A variety of stresses on the heart initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and cause myocyte hypertrophy with heart failure. However, common nuclear pathways that lead to this state are unknown. A zinc finger protein, GATA-4, is one of the transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. p300 not only acts as a transcriptional coactivator of GATA-4, but also possesses an intrinsic histone acetyltransferase activity. In primary cardiac myocytes derived from neonatal rats, we show that stimulation with phenylephrine increased an acetylated form of GATA-4 and its DNA-binding activity, as well as expression of p300. A dominant-negative mutant of p300 suppressed phenylephrine-induced nuclear acetylation, activation of GATA-4-dependent endothelin-1 promoters, and hypertrophic responses, such as increase in cell size and sarcomere organization. In sharp contrast to the activation of cardiac MEK-1, which phosphorylates GATA-4 and causes compensated hypertrophy in vivo, p300-mediated acetylation of mouse cardiac nuclear proteins, including GATA-4, results in marked eccentric dilatation and systolic dysfunction. These findings suggest that p300-mediated nuclear acetylation plays a critical role in the development of myocyte hypertrophy and represents a pathway that leads to decompensated heart failure.Heart failure arises from a number of diverse primary cardiovascular disorders and is associated with significant morbidity and mortality. Therefore, elucidating the mechanisms of this disease is of clinical importance. Previous studies have demonstrated that a variety of stresses on the heart activate neuronal and hormonal factors, such as the renin-angiotensin system and factors regulating the sympathetic nervous systems. These factors initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and change the pattern of gene expression associated with hypertrophy (reviewed in references 14 and 53). In order to establish appropriate therapy for heart failure, it is critical to identify a common nuclear pathway which can be targeted by pharmacological agents in the future.We have been interested in transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. A zinc finger protein, GATA-4, is one such factor and is required for transcriptional activation of cardiac genes whose expression is upregulated during myocardial-cell hypertrophy (22, 25; reviewed in reference 46). While overexpression of GATA-4 in cardiac myocytes causes hypertrophy, expression of a dominant-negative form of GATA-4 inhibits Gq protein-coupled receptor agonist-induced hypertrophy (37). During myocardial-cell hypertrophy, GATA-4 is phosphorylated at a serine residue and shows increased DNA-binding ability (38,47). Phosphorylation of cardiac GATA-4 requires activation of MEK1/extracellular signal-regulated kinase (ERK) 1/2. On the other hand, activation of MEK1 in...

The expression of endothelin-1 (ET-1) in cardiac myocytes is markedly induced during the development of heart failure in vivo and by stimulation with the ␣ 1 -adrenergic agonist phenylephrine in culture. Although recent studies have suggested a role for cardiac-specific zinc finger GATA factors in the transcriptional pathways that modulate cardiac hypertrophy, it is unknown whether these factors are also involved in cardiac ET-1 transcription and if so, how these factors are modulated during this process. Using transient transfection assays in primary cardiac myocytes from neonatal rats, we show here that the GATA element in the rat ET-1 promoter was required for phenylephrine-stimulated ET-1 transcription. Cardiac GATA-4 bound the ET-1 GATA element and activated the ET-1 promoter in a sequencespecific manner. Stimulation by phenylephrine caused serine phosphorylation of GATA-4 and increased its ability to bind the ET-1 GATA element. Inhibition of the extracellularly responsive kinase cascade with PD098059 blocked the phenylephrine-induced increase in the DNA binding ability and the phosphorylation of GATA-4. These findings demonstrate that serine phosphorylation of GATA-4 is involved in ␣ 1 -adrenergic agonist-responsive transcription of the ET-1 gene in cardiac myocytes and that extracellularly responsive kinase 1/2 activation plays a role upstream of GATA-4. Endothelin-1 (ET-1)1 was initially identified as a 21-amino acid vasoconstrictive peptide in porcine vascular endothelial cells (1). Later work showed that it acts not only as a vasoconstrictor but also as a potent growth-promoting peptide. For example, ET-1 can induce myocyte hypertrophy (2, 3) through coupling of ET receptors with G q protein. ET-1 signaling is also coupled with G i protein. Therefore, it is able to decrease intracellular cAMP levels (4). Although ET-1 is mainly produced by endothelial cells in the basal state, a number of cell types can synthesize ET-1 in response to various stimuli (5-8). ET-1 expression in cardiac myocytes is induced by myocardial stretch, angiotensin II, and norepinephrine (6 -8). Left ventricular levels of ET-1 increase markedly in close association with the deterioration of systolic function following myocardial infarction and pressure overload (9, 10). Immunohistochemical studies have demonstrated that ET-1 in the failing heart is localized in cardiac myocytes. ET receptor antagonists bosentan and BQ123 prevent the remodeling of the heart and have been shown to improve survival following myocardial infarction and pressure overload (9, 10). These findings demonstrate that up-regulated expression of ET-1 in cardiac myocytes plays a critical role in the development of heart failure in vivo. However, the molecular mechanisms leading to this up-regulation in the failing heart are unclear at present.The mechanisms regulating the transcription of the ET-1 gene have been studied in endothelial cells. The 204-bp sequences proximal to the transcription starting site is sufficient to drive high levels of expression in these...

Abstract-Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide-induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling-positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide-induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A-dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1-stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress-induced apoptosis in cardiac myocytes.

Cardiac myocytes within the adult heart are terminally differentiated and do not undergo cell division. In response to stimuli that affect the mechanical load on the heart or in response to various neurohumoral factors, the heart adapts through the activation of a hypertrophic response in individual cardiac muscle cells. This response is characterized by an increase in myocyte size, accumulation of contractile proteins within individual cardiac cells, and activation of embryonic gene marker expression (for reviews, see Refs. 1-3). For example, genes such as ␤-myosin heavy chain (MHC) 1 and atrial natriuretic factor become highly expressed within ventricular myocytes (4 -6). Studies focused on elucidating the mechanisms of transcriptional regulation of these genes have identified a group of DNA-binding factors that might mediate the nuclear response to hypertrophic stimuli. These factors include the GATA family of zinc finger transcription factors, which mediate transcriptional activation of the genes for ␤-MHC and angiotensin II type 1a receptor during pressure overload-induced hypertrophy in vivo (7-9). GATA factors are also required for the transcriptional activation of the endothelin-1

Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle–myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, we showed that NFATc1 interacted with GATA-6. Consistent with this, NFATc1 further potentiated GATA-6–activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype caused nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulated the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs.