calcimycin and Cardiomegaly

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

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

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, we investigated the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling in vitro and in vivo. We show that calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart were characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrated reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreased p38 phosphorylation and increased MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreased MKP-1 protein levels and increased p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes, which is associated with p38 inactivation.

Topics: Animals; Animals, Newborn; Calcimycin; Calcineurin; Cardiomegaly; Cell Cycle Proteins; Cell Line; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cyclosporine; Dual Specificity Phosphatase 1; Fibroblasts; Gene Expression Regulation, Enzymologic; Heart; Immediate-Early Proteins; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; Myocardium; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transfection

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Animals, Newborn; Calcimycin; Calcineurin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cells, Cultured; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Heart; Ionomycin; Kinetics; Models, Cardiovascular; Myocardium; Myosin Heavy Chains; Promoter Regions, Genetic; Rats; Rats, Wistar; Sulfonamides; Tetradecanoylphorbol Acetate; Transfection

Angiotensin II-induced phosphorylation of proteins was examined in isolated myocytes from hearts of Dahl rats. A high salt diet induced cardiac hypertrophy in Dahl salt-sensitive rats. Angiotensin II-induced phosphorylation of a 42-kd protein (pp42) was detected by two-dimensional electrophoresis in hypertrophic but not normal ventricular myocytes. Angiotensin II stimulation was time-dependent, with a peak effect at 30 minutes. The half-maximal and maximal concentrations of angiotensin II that stimulated pp42 phosphorylation were 1 and 10 nM, respectively. Phosphorylation of pp42 was a function of cardiac hypertrophy. Phorbol 12-myristate 13-acetate-induced phosphorylation of pp42 indicates the possibility of an association between protein kinase C and the signal transduction pathway of angiotensin II-induced pp42 phosphorylation. Ionomycin and A23187 (both at 1 microM) did not stimulate phosphorylation of pp42. Angiotensin II produced a small increase in the synthesis of myocyte proteins in both normal and hypertrophic cells as shown by [35S]methionine incorporation. However, this increase could not account for the increase in the phosphate content of pp42. This protein was not an isoform of actin nor was it of platelet origin. These results raise the possibility that angiotensin II may play a role in the activation of factors in hypertrophic myocytes; however, further study is required to define a link between phosphorylation of pp42 and the hypertrophic process.

Topics: Angiotensin II; Animals; Calcimycin; Cardiomegaly; Cell Separation; Drug Resistance; Electrophoresis; Ionomycin; Methionine; Muscle Proteins; Myocardium; Phosphoproteins; Phosphorylation; Rats; Rats, Inbred Strains; Sodium Chloride; Tetradecanoylphorbol Acetate

We recently described the early appearance of pulmonary hypertension in the fawn-hooded rat (FHR), an animal with platelet storage pool disease also known to develop systemic hypertension at later ages. Since mediators released from aggregating platelets influence vascular tone, we hypothesized that platelet-mediated pulmonary vascular responses in FHR may be abnormal and potentially linked to the mechanism of pulmonary hypertension. To test this we examined reactivity of isolated pulmonary arteries (PA) and thoracic aortas (Ao) from young FHR with moderately severe pulmonary hypertension but normal systemic pressures. These vessels were compared with PA and Ao from control Sprague-Dawley rat (SDR). Aggregating platelets (1,000-40,000 platelets/mm3) from FHR caused dilation of SDR PA and Ao but constriction of FHR PA and Ao. Qualitatively similar responses were also observed with platelets isolated from SDR implying that abnormal responses were not simply due to the storage pool deficiency in FHR. Response to the platelet-derived endothelium-dependent vasodilator ADP was markedly impaired in FHR PA and mildly impaired in FHR Ao. Endothelium-dependent dilation to acetylcholine, but not to A23187, was mildly impaired in FHR PA while responses to both dilators were normal in FHR Ao. Endothelium-independent dilation to sodium nitroprusside was normal in both FHR PA and Ao. Constrictor sensitivity to serotonin, but not to the thromboxane A2 mimetic U-46619, was increased in FHR PA while responses to both constrictors were normal in FHR Ao. In summary, PAs from FHR with spontaneous pulmonary hypertension exhibit paradoxical constriction to both normal and storage pool deficient platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adenosine Diphosphate; Animals; Aorta; Blood Platelets; Calcimycin; Cardiomegaly; Endothelium, Vascular; Hypertension, Pulmonary; Male; Meclofenamic Acid; Nitroprusside; Platelet Aggregation; Platelet Storage Pool Deficiency; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Inbred Strains; Serotonin; Vasoconstriction; Vasodilation

The effect of changes in left ventricular (LV) systolic force generation on cardiac c-fos and c-jun protooncogene expression was studied by using isolated beating hearts from male Wistar rats. An isovolumic buffer-perfused heart preparation was utilized in which coronary flow and heart rate were held constant and increments in LV balloon volume were used to generate defined levels of LV systolic wall stress. Using Northern and slot-blot analyses, we found that LV tissue from control hearts that generated high levels of LV systolic wall stress expressed 3- to 4.4-fold higher c-fos and c-jun mRNA levels in comparison with tissue from the respective flaccid right ventricles, and in comparison with LV tissue from hearts that generated minimal LV systolic wall stress. To distinguish the role of passive LV diastolic wall stretch from active LV force generation, we found that distension of the LV balloon per se did not have a significant effect on protooncogene induction in hearts perfused with 2,3-butanedione monoxime, which prevents systolic cross-bridge cycling and force generation. In additional hearts studied at a constant LV balloon volume to generate an LV end-diastolic pressure of 10 mm Hg, c-fos mRNA levels were proportional to the magnitude of peak LV systolic wall stress (r = 0.823, P less than 0.05). In these protocols, Fos protein was localized by immunohistochemistry in myocyte nuclei with minimal staining in fibroblasts and vascular smooth muscle. When c-fos and c-jun mRNA expression was compared in hearts with chronic LV hypertrophy due to ascending aortic banding and age-matched control hearts that generated similar incremental levels of LV systolic wall stress, significantly lower levels of c-fos and c-jun mRNA were measured in the hypertrophied hearts. However, there was no difference in protooncogene mRNA expression in response to stimulation by the Ca2+ ionophore A23187. These data suggest that, in this isolated isovolumic beating heart preparation, the active generation of an acute increment in LV systolic force independent of passive diastolic myocardial stretch causes a rapid induction of both c-fos and c-jun, which is down-regulated in the presence of established LV hypertrophy.

Topics: Animals; Aortic Valve Stenosis; Calcimycin; Cardiomegaly; Diastole; Gene Expression Regulation; Genes, fos; Genes, jun; Heart; In Vitro Techniques; Male; Perfusion; Rats; Rats, Inbred Strains; RNA, Messenger; Stress, Mechanical; Systole

Stimulation of ornithine decarboxylase (ODC) activity was examined in cultured heart cells from neonatal rats. Fetal bovine serum had a concentration-dependent effect on ODC activity with maximum response obtained at 10% serum. ODC activity peaked 4 h after the addition of serum and returned to initial levels at 8 h. In the absence of serum, non-cytotoxic concentrations of the adrenergic agonists epinephrine, norepinephrine or isoproterenol did not stimulate ODC activity. Co-administration of serum and catecholamines at 10(-5) M induced an ODC response that was significantly greater than that induced by serum alone. A screen of various constituents of serum revealed that insulin, though relatively ineffective alone, acted cooperatively with catecholamines to produce an ODC response equivalent to that induced by 10% serum. Propranolol effectively blocked the cooperative effect of insulin on catecholamine stimulation of ODC activity, and markedly inhibited the stimulation of ODC by 10% serum. Insulin also acted cooperatively with the second-messenger analogue dibutyryl-cAMP. The calcium ionophore A23187 significantly increased ODC activity and this response was potentiated by the presence of insulin. The present findings are concordant with in vivo observations in that beta-adrenergic activation stimulates ODC activity in cultured heart cells, but it also demonstrates that the cooperative action of other factors present in serum, such as insulin, are required.

Topics: Animals; Blood Proteins; Calcimycin; Cardiomegaly; Catecholamines; Cells, Cultured; Cyclic AMP; Drug Synergism; Fibroblasts; Heart; Hypertrophy; Insulin; Isoproterenol; Myocardium; Ornithine Decarboxylase; Rats