weeks in both strains resulted in a decrease in mean arterial pressure and increases in ventricular weight to body weight ratios, plasma IR-ANP concentrations (in WKY from 133 +/- 20 to 281 +/- 34 pg ml-1, P less than 0.01; in SHR from 184 +/- 38 to 339 +/- 61 pg ml-1, P less than 0.05), and in ventricular IR-ANP contents (in WKY: 53%; in SHR: 41%). A highly significant correlation was found between ventricular IR-ANP content and ventricular weight to body weight ratio (r = 0.59, P less than 0.001, n = 26). 3. When studied in vitro, in isolated perfused heart preparations, the hypertrophied ventricular tissue after atrialectomy secreted more ANP into the perfusate than ventricles of the control hearts; ventricles contributed 28%, 22%, 18% and 15% of the total ANP release to perfusate in the minoxidil-treated SHR, control SHR, minoxidil-treated WKY and control WKY, respectively. A significant correlation was found between the IR-ANP release from ventricles and ventricular weight to body weight ratio (r = 0.56, P < 0.01, n = 24). 4. These studies demonstrate that the ventricles contribute substantially to the circulating level of ANP, and that the amount released depends on the degree of ventricular hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Body Weight; Cardiomegaly; Hemodynamics; In Vitro Techniques; Kidney; Male; Minoxidil; Myocardium; Norepinephrine; Organ Size; Osmolar Concentration; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Sodium

The beneficial effect of angiotensin-converting enzyme (ACE) inhibitors on myocardial mass and contractility in hypertension and, possibly, congestive heart failure (CHF) may be related to their ability to induce a decreased afterload. This has been assessed in four experimental models--renovascular hypertension, DOCA-salt hypertension, spontaneously hypertensive rats (SHR) and myocardial infarction (MI)--and in normotensive mature rats. In renovascular hypertension, ACE inhibitors normalized blood pressure as well as left ventricular hypertrophy and hypocontractility. In the DOCA-salt model, blockade of the renin-angiotensin system by ACE inhibitors did not decrease blood pressure and therefore had no effect on cardiac mass and contractility. In the SHR model, the arterial smooth muscle cell is functionally and structurally abnormal; as a result, cardiac overload led, over time, to a terminal, decompensated phase of CHF. ACE inhibitors, by decreasing blood pressure, reversed cardiac hypertrophy, hyperfibrosis and atrial natriuretic factor (ANF) oversecretion and prevented overload and time-induced CHF. In the MI model, ACE inhibitors decreased blood pressure and thereby decreased overload and reversed cardiac hypertrophy, hypocontractility, hyperfibrosis and ANF oversecretion. In normal ageing, heart function and structure are modified over time. ACE inhibitors, by blocking a 'normal' signal upstream, allowed a 'normal' effector system to decrease blood pressure and prevented the development of age-dependent cardiac hypertrophy.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Collagen; Disease Models, Animal; Endomyocardial Fibrosis; Gene Expression; Heart Failure; Hypertension; Indoles; Male; Myocardial Contraction; Myocardial Infarction; Myosins; Organ Size; Perindopril; Rats; Rats, Inbred SHR; Rats, Inbred Strains; RNA, Messenger

A radioimmunoassay was used to study the structural and functional status of the myocardium and blood natriuretic factor levels in 45 patients with hypertensive disease and 17 healthy subjects. Left atrial dysfunction was revealed to affect natriuretic factor levels in the blood of hypertensive patients with moderate left ventricular hypertrophy. As myocardial contractility became lower, natriuretic factor levels were reduced. The changes found in the levels in left ventricular hypertrophic variants may be accounted for by various values of intramyocardial stress. The findings suggest that cardiohemodynamic abnormalities may affect the secretion of natriuretic factor.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Female; Heart Ventricles; Hemodynamics; Humans; Hypertension; Male; Middle Aged; Myocardial Contraction; Myocardium

In order to define the factors responsible for cardiac hypertrophy in elderly hypertension, blood pressures and non-hemodynamic humoral parameters were correlated with echocardiographic left ventricular mass (LVM) in 30 elderly hypertensive patients and 30 age-matched normotensive subjects with a mean age of 65 years. Arterial blood pressures were obtained either at the clinic, after supine rest, during maximal exercise test, or with a 24 hour ambulatory monitoring device at 10 minute intervals. Interventricular septum and posterior wall thickness, but not end-diastolic diameter, were significantly increased in the hypertensives than in the normotensives. LVM was significantly correlated with all of te blood pressure parameters, having the strongest association with the mean of ambulatory systolic blood pressures (r = 0.65, p less than 0.001). On the other hand, plasma norepinephrine (r = 0.09), plasma renin activity (r = -0.14), and atrial natriuretic factor (r = -0.08), though known to influence cardiac adaptation to hypertrophy in young or middle aged hypertensives, were not correlated wit LVM. These results suggest that the heart in elderly hypertension is characterized by concentric hypertrophy, the only significant determinant of which appears to be a hemodynamic factor. Unlike younger patients, sympathetic nervous and renin-angiotensin systems may not play an important role in the development of cardiac hypertrophy in the elderly.

Topics: Aged; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Echocardiography; Female; Heart Ventricles; Humans; Hypertension; Male; Norepinephrine; Renin

The role of the calcium antagonist nisoldipine and the arteriolar vasodilator minoxidil on plasma levels of atrial natriuretic peptides (ANP), systolic blood pressure and heart weight was estimated in inbred Dahl salt sensitive (S) rats and inbred Dahl resistant (R) rats in long-term experiments. S rats develop quickly malignant hypertension, cardiac hypertrophy and have increased ANP plasma levels when fed a high salt diet (8% NaCl), while R rats on a high salt stay normotensive. In S rats 5 weeks on a high salt diet, therapeutic treatment with nisoldipine for 5 weeks not only decreased blood pressure but also produced a regression in cardiac hypertrophy and a reduction in elevated ANP plasma levels in comparison to the untreated salt-loaded S controls. Similar results were achieved in a preventive trial. In contrast with nisoldipine, therapeutic treatment with minoxidil in salt-loaded S rats lead to no reduction in cardiac hypertrophy and produced an additional increase in plasma ANP despite a reduction in blood pressure. The increase in plasma ANP level in this model of hypertension and its modulation by antihypertensive treatment with a calcium antagonist or an arteriolar vasodilator show that the changes in ANP plasma levels are probably secondary to hypertensive disease and the associated cardiac volume overload.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Male; Minoxidil; Nisoldipine; Rats; Renin; Sodium, Dietary

We have measured the rate of release of atrial natriuretic peptide in response to stretch and to isoproterenol from superfused atria isolated from rats after chronic in vivo infusion of either atrial natriuretic peptide or isoproterenol. The infusion lasted seven days, using minipumps filled with: 1) saline; 2) synthetic atrial natriuretic peptide to release 3 micrograms/kg/h; 3) l-isoproterenol HCl to release 400 micrograms/kg/h. Infusion of isoproterenol caused hypertrophy of the left chambers of the heart, a decrease of atrial content of atrial peptide with increased plasma levels. Infusion of atrial natriuretic peptide resulted only in elevated plasma levels of the peptide. Atria from animals infused with atrial natriuretic peptide responded to stretch and to isoproterenol (10(-9) M) with a prompt and transient release of peptide. Atria from animals infused with isoproterenol showed a severe reduction in sensitivity to the inotropic and chronotropic action of the drug. Their basal release of atrial peptide was extremely reduced and they were unable to respond to stretch or to isoproterenol. These data indicate that high circulating levels of atrial natriuretic peptide do not influence the release of the peptide from the atria, while high levels of isoproterenol drastically reduce it. Beta adrenoceptor desensitization, atrial hypertrophy and decrease of stores of atrial peptide are likely to account for this phenomenon.

Topics: Animals; Atrial Function; Atrial Natriuretic Factor; Cardiomegaly; Exocytosis; Heart Atria; Heart Failure; Isoproterenol; Male; Rats; Rats, Inbred Strains; Time Factors

1. To investigate the mechanisms leading to enhanced synthesis and release of atrial natriuretic factor during chronic hypoxia, we measured immunoreactive plasma atrial natriuretic factor, blood gases, packed cell volume, pulmonary artery pressure and systemic artery pressure in male Sprague-Dawley rats exposed to 1, 2 or 3 weeks of normobaric hypoxia. Rats were implanted with pulmonary and carotid artery catheters and studied conscious, 23 h after return to hypoxia. 2. The concentration of atrial natriuretic factor messenger RNA was measured in the right and left ventricular free walls of rats exposed to 3 weeks of hypoxia and in normoxic control rats. 3. There was a trend for plasma atrial natriuretic factor to increase with the duration of exposure to hypoxia but only the 3-week hypoxic rats had a significantly higher level (1080 +/- 193 pg/ml) than the normoxic control rats (318 +/- 46 pg/ml, P less than 0.05, mean +/- SEM). When all the data from normoxic and hypoxic rats were considered together, plasma atrial natriuretic factor was positively correlated with packed cell volume (r = 0.66, P less than 0.001), pulmonary artery pressure (r = 0.68, P less than 0.002), and the index of right ventricular hypertrophy (r = 0.54, P less than 0.01), but after analysis of partial correlation, packed cell volume was the only independent contributing factor to the variance in the level of plasma atrial natriuretic factor (r2 = 0.24).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Heart Ventricles; Hematocrit; Hypoxia; Male; Rats; Rats, Inbred Strains; RNA, Messenger

1. Atrial natriuretic peptide (ANP) levels were measured in cardiac tissues and in plasma from adult rats exposed to chronic alveolar hypoxia for periods of 2 h, 24 h and 7 days. Levels were also measured in rats that were maintained in hypoxia for 7 days and then returned to air for 24 h. 2. Plasma ANP was not altered at 2 h but was significantly increased at both 24 h and at 7 days. Plasma ANP in animals exposed to hypoxia for 7 days was normal 24 h after returning to air breathing, despite the persistence of indices of pulmonary hypertension. 3. No significant right atrial hypertrophy was observed under these conditions of chronic hypoxia. A reduction in right atrial ANP content was found at 24 h and was accompanied by a decrease in the number of electrondense granules per right atrial muscle cell. After exposure to hypoxia for 7 days, right atrial ANP and granule number was not different from control, and no alteration was found in right atrial ANP level after removal from the hypoxic environment. 4. No significant right ventricular hypertrophy was produced by exposure to hypoxia for 2 or 24 h. In the former group ventricular ANP had decreased significantly compared with control. Right ventricular hypertrophy was found in both the hypoxic groups after exposure for 7 days, when selective increases in right ventricular ANP content were found. 5. These findings are consistent with the hypothesis that ANP release occurs on exposure to chronic hypoxia and is independent of the associated cardiac hypertrophy and pulmonary vascular remodelling. The findings may have relevance to the natriuresis and reported changes in the renin-angiotensin-aldosterone axis under hypoxic conditions.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cytoplasmic Granules; Hypoxia; Male; Myocardium; Organ Size; Rats; Rats, Inbred Strains; Time Factors

Left ventricular hypertrophy is characterized by stimulation of ventricular synthesis of atrial natriuretic peptide (ANP). This study was designed to test the hypothesis that the increased ventricular ANP levels participate in the release of ANP into the circulation. Swimming was used as a physiologic model to induce ANP release from the heart, and atrial and ventricular levels of immunoreactive ANP (IR-ANP) and ANP messenger RNA (mRNA) were measured simultaneously in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at rest and after swimming. IR-ANP concentration in the left ventricle of 1-year-old SHR with severe left ventricular hypertrophy was increased in association with the augmentation of ANP mRNA levels, whereas right ventricular levels of ANP were reduced in SHR compared with normotensive controls. A 30-minute exercise in hypertensive and in normotensive rats resulted in marked increases in mean arterial pressure, heart rate, plasma catecholamine levels, blood lactate levels, and plasma IR-ANP concentration. The increased ANP secretion was associated with a decrease in left (34-39%) and right (24%) ventricular concentration of IR-ANP; transmurally, this depletion of ventricular IR-ANP was greatest (28%) in the endocardial layer of the left ventricle of SHR. No significant differences were noted in total atrial and left or right auricular IR-ANP concentration between SHR and WKY rats or between the resting and swimming rats. When studied in vitro with an isolated, perfused heart preparation, the hypertrophic ventricular tissue after atrialectomy secreted more ANP into the perfusate than did control hearts; in SHR, ventricles contributed 28% of the total ANP release to perfusate, and in normotensive control rats, ventricles contributed 8%. These studies show that stimulated release of ANP is associated with depletion of endocardial left ventricular stores. The amount of ANP released in vitro and in vivo correlated with the degree of hypertrophy of the ventricle. Finally, the phorbol ester, known to increase ANP secretion from intact perfused hearts, had only a limited effect on ANP release after atrialectomy, suggesting that the secretion of ANP from ventricular cells may be mainly of the constitutive type.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Catecholamines; Heart Ventricles; Hypertension; Lactates; Lactic Acid; Male; Myocardium; Physical Exertion; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Swimming

To evaluate determinants of elevated plasma atrial natriuretic factor levels in patients with hypertension, immunoreactive plasma atrial natriuretic factor in 54 normal subjects and 40 untreated hypertensive patients was compared with echocardiographic measurements of cardiac size, function and systemic hemodynamics. In normal subjects, plasma atrial natriuretic factor was related to age, systolic blood pressure and left atrial and ventricular chamber sizes, but only age and ventricular size were independent predictors. In untreated hypertensive patients, atrial natriuretic factor was directly related to age, atrial size, systolic pressure, peripheral resistance and ventricular systolic performance; age, atrial size and peripheral resistance were independent predictors. Eight patients with elevated atrial natriuretic factor values (greater than 25 fmol/ml) were significantly (p less than 0.01) older and had greater atrial and ventricular size and higher systolic pressure and function than normal subjects or patients with normal natriuretic factor levels. Plasma atrial natriuretic factor was inversely related to peak diastolic filling rate in normal subjects (r = -0.59; p less than 0.001), whereas it was positively related to the proportional contribution of atrial systole to left ventricular filling in hypertensive patients (r = 0.77; p less than 0.001). These findings suggest that in normal subjects, impairment of ventricular relaxation with age may contribute to atrial natriuretic factor secretion by increasing left atrial afterload; the correlation with left ventricular size may reflect physiologic fluctuations in plasma volume. In patients with uncomplicated hypertension, left atrial enlargement and consequent stronger atrial contraction contributed to increased atrial natriuretic factor release, whereas no independent relation existed with left ventricular hypertrophy or systolic function. Because ventricular relaxation was normal and ventricular size and systolic performance were increased in hypertensive patients with high atrial natriuretic factor levels, the observed increase in left atrial size and atrial contribution to ventricular filling might reflect a primary increase in venous return in this subset of hypertensive patients.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Diastole; Female; Hemodynamics; Humans; Hypertension; Male; Middle Aged; Systole

Elevation of the plasma concentrations of immunoreactive atrial natriuretic peptide (ANP) was observed in canine chronic right heart failure (RHF) secondary to right ventricular (RV) pressure overload. Pressure overload on the right heart was gradually increased using an inflatable cuff. The interval between banding and the onset of RHF was 152 +/- 52 days. Seventeen RHF dogs were produced and divided into Group I (n = 11) and Group II (n = 6). At the onset of RHF, Group I dogs were either sacrificed for study of the heart, or unbanded to allow recovery from RHF. The dogs in Group II were maintained in RHF for 3 additional months before being either sacrificed or unbanded. Following unbanding, the ANP level of Group I recovered from 108 +/- 36 (n = 11) to 20 +/- 6 pg/ml (n = 6) at 1 month and was maintained at 27 +/- 7 pg/ml (baseline, 21 +/- 5 pg/ml, n = 11) at 4 months. ANP levels of Group II declined from 165 +/- 55 (n = 6) to 87 +/- 2 pg/ml (n = 3) at 1 month and further decreased to 42 +/- 14 pg/ml (n = 3) 4 months after unbanding. Thus, compared to Group I, Group II had a high ANP level before unbanding and a delay in recovery of the ANP levels despite normalization of the right atrial pressure (RAP). Four months after release of pressure overload, the right atrial hypertrophy persisted in the unbanded dogs; however, Group I unbanded dogs showed a better reversal of the right atrial hypertrophy than Group II unbanded dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Biomarkers; Cardiomegaly; Disease Models, Animal; Dogs; Heart Failure; Heart Ventricles; Organ Size; Ventricular Function

Activation of atrial natriuretic factor (ANF) gene expression has been reported in the rat ventricle in several models of hemodynamic overload, including hypertension. However, nothing is known about the potential trigger(s) and the time course of this activation during the development of hypertension. We measured aortic blood pressure, left ventricular hypertrophy (LVH), and left ventricular ANF mRNA concentration (LV ANF mRNA) in a first group of rats (study A) killed at 5 and 18 h and 2, 4, 6, 9, 15, and 30 days after suprarenal coarctation of the abdominal aorta. Coarctation induced a progressive rise in aortic blood pressure and left ventricular mass. We observed a biphasic accumulation of ANF mRNA in the left ventricle with a peak at day 4 averaging 20 times the control value long before stable hypertension and hypertrophy were achieved, followed by a decrease until day 9. This decrease was followed by a new rise, which stabilized around 10 times the control value seen during stable hypertension and hypertrophy. In a second group of rats killed at days 4 and 30 (study B), we determined, in addition to the previous parameters, left ventricular end-diastolic pressure (LVEDP), plasma renin (PRC), and plasma ANF concentrations. LVEDP and PRC were markedly increased at day 4, but at day 30, during stable hypertension and hypertrophy, these parameters returned to control values, whereas plasma ANF was increased. Using immunocytochemistry, we looked in a third group of rats (study C) for the presence of the immunoreactive peptide at days 4 and 30.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta; Aortic Coarctation; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Gene Expression Regulation; Heart; Heart Ventricles; Hypertension; Immunohistochemistry; Male; Myocardium; Rats; Renin; RNA, Messenger; Time Factors

To investigate whether or not hypertension with left ventricular hypertrophy (LVH) modifies the mechanisms underlying the vascular adjustments to orthostatic stress, we evaluated the hemodynamic and hormonal effects of graded lower-body negative pressure (LBNP) (-10 and -40 mm Hg) before and after sympathetic blockade in 10 hypertensive patients with LVH and in five age- and sex-matched normotensive subjects. In control conditions, LBNP elicited comparable vasoconstrictor responses in the forearm in the two groups. In normotensive subjects, graded increases in plasma norepinephrine and plasma renin activity (PRA) and reductions in plasma immunoreactive atrial natriuretic factor (irANF) were recorded. In hypertensive patients, a significant increase in plasma norepinephrine and plasma renin activity was obtained only with the higher level of LBNP, whereas irANF plasma levels decreased progressively. In both groups, sympathetic blockade abolished the increase in plasma renin activity and did not modify the changes in plasma irANF induced by both levels of LBNP in control conditions. The vascular response to -10 mm Hg LBNP remained unchanged after sympathetic blockade in both groups. However, after sympathetic blockade, the vasoconstrictor response to -40 mm Hg LBNP in normal subjects was no longer different from that elicited by -10 mm Hg LBNP, whereas in hypertensive patients the vasoconstrictor response was still significantly higher than that induced by -10 mm Hg LBNP. Direct correlations between the percent changes in forearm vascular resistance and those in plasma norepinephrine and plasma renin activity were found only in normal subjects in control conditions but were not observed after sympathetic blockade. On the contrary, the inverse correlation between changes in irANF plasma levels and in forearm vascular resistance found in control conditions in both groups was still observed after sympathetic blockade. In a separate group of hypertensive patients with left ventricular hypertrophy, exogenous infusion of ANF induced an increase in venous irANF plasma levels of the same magnitude of the decrease evoked by LBNP and significantly reduced forearm vascular resistance. These data show that in hypertensive patients with left ventricular hypertrophy, sympathetic activation does not contribute to the vascular response to cardiopulmonary receptor unloading (-10 mm Hg LBNP). They also suggest that in these patients inhibition of ANF secretion may play a rol

Topics: Adaptation, Physiological; Adult; Atrial Natriuretic Factor; Autonomic Nerve Block; Cardiomegaly; Female; Heart; Hemodynamics; Hormones; Humans; Hypertension; Lower Body Negative Pressure; Male; Middle Aged; Pressoreceptors; Pulmonary Artery; Sympathetic Nervous System

To investigate the mechanism of expression of atrial natriuretic polypeptide (ANP) in human ventricles, we conducted an immunohistochemical study of ANP in biventricular endomyocardial biopsy specimens obtained from a total of 49 patients with cardiac dilatation due to dilated cardiomyopathy (21 patients), postmyocarditis (18 patients), or volume overload (five patients) and subjects with no dilatation as controls (five patients). Four-micron thick sections were stained by an indirect immunoperoxidase method using monoclonal antibody to alpha-human ANP as the primary antibody. The frequency of ANP-present myocytes was calculated in each specimen and compared with clinical, echocardiographic, hemodynamic, angiographic, and histologic parameters. ANP-present myocytes were noted in all of the 21 patients with dilated cardiomyopathy, in 11 of the 18 patients with postmyocarditis, in four of the five patients with volume overload, and in zero of the five controls. The mean percentage of ANP-present myocytes was significantly greater in the left-side specimens (35 +/- 37%) than in the right-side ones (2 +/- 4%). The percentage of ANP-present myocytes in the left-side specimens significantly correlated with peak systolic or end-diastolic wall stress (r = 0.67 and 0.58), left ventricular end-systolic or end-diastolic volume index (r = 0.75 and 0.69), or left ventricular end-diastolic pressure (r = 0.42) and inversely correlated with ejection fraction (r = -0.73), systolic left ventricular wall thickness (r = -0.58), or cardiac index (r = -0.30). Expression of ANP was rarely seen in the cases with normal wall stresses, normal ejection fraction, normal volume, or normal myocyte size. However, it was seen frequently even in hearts with normal levels of left ventricular end-diastolic pressure and cardiac index (compensated hearts). The percent of ANP-present myocytes in both sides significantly correlated with size of myocytes (r = 0.48 at right and r = 0.57 at left side) or degree of fibrosis (r = 0.45 at right and r = 0.48 at left side). These results suggest that ANP expression is augmented in the dilated ventricles regardless of the causes of dilatation and that the augmentation is a compensatory mechanism as prevention against decompensation responding to reduced contractility, excess of wall stresses, or both, concomitantly occurring with cardiac dilatation and myocardial hypertrophy.

Topics: Adult; Atrial Natriuretic Factor; Biopsy; Cardiomegaly; Cardiomyopathy, Dilated; Female; Heart Ventricles; Hemodynamics; Humans; Immunoenzyme Techniques; Male; Myocarditis; Myocardium

We report a rare case of giant right atrial dilatation in a patient with Marfan's syndrome complicated by severe tricuspid regurgitation. Interestingly, the right atrial pressure was normal, but the atrial natriuretic peptide level was markedly elevated. We suggest that the underlying connective tissue abnormality had allowed extensive atrial distension which by causing an increased intramural tension according to Laplace's law stimulated atrial natriuretic peptide secretion.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Female; Heart Atria; Humans; Marfan Syndrome; Tricuspid Valve Insufficiency

Cardiac hypertrophy secondary to chronic hemodynamic overload is associated with an increase in the ventricular concentration of the messenger ribonucleic acid (mRNA) coding for the atrial natriuretic factor (ANF). We have compared, in male Wistar rats (10 week old, 200-220 g), using dot blot hybridization and a specific oligonucleotide probe, the left ventricular concentration of ANF mRNA (LV ANF mRNA) in 4 models of chronic hemodynamic overload inducing various patterns of left ventricular hypertrophy (LVH): a model of volume overload, the aortocaval fistula (ACF, n = 15); a model of pressure overload, coarctation of the abdominal aorta (CoA, n = 13) and 2 models of mixed overload, aortic regurgitation (AR, n = 7) and myocardial infarction (INF, n = 18). A month after surgery, LVH was 49 p. 100 for AR, 41 p. 100 for Co A and 21 p. 100 for ACF. Instead of a severe infarction, LVH was 6 p. 100 in INF demonstrating a marked hypertrophy of the non infarcted myocardium. For each model, LV ANF mRNA was compared to that in a corresponding group of sham-operated control rats and expressed as the percentage of ANF mRNA concentration in the pooled atria of the controls. In the 4 control groups LV ANF mRNA was 1 +/- 0.5 p. 100 that in the corresponding atria and the sham-operated animals were thus pooled in a single group (n = 19). In the 4 models of LVH, LV ANF mRNA markedly increased as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart Ventricles; Humans; Male; Models, Biological; Rats; Rats, Inbred Strains; RNA, Messenger

Rats given monocrotaline develop severe right ventricular hypertrophy often accompanied by ascites and pleural effusions. In rats with right ventricular hypertrophy and no serous effusions ("hypertrophy" group), ventricular concentrations of noradrenaline were reduced but ventricular contents were unchanged. Atrial concentrations of noradrenaline were unaffected. Those with more severe right ventricular hypertrophy and serous effusions ("failure" group) had greatly reduced concentrations of noradrenaline in all four chambers, particularly on the right side; the right and left ventricular contents of noradrenaline were also diminished. The distributions of ir-ANP, ir-bombesin and ir-neurotensin in the normal rat heart are presented. ANP concentration fell to 33% in the right atrium and 46% in the left atrium of "failure" animals and to 57% in the right atrium of "hypertrophy" animals. Right ventricular content of ANP, normally low, increased more than two-fold in both groups, the concentration remaining unchanged. Left ventricular content of ANP decreased in the "failure" group. Concentrations of bombesin and neurotensin fell in both ventricles of both groups. Ventricular contents of bombesin did not change, but ventricular contents of neurotensin decreased, especially on the right side. Plasma ANP rose nearly six-fold while plasma bombesin and neurotensin fell in the "failure" group. Plasma peptide concentrations were unchanged in the "hypertrophy" group. The studies show the utility of the monocrotaline model in distinguishing between the effects of hypertrophy and those associated specifically with the syndrome of congestive cardiac failure.

Topics: Animals; Atrial Natriuretic Factor; Bombesin; Cardiomegaly; Heart Atria; Heart Failure; Heart Ventricles; Monocrotaline; Myocardium; Neurotensin; Norepinephrine; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Strains

The influence of physiological cardiac hypertrophy on the concentration of plasma atrial natriuretic peptide was studied in six male athletes and six normally active, matched control men. They were examined by echocardiography during a graded exercise test on a bicycle ergometer. Plasma atrial natriuretic peptide was measured at rest, at each workload until exhaustion, and 15 and 30 minutes after the exercise test. Echocardiography showed that the athletes had a significantly larger left atrium, left ventricular end diastolic diameter, left ventricular posterior wall, interventricular septum, left ventricular ejection fraction, and left ventricular mass than the controls. The athletes performed significantly more work than the control group--325 W v 277 W. The plasma concentration of atrial natriuretic peptide rose by a mean factor of 2.76 (range 1.78-4.28) in all men from rest to maximum exercise. There were no differences between the athletes and the controls in the concentrations of plasma atrial natriuretic peptide at rest, at any workload, or at maximum workload. Neither was there any difference in the increase in plasma atrial natriuretic peptide between the groups. There was no correlation between the plasma concentrations of atrial natriuretic peptide and any of the variables measured by echocardiography. In healthy young men plasma atrial natriuretic peptide rises by a factor of about 2.8 during maximum exercise and the size of the chambers on the left side of the heart or left ventricular hypertrophy does not seem to influence the concentration of plasma atrial natriuretic peptide at rest or during exercise.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Humans; Male; Physical Exertion; Rest

In a model of pulmonary hypertension induced by a single injection of monocrotaline (MCT), we observed a time-dependent right ventricular hypertrophy, which became apparent in treated rats 21 days after administration of MCT and progressed through day 45. Associated with this right ventricular hypertrophy were time-dependent increases in ventricular levels of immunoreactive atrial natriuretic peptide (iANP). Forty-five days after MCT treatment, treated rats exhibited a 72-fold increase in right ventricular iANP levels and a 7-fold increase in left ventricular iANP levels. Hybridization analysis of total RNA extracted from cardiac tissue indicated that both atrial and ventricular ANP mRNA levels were elevated in treated rats. These data suggest that during pulmonary hypertension and cardiac hypertrophy the endocrine activity of the heart expands to include ventricular tissue. ANP binding site autoradiography revealed decreased binding site density in the kidney and hearts of treated rats at 49 days, consistent with the occurrence of desensitization/down-regulation. Enhanced ventricular ANP production may serve as a compensatory response to sustained elevation of pulmonary arterial pressure or may function as an autocrine/paracrine system regulating cardiac function. In either case, the effects of augmented ANP production may be subject to modulation by the status of ANP receptors in target organs and cells.

Topics: Animals; Atrial Natriuretic Factor; Autoradiography; Cardiomegaly; Hypertension, Pulmonary; Immunoblotting; Male; Monocrotaline; Myocardium; Pyrrolizidine Alkaloids; Radioimmunoassay; Rats; Rats, Inbred Strains; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; RNA, Messenger

The plasma concentration of atrial natriuretic peptide (ANP) and the density and affinity of binding sites for ANP in platelets was investigated in patients with essential hypertension. Severe hypertensives were studied whilst still on medication. All subjects were ambulatory and were investigated after 3 days on a 135 mmol/day sodium intake. Plasma ANP levels were significantly increased from 13 +/- 1 fmol/ml in healthy normotensive controls to 39 +/- 5 fmol/ml (P less than 0.01) in moderate or severe hypertensives uncontrolled by treatment. Platelet binding sites varied in a non-linear inverse relationship to plasma concentration of ANP (r = -0.76), from 14 +/- 1 fmol per 10(9) platelets in healthy subjects to 8 +/- 1 fmol per 10(9) platelets in severe hypertensives, uncontrolled by treatment, (P less than 0.05). The latter group with elevated plasma ANP and reduced density of ANP platelet sites, had a high incidence of left ventricular hypertrophy and increased left ventricular mass index by echocardiography. When a diuretic was added or stopped, plasma ANP and ANP sites in platelets varied inversely, with lower ANP concentration in plasma and higher density of ANP receptors in platelets when receiving the diuretic. Plasma concentrations of ANP are increased and the density of ANP receptors is decreased in moderate to severe uncontrolled essential hypertensives with left ventricular hypertrophy, but neither parameter differs from those of healthy age-matched volunteers in mild essential hypertension.

Topics: Adult; Atrial Natriuretic Factor; Binding Sites; Blood Platelets; Cardiomegaly; Echocardiography; Female; Humans; Hypertension; Male; Middle Aged; Radioimmunoassay; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface

To evaluate the role of extra-atrial atrial natriuretic peptide (ANP) in volume and blood pressure regulation, the plasma, atrial, ventricular, and hypothalamic levels of immunoreactive atrial natriuretic peptide (IR-ANP) were measured simultaneously in the spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at the ages of 2, 6, and 12 months. Plasma IR-ANP in the 12-month-old, conscious SHR was significantly higher than that of the WKY (300 +/- 18 versus 200 +/- 20 pg/ml, p less than 0.05, n = 9), while no differences in plasma IR-ANP levels were found between the strains in younger rats. Acute volume expansion with saline (1.1 ml/100 g body wt) in hypertensive as well as in normotensive rats resulted in marked increases in right atrial pressure and plasma IR-ANP concentration. The older SHR had attenuated ANP release to volume loading as shown by the shift of the ANP versus right atrial pressure curve to the right. Right auricular IR-ANP concentration decreased, while that of left auricle increased with increasing age in both strains. No substantial differences were noted in auricular ANP concentration between SHR and WKY. However, the total atrial IR-ANP content (micrograms/atria) was consistently lower in SHR compared with WKY. In both ventricles, IR-ANP concentrations and contents increased with increasing age in WKY and SHR, but the ventricular levels of ANP were reduced in ventricles of the SHR heart compared with normotensive controls. The depletion of total ventricular IR-ANP was greatest in SHR with greatest ventricular hypertrophy and coincided with the attenuated ANP release to acute volume load. The increase of left but not right ventricular weight occurring secondary to 6 weeks minoxidil treatment was accompanied by higher ANP concentration in both strains. In contrast to the ventricles, the hypothalamic IR-ANP concentration was significantly increased in SHR compared with that of WKY and decreased in both strains after 6 weeks' treatment with antihypertensive drugs. Thus, ventricular and hypothalamic, as well as atrial, ANP respond to increased pressure overload in genetically hypertensive rats. Our results suggest that chronic stimulation of ANP release from ventricles is associated with depleted stores of ANP from both ventricles and reduced response to acute volume load. Our findings that ventricular ANP increased with increasing weight and in response to a hypertrophic stimulus in WKY and was decreased in SHR with severe v

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Heart Rate; Hypertension; Hypothalamus; Kidney; Male; Methyldopa; Minoxidil; Myocardium; Organ Size; Rats; Rats, Inbred SHR; Rats, Inbred WKY

A subset of Wistar-Kyoto (WKY) rats that spontaneously develops biventricular hypertrophy (BVH) in response to increased cardiac output was evaluated for ventricular expression of the atrial natriuretic factor (ANF) gene. Normal WKY rats had low levels of left ventricular ANF mRNA and minimally detectable ANF transcripts in the right ventricle. In contrast, BVH rats showed a sixfold greater ANF mRNA concentration in the left ventricle than age-matched WKY controls. BVH right ventricular ANF mRNA levels equaled those found in BVH left ventricles and were dramatically greater than WKY right ventricular controls. Unlike experimental models of hypertrophy, both left and right ventricles significantly increase ANF gene transcripts in the natural development of BVH. The left and right ventricles can concordantly respond to hypertrophy and increase ANF gene transcription.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Gene Expression Regulation; Heart Ventricles; Radioimmunoassay; Rats; RNA, Messenger

Hypertrophy, an increase in cell size without cell division, is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle cells. Cardiac myosins undergo an adult-to-fetal isoform transition in various models of hypertrophy. Using gene-specific cDNA probes, we show here that in the adult myocardium the mRNAs encoding the fetal (skeletal muscle type) isoforms of alpha-actin and sarcomeric tropomyosin are re-expressed within 2 days in response to pressure overload. In addition, atrial natriuretic factor mRNA, so far believed to be expressed primarily in the atria, was readily detectable in the ventricles of neonates and was induced to markedly high levels in pressure-overloaded adult ventricles. In contrast, cardiac hypertrophy produced by thyroid hormone excess was not associated with induction of the atrial natriuretic factor gene or fetal contractile protein isogenes. Furthermore, the c-fos and c-myc protooncogenes and a major heat shock protein gene (hsp70) are induced in the ventricular myocardium within 1 hr after imposition of pressure overload. These results suggest that induction of cellular protooncogenes and heat shock (stress) protein genes is an early response to pressure overload, whereas reinduction of the genes normally expressed only in perinatal life, such as fetal isoforms of contractile proteins and atrial natriuretic factor, is a later event. These two types of responses might represent the general pattern of growth induction to work overload by terminally differentiated cells that have lost the ability to undergo DNA replication.

Topics: Aging; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Fetus; Gene Expression Regulation; Genes; Heart; Hyperthyroidism; Myocardium; Myosins; Proto-Oncogenes; Rats; Rats, Inbred Strains; Reference Values; RNA, Messenger; Transcription, Genetic; Triiodothyronine

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cardiomyopathy, Hypertrophic; Cricetinae; Genetic Markers; Humans

Right ventricular hypertrophy produced in rats exposed to 10% oxygen for 3 weeks resulted in a ninefold increase in atriopeptin immunoreactivity (APir) and a 160-fold increase in atriopeptin messenger RNA (AP mRNA) in the right ventricular myocardium. A small but significant increase in left ventricular APir and AP mRNA was also present, probably representing the interventricular septum. Right atrial APir was decreased by 50%, but left atrial APir was not different from normoxic controls. Purification of ventricular tissue extracts by high-performance liquid chromatography revealed primarily the high molecular weight prohormone. The development of right ventricular hypertrophy and right ventricular APir content followed a similar time course, each evident at 7 days of hypoxia and reaching a plateau at 14 days. Hypoxia followed by normoxia caused right ventricular APir to fall to control levels within 3 days, despite persistent right ventricular hypertrophy. This data demonstrates that hypoxia can reversibly induce extra-atrial expression of atriopeptin synthesis in the cardiac ventricle.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart Ventricles; Hypoxia; Male; Myocardium; Rats; RNA, Messenger

Topics: Atrial Natriuretic Factor; Cardiomegaly; Heart Atria; Humans; Tachycardia, Supraventricular

Atrial natriuretic peptide (ANP) is a newly discovered peptide hormone present mainly in the atria. We investigated the occurrence and distribution of ANP immunoreactivity in the myocardiocytes of the ventricles of spontaneously hypertensive rats by use of immunocytochemistry at both light and electron microscopic level. ANP immunoreactivity was found in the specific granules in the cytoplasm of the cardiocytes in the subendocardium and the myocardium of the ventricles, as well as in the atria. The specific granules found in the ventricles of hypertensive rats were similar in size, shape, and ANP immunoreactive content to those in the atria. The abundance of ANP immunoreactivity in the left ventricle is greater than that in the right, and appears to increase with increasing severity of hypertension. Conversely, the overall content of ANP in the atria of hypertensive rats was decreased when compared with that in age-matched normotensive rats. The present findings indicate that ventricles may become a major source for ANP synthesis and release during hypertension, and may play important roles in cardiac endocrine pathology and cardiac hypertrophy.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart Atria; Heart Ventricles; Hypertension; Immunohistochemistry; Microscopy, Electron; Myocardium; Rats; Rats, Inbred SHR; Rats, Inbred WKY

In order to investigate the role of cardiac hypertrophy in atrial natriuretic peptide (ANP) secretion in patients with essential hypertension, plasma levels of ANP were measured after overnight rest in 36 patients with untreated hypertension and in 31 normotensive controls. In the hypertensive subjects, plasma levels were correlated with left ventricular (LV) and left atrial abnormalities detected by chest X-ray, electrocardiogram (ECG) and M-mode echocardiography. Plasma ANP levels in patients with hypertension averaged 146 +/- 27 pg/ml compared to 46 +/- 7 pg/ml in the normotensive subjects (P less than 0.001). In patients with hypertension a significant correlation was found between ANP and supine systolic blood pressure (r = 0.54, P less than 0.001) and between ANP and diastolic blood pressure (r = 0.38, P less than 0.05). Furthermore, plasma ANP levels were correlated with total heart volume (r = 0.68, P less than 0.01), LV mass (r = 0.525, P less than 0.001), LV posterior wall thickness (r = 0.39, P less than 0.05), Sokolow-Lyon index (r = 0.721, P less than 0.001) and end-diastolic diameter of the left atrium (r = 0.334, P less than 0.05). The results suggest a contribution of LV and left atrial abnormalities to ANP secretion in essential hypertension.

Topics: Adult; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Electrocardiography; Female; Humans; Hypertension; Male; Middle Aged

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Cloning, Molecular; Gene Expression Regulation; Genes; Humans; Mice; Myocardium; Nucleic Acid Hybridization; Polymorphism, Genetic; Rats

The effects of acute exercise on plasma concentrations of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), and plasma renin activity (PRA) were studied in 13 patients with previously untreated essential hypertension, and 8 matched normotensive control subjects. Resting levels of ANP and PRA were similar in the two groups, while resting AVP concentrations were 1.4 times higher in hypertensive subjects. Graded exercise was performed on a bicycle ergometer with workload increased each minute until exhaustion (Wmax). Wmax was higher in normal subjects than in hypertensive patients. Blood pressure and heart rate rose more steeply in hypertensive patients. Plasma ANP increased during acute exercise in both groups, but the average increase in hypertensives was substantially greater than in normal subjects (P less than 0.05). The increase in ANP during exercise was greater in hypertensives with left ventricular (LV) hypertrophy, and there was a positive correlation between LV mass and the percentage rise in ANP during exercise (r = 0.56, P less than 0.005). Plasma AVP did not alter during exercise. Plasma renin concentrations showed a small rise during exercise in both groups, which was 16% less in hypertensive subjects (P less than 0.05). The enhancement of ANP release during exercise in hypertensive subjects may reflect both cardiac structural changes and increased redistribution of blood to the cardiopulmonary compartment.

Topics: Arginine Vasopressin; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Humans; Hypertension; Middle Aged; Physical Exertion; Renin

The relationship between plasma atrial natriuretic factor (ANF), blood pressure (BP), age, plasma renin activity (PRA) and urinary sodium excretion was studied in 64 normal subjects (mean age 48.7 +/- 2.1 yrs; BP: 126.5 +/- 1.6/79.5 +/- 0.9 mmHg) and in 104 untreated uncomplicated essential hypertensives (50.8 +/- 1.1 yrs; BP: 164.7 +/- 1.6/105.2 +/- 0.6 mmHg). ANF was measured by radioimmunoassay after extraction on C18 columns. ANF was significantly higher in the hypertensives than in the normal subjects (37.1 +/- 1.2 vs 29.7 +/- 1.5 pg/ml, P less than 0.01). In normals plasma ANF was significantly correlated with age (r = 0.72, P less than 0.001), Na excretion (r = 0.42, P less than 0.001) and PRA (r = -0.71, P less than 0.001) whereas in the hypertensives ANF plasma levels correlated only with systolic (r = 0.46, P less than 0.001) and diastolic (r = 0.51, P less than 0.001) BP. In addition in hypertensive patients, by multivariate linear regression analysis, a significant correlation was found between age, known duration of hypertension and plasma ANF. The partial correlation coefficient between duration of hypertension and plasma ANF was highly significant (r = 0.80, P less than 0.001). These findings suggest that in essential hypertension the level of arterial BP is a main determinant of the ANF plasma values offsetting the ability of other physiological factors to regulate plasma ANF levels.

Topics: Aged; Atrial Natriuretic Factor; Cardiomegaly; Female; Humans; Hypertension; Male; Middle Aged; Radioimmunoassay; Renin-Angiotensin System

Echocardiographically determined left ventricular function and cardiovascular hormone balance were assessed before and after hemodialysis in 10 patients who had been on hemodialysis for 4 months to 15 years. Plasma levels of atrial natriuretic peptide (ANP), antidiuretic hormone (ADH), renin activity and aldosterone were determined. All patients had vector- and echocardiographic evidences of slight to moderate left ventricular hypertrophy. The body weight decreased 2.0 kg (3.3 +/- 0.5%) with dialysis. Nine out of ten patients showed a slightly reduced ejection fraction that normalized after dialysis (p less than 0.05). Left atrial and ventricular systolic dimensions were around the upper reference limit before dialysis with a decrease after dialysis (p less than 0.05 and p less than 0.02, respectively). The levels of ANP decreased with dialysis from 2-17 times to 1 to 15 times the upper reference value in nine out of the ten patients. In the whole group the decrease was 117 +/- 35% (p less than 0.005). A significant regression was obtained between percentage decrease of body weight and percentage change of ANP (r = 0.67; p less than 0.05). The plasma concentration of ADH did not change following dialysis but the mean value was significantly higher than the mean value of the reference group of the laboratory (p less than 0.05 before and p less than 0.005 after dialysis). Renin activity and aldosterone levels were low and did not change during dialysis. In conclusion, the slight left ventricular hypertrophy may partly be a response to volume overload with hyperdynamic circulation and partly to metabolically depressed myocardial function.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Aldosterone; Atrial Natriuretic Factor; Cardiomegaly; Echocardiography; Female; Heart; Humans; Kidney Failure, Chronic; Male; Middle Aged; Renal Dialysis; Renin; Vasopressins

Left ventricular hypertrophy or treatment with dexamethasone caused a 2.5-fold to threefold increase in both immunoreactive atriopeptin (AP) and AP messenger RNA (mRNA), primarily in left ventricular tissue. The combined treatments increased immunoreactive AP and AP mRNA more than either treatment alone. In the animals in which cardiac hypertrophy had been produced by abdominal aortic constriction, there was a decrease in atrial levels of AP and an increase in plasma levels of immunoreactive AP. The increase in left ventricular immunoreactive AP was confirmed by immunohistochemical staining of tissue from hypertrophied and/or dexamethasone-treated rats. The mRNA accumulated in the left ventricle was identical to atrial AP mRNA, as judged by transcriptional start site and by size on Northern blots. Because the mass of ventricular tissue is substantially greater than that of atrial tissue, the induced mRNA levels may represent a total abundance approaching one third of the total AP mRNA in the atria. High performance liquid chromatographic purification of ventricular extracts primarily demonstrated the presence of the high molecular precursor and small amounts of C-terminal peptide AP. Induction of ventricular AP (mRNA and peptide) may represent regression of the tissue to an earlier developmental form. These data provide a unique example of regulation of AP biosynthesis in nonatrial tissue.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Chromatography, High Pressure Liquid; Dexamethasone; Fluorescent Antibody Technique; Histocytochemistry; Male; Myocardium; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger; Templates, Genetic

We have compared atrial and plasma concentration of atrial natriuretic factor (ANF) in 4 models of non spontaneous experimental hypertension with different pathogenic mechanisms in the rat: two-kidney, one-clip (2-K, 1-C), one-kidney, one-clip (1-K, 1-C), DOCA-NaCl and adrenal regeneration hypertension (ARH) and their respective normotensive controls. All hypertensive groups developed cardiac hypertrophy. In all hypertensive groups plasma ANF was higher than in controls. Atrial ANF concentration was lower in the right and left atrium of 1-K, 1-C rats and in the left atrium of ARH. A good correlation was found between systolic BP and plasma ANF in 2-K, 1-C (r = 0.82; p less than 0.01) and 1-K, 1-C animals (r = 0.70; p less than 0.01). This correlation was less good in DOCA-NaCl (r = 0.41; p less than 0.05) and non existent in ARH (r = 0.28; NS). A negative correlation between plasma ANF and atrial ANF concentrations was found only in the 1-K, 1-C group (r = 0.41; p less than 0.05). A good correlation between plasma ANF levels and cardiac weight was found in all groups: 2-K, 1-C (r = 0.83; p less than 0.01), 1-K, 1-C (r = 0.73; p less than 0.01), DOCA-NaCl (r = 0.69; p less than 0.01) and ARH (r = 0.71; p less than 0.01). We suggest that the release of ANF in experimental hypertension depends of the pathogenesis and could be related either to the level of BP (hence the magnitude of the left ventricular end-diastolic pressure) or to the existence of an expanded blood volume. The correlation between plasma ANF levels and cardiac hypertrophy suggests that ANF could be partially released by the ventricles.

Topics: Adrenal Glands; Animals; Atrial Natriuretic Factor; Cardiomegaly; Desoxycorticosterone; Hypertension; Hypertension, Renal; Male; Nephrectomy; Rats; Rats, Inbred Strains; Regeneration; Sodium Chloride

The atria produce several peptides that have natriuretic and vasoactive properties, collectively called atrial natriuretic factor. All these peptides share a single messenger ribonucleic acid, the amount of which greatly increases in the rat left ventricle when the latter is submitted to chronic volume overload. Using the molecular hybridization technique and a desoxyribonucleic acid probe complementary to the atrial natriuretic factor messenger ribonucleic acid, we now report that a very important increase in the amount of this messenger ribonucleic acid is also observed in rat ventricle at at the compensatory stage of a pressure overload induced cardiac hypertrophy. This result suggests that the pressure overload hypertrophied rat ventricle also has the potential to itself regulate it's loading conditions via the regulation of extracellular fluid volume and vascular resistance.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Heart; Heart Ventricles; Myocardium; Organ Size; Rats; Rats, Inbred Strains; RNA, Messenger

The effect of long-term treatment with the calcium antagonist nisoldipine on development of hypertension, cardiac hypertrophy, and plasma levels of atrial natriuretic peptides (ANP) was determined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) of the same age. Measurement of immunoreactive ANP in plasma provided a sensitive marker for the severity of hypertension and the associated cardiac overload. Long-term treatment with nisoldipine prevented the development of hypertension, the associated heart failure, and the increase of plasma levels of ANP in SHR but had no effect on systolic blood pressure, heart weight, and plasma levels of ANP in WKY. In addition, nisoldipine had a therapeutic effect in old SHR with manifest cardiac failure in end-stage hypertension, as evidenced not only by the reduction of blood pressure but also by the reduction of cardiac hypertrophy, of elevated immunoreactive ANP in plasma, and of increased plasma renin activity.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Hypertension; Male; Nifedipine; Nisoldipine; Rats; Rats, Inbred SHR

Plasma concentrations of atrial natriuretic factor (ANF) were compared in normotensive subjects and subjects with untreated, uncomplicated essential hypertension (n = 21 pairs) matched for age, sex, and race. Plasma peptide values were slightly greater (45 +/- 3 vs 36 +/- 3 pg/ml; p less than 0.05) in the hypertensive group. On univariate analysis, age (r = 0.52, n = 47, p less than 0.001) and creatinine clearance (r = -0.30, n = 47, p less than 0.05) were significantly related to plasma ANF concentrations, but arterial pressure was not (r = 0.14, n = 47), in an extended group of normal subjects. In contrast, plasma ANF values were related to arterial pressure in both an extended group of subjects with untreated essential hypertension (r = 0.54, n = 38, p less than 0.001) and in our total heterogeneous pool of hypertensive patients (r = 0.46, n = 79, p less than 0.001), but weak positive associations with age and inverse relationships with creatinine clearance were not statistically significant in either hypertensive group. Similar weak inverse relationships between plasma ANF values and renin-angiotensin-aldosterone system activity were found in both normal and hypertensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Age Factors; Atrial Natriuretic Factor; Blood Pressure; Cardiomegaly; Female; Humans; Hypertension; Male; Middle Aged; Renin-Angiotensin System

To assess the relation between atrial natriuretic peptide and ventricular dysfunction, we simultaneously measured both atrial and plasma immunoreactive atrial natriuretic peptide concentrations in rats 4 weeks after myocardial infarction induced by left coronary artery ligation. When compared to controls (n = 39), rats with infarction (n = 16) had markedly elevated plasma immunoreactive atrial natriuretic peptide concentrations (1205.8 +/- 180.9 vs. 126.7 +/- 8.9 pg/ml, p less than 0.001) and reduced immunoreactive atrial natriuretic peptide concentrations in right and left atria (31.4 +/- 4.6 vs. 61.2 +/- 3.2 ng/mg, p less than 0.001; 14.9 +/- 2.2 vs. 32.7 +/- 2.4 ng/mg, p less than 0.001, respectively). Right ventricular weight increased in proportion to infarct size, and both were correlated with plasma immunoreactive atrial natriuretic peptide levels (r = 0.825, p less than 0.001 and r = 0.816, p less than 0.001, respectively). Right atrial immunoreactive atrial natriuretic peptide content was significantly higher than left in both controls and rats with infarction. Both right and left atrial immunoreactive atrial natriuretic peptide concentrations were negatively correlated with both right ventricular weight as well as plasma immunoreactive atrial natriuretic peptide concentrations (right atrium: r = -0.816, p less than 0.001, r = -0.708, p less than 0.01; left atrium: r = -0.687, p less than 0.01, r = -0.644, p less than 0.01, respectively). These results suggest that chronic stimulation of atrial natriuretic peptide release from both atria is associated with increased turnover and depleted stores of atrial natriuretic peptide in atria in proportion to the severity of heart failure. It also suggests that plasma atrial natriuretic peptide levels may be used as a reliable index of cardiac decompensation in chronic heart failure.

Topics: Animals; Atrial Natriuretic Factor; Cardiomegaly; Female; Heart Atria; Heart Failure; Myocardial Infarction; Rats; Rats, Inbred Strains

Topics: Adult; Aged; Atrial Natriuretic Factor; Cardiomegaly; Female; Heart Atria; Humans; Male; Middle Aged; Renin; Vasopressins