thapsigargin and Cardiomegaly

Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice.. We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate.. These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

Topics: Animals; Cardiomegaly; Diastole; Disease Models, Animal; Echocardiography; Heart Diseases; Heart Failure; Mice, Inbred C57BL; Observer Variation; Pressure; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Stroke Volume; Systole; Thapsigargin

Cardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca2+ entry, a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+ entry with respect to cardiac hypertrophy in vitro and in vivo.. Consistent with earlier reports, we found drug-inducible store-operated Ca2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca2+ entry was only marginal in adult cardiomyocytes isolated from control hearts, it increased significantly in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (i.e., in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, we found STIM1 to be both sufficient and necessary for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo. Stim1 silencing by adeno-associated viruses of serotype 9-mediated gene transfer protected rats from pressure overload-induced cardiac hypertrophy.. By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.

Topics: Adenoviridae; Age Factors; Animals; Animals, Newborn; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Cardiomegaly; Cells, Cultured; Enzyme Inhibitors; Gene Silencing; Gene Transfer Techniques; Membrane Glycoproteins; Mice; Mice, Transgenic; Myocytes, Cardiac; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Rats; Sarcolemma; Stromal Interaction Molecule 1; Thapsigargin

Endoplasmic reticulum (ER) stress (ERS) is involved in various cardiovascular diseases. Our previous study verified that ERS took part in the development of cardiac hypertrophy; however, its mechanism is still unclear. This study aimed to investigate the roles of the calcineurin (CaN) signal pathway in hypertrophy induced by the ERS inductor thapsigargin (TG) in neonatal cardiomyocytes from Sprague-Dawley rats. Investigation of ER chaperone expression, ER staining, and calreticulin immunofluorescence were used to detect the ERS response. mRNA expression of atrial natriuretic peptide and brain natriuretic peptide, total protein synthesis rate, and cell surface area were used to evaluate cardiac hypertrophy induced by TG. TG induced a significant ERS response along with hypertrophy in a dose- and time-dependent manner in cardiomyocytes, which was verified by treatment with tunicamycin, another ERS inducer. Furthermore, TG induced a significant elevation of the intracellular Ca(2+) level, CaN activation, and myocyte enhancer factor 2c (MEF2c) expression in a dose- and time-dependent manner in cardiomyocytes. Cyclosporine A, a CaN inhibitor, markedly suppressed MEF2c nuclear translocation and inhibited TG-induced hypertrophy. These results demonstrate that ERS induces cardiac hypertrophy and that the CaN-MEF2c pathway is involved in ERS-induced hypertrophy in cardiomyocytes.

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Calcineurin; Calcium; Cardiomegaly; Cell Size; Cells, Cultured; Cyclosporine; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Fluorescent Antibody Technique; L-Lactate Dehydrogenase; MEF2 Transcription Factors; Myocytes, Cardiac; Myogenic Regulatory Factors; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Tunicamycin

Excessive endoplasmic reticulum stress (ERS) triggers apoptosis in various conditions including diabetic cardiomyopathy and pressure overload-induced cardiac hypertrophy and heart failure. The primary function of 14-3-3 protein is to inhibit apoptosis, but the roles of this protein in protecting against cardiac ERS and apoptosis are largely unknown.. We investigated the roles of 14-3-3 protein in vivo during cardiac ERS and apoptosis induced by pressure overload or thapsigargin injection using transgenic (TG) mice that showed cardiac-specific expression of dominant negative (DN) 14-3-3eta.. Cardiac positive apoptotic cells and the expression of glucose-regulated protein (GRP)78, inositol-requiring enzyme (Ire)1alpha, tumor necrosis factor receptor (TNFR)-associated factor (TRAF)2, CCAAT/enhancer binding protein homology protein (CHOP), caspase-12, and cleaved caspase-12 protein were significantly increased in the pressure-overload induced DN 14-3-3eta mice compared with that in the WT mice. Furthermore, thapsigargin injection significantly increased the expression of GRP78 and TRAF2 expression in DN 14-3-3eta mice compared with that in the WT mice.. The enhancement of 14-3-3 protein may provide a novel protective therapy against cardiac ERS and ERS-initiated apoptosis, at least in part, through the regulation of CHOP and caspase-12 via the Ire1alpha/TRAF2 pathway.

Topics: 14-3-3 Proteins; Animals; Apoptosis; Cardiomegaly; Caspase 12; CCAAT-Enhancer-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Heat-Shock Proteins; Injections, Intraperitoneal; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Pressure; Protein Serine-Threonine Kinases; Stress, Physiological; Thapsigargin; TNF Receptor-Associated Factor 2

Neonatal rat cardiac myocytes were exposed to 10 nM thapsigargin (TG) or 20 muM phenylephrine (PE) to compare resulting alterations of Ca(2+) homeostasis. Either treatment results in resting cytosolic [Ca(2+)] rise and reduction of Ca(2+) signals in myocytes following electrical stimuli. In fact, ATP-dependent Ca(2+) transport is reduced due to catalytic inhibition of sarcoplasmic reticulum ATPase (SERCA2) by TG or reduction of SERCA2 protein expression by PE. A marked rise of nuclear factor of activated T cells (NFAT)-dependent expression of transfected luciferase cDNA is produced by TG or PE, which is dependent on increased NFAT dephosphorylation by activated calcineurin and reduced phosphorylation by inactivated glycogen synthase kinase 3beta. Expression of SERCA2 (inactivated) protein is increased following exposure to TG, whereas no hypertrophy is produced. On the contrary, SERCA2 expression is reduced, despite high CN activity, following protein kinase C (PKC) activation by PE (or phorbol 12-myristate 13-acetate) under conditions producing myocyte hypertrophy. Both effects of TG and PE are dependent on NFAT dephosphorylation by CN, as demonstrated by CN inhibition with cyclosporine (CsA). However, the hypertrophy program triggered by PKC activation bypasses SERCA2 transcription and expression due to competitive recruitment of NFAT and/or other transcriptional factors. A similar dependence on CN activation, but relative reduction under conditions of PKC activation, involves transcription and expression of the Na(+)/Ca(2+) exchanger-1. On the other hand, significant upregulation of transient receptor potential channel proteins is noted following PKC activation. The observed alterations of Ca(2+) homeostasis may contribute to development of contractile failure.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcineurin; Calcium Signaling; Cardiomegaly; Cardiotonic Agents; Cell Division; Cells, Cultured; Cytosol; Enzyme Inhibitors; Gene Expression; Myocytes, Cardiac; NFATC Transcription Factors; Phenylephrine; Protein Kinase C; Rats; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Sodium-Calcium Exchanger; Thapsigargin; TRPC Cation Channels

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

Topics: Animals; Animals, Newborn; Calcium Signaling; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Silencing; Myocytes, Cardiac; Phenylephrine; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors

The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear.. Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12-, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels.. These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Aortic Valve Stenosis; Apoptosis; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Disease Progression; Endoplasmic Reticulum; Gene Expression Regulation; Heart Failure; Humans; Imidazoles; Ligation; Male; Mice; Mice, Inbred C57BL; Molecular Chaperones; Myocytes, Cardiac; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Rats; Rats, Inbred WKY; RNA, Messenger; Signal Transduction; Stress, Physiological; Tetrazoles; Thapsigargin; Tunicamycin

Hyperglycemia alters cardiac function and often leads to diabetic cardiomyopathy as cardiomyocyte apoptosis causes a hypertrophied heart to deteriorate to dilation and failure. Paradoxically, many short-term animal models of hyperglycemia protect against ischemia-induced damage, including apoptosis, by limiting Ca(2+) overload. We have determined that, like nonexcitable cells, both neonatal and adult cardiomyocytes respond to depletion of sarcoplasmic/endoplasmic reticulum Ca(2+) stores with an influx of extracellular Ca(2+) through channels distinct from voltage-gated Ca(2+) channels, a process termed capacitative Ca(2+) entry (CCE). Here, we demonstrate that in neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by angiotensin II or the Ca(2+)ATPase inhibitor thapsigargin. Hyperglycemia also significantly blunted Ca(2+)-dependent hypertrophic responses by approximately 60%, as well as the Ca(2+)-sensitive nuclear translocation of a chimeric protein bearing the nuclear localization signal of a nuclear factor of activated T-cells transcription factor. The attenuation of CCE by hyperglycemia was prevented by azaserine, an inhibitor of hexosamine biosynthesis, and partially by inhibitors of oxidative stress. This complements previous work showing that increasing hexosamine metabolites in neonatal cardiomyocytes also inhibited CCE. The inhibition of CCE by hyperglycemia thus provides a likely explanation for the transition to diabetic cardiomyopathy as well as to the protection afforded to injury after ischemia/reperfusion in diabetic models.

Topics: Animals; Animals, Newborn; Biological Transport; Calcium; Calcium Channels; Cardiomegaly; Cell Nucleus; DNA-Binding Proteins; Glucose; Hexosamines; Hyperglycemia; Myocytes, Cardiac; NFATC Transcription Factors; Nuclear Proteins; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Thapsigargin; Transcription Factors

Modifications to cell relaxation and handling of intracellular Ca have been demonstrated in animals with cardiac cell hypertrophy leading to decompensated heart failure. A previously described model of renal hypertension leading to cardiac cell hypertrophy in the guinea pig, produced using the Goldblatt 2-kidney, 1-clip technique, was used to investigate which of the main mechanisms causing cell relaxation (the sarcoplasmic reticulum Ca-adenosinetriphosphatase and Na/Ca exchanger) are altered in hypertrophy. Relaxation upon rewarming from a rapid cooling contracture was slowed in hypertrophied (H) compared with control (C) cells. Relaxation was further slowed in H compared with C cells when Na/Ca exchange was inhibited by rewarming in a Na-free, Ca-free solution and slowed most markedly in H cells in the presence of 10 mM caffeine. Hypertrophy led to greater modification of cell length relaxation in comparison with the decline in the indo-1 transient, but the force-pCa relationship in skinned muscles showed that myofilament sensitivity was unchanged. Such results indicate that cell relaxation and Ca handling are affected in hypertrophy, possibly involving modifications of Na/Ca exchange activity.

Topics: Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Cardiomegaly; Cells, Cultured; Fluorescent Dyes; Guinea Pigs; Heart; In Vitro Techniques; Indoles; Male; Myocardial Contraction; Papillary Muscles; Reference Values; Temperature; Terpenes; Thapsigargin