In overloaded heart the cardiomyocytes adapt to increased mechanical and neurohumoral stress by activation of hypertrophic program, resulting in morphological changes of individual cells and specific changes in gene expression. Accumulating evidence suggests an important role for the zinc finger transcription factor GATA-4 in hypertrophic agonist-induced cardiac hypertrophy. However, its role in stretch-induced cardiomyocyte hypertrophy is not known. We employed an in vitro mechanical stretch model of cultured cardiomyocytes and used rat B-type natriuretic peptide promoter as stretch-sensitive reporter gene. Stretch transiently increased GATA-4 DNA binding activity and transcript levels, which was followed by increases in the expression of B-type natriuretic peptide as well as atrial natriuretic peptide and skeletal ␣-actin genes. The stretch inducibility mapped primarily to the proximal 520 bp of the B-type natriuretic peptide promoter. Mutational studies showed that the tandem GATA consensus sites of the proximal promoter in combination with an Nkx-2.5 binding element are critical for stretch-activated B-type natriuretic peptide transcription. Inhibition of GATA-4 protein production by adenovirus-mediated transfer of GATA-4 antisense cDNA blocked stretch-induced increases in B-type natriuretic peptide transcript levels and the sarcomere reorganization. The proportion of myocytes with assembled sarcomeres in control adenovirus-infected cultures increased from 14 to 59% in response to stretch, whereas the values for GATA-4 antisense-treated cells were 6 and 13%, respectively. These results show that activation of GATA-4, in cooperation with a factor binding on Nkx-2.5 binding element, is essential for mechanical stretchinduced cardiomyocyte hypertrophy.

The expression of cardiac hormones, atrial natriuretic peptide and B-type natriuretic peptide, is induced by cardiac wall stretch and responds to various hypertrophic agonists such as endothelin-1. In cardiac myocytes, endothelin-1 induces GATA-4 binding to the B-type natriuretic peptide gene, but the signaling pathways involved in endothelin-1-induced GATA-4 activation are unknown. Mitogen-activated protein kinase pathways are stimulated in response to various extracellular stimuli, and they modulate the function of several transcription activators. Here we show that inhibition of p38 kinase with SB203580 inhibited endothelin-1-induced GATA-4 binding to B-type natriuretic peptide gene and serine phosphorylation of GATA-4. Inhibition of extracellular signal-regulated protein kinase with MEK1 inhibitor PD98059 reduced basal and p38-induced GATA-4 binding activity, but it had no significant effect on endothelin-1-induced GATA-4 binding activity. Overexpression of p38 kinase pathway, but not extracellular signal-regulated kinase or c-Jun Nterminal protein kinase, activated GATA-4 binding to B-type natriuretic peptide gene and induced rat B-type natriuretic peptide promoter activity via proximal GATA binding sites. In conclusion, these findings demonstrate that activation of p38 kinase is necessary for hypertrophic agonist-induced GATA-4 binding to B-type natriuretic peptide gene and sufficient for GATA-dependent B-type natriuretic peptide gene expression.Cardiac hypertrophy is a physiological process adapting heart to increased hemodynamic workload. In early stages, hypertrophy is a compensatory mechanism, but if prolonged, it leads to pathologic myocyte hypertrophy characterized by increase in cell size, enhanced sarcomeric organization, and induction of the fetal gene program (1). Myocyte hypertrophy can be induced by pressure or volume overload and by different neurohumoral factors, including endothelin-1 (ET-1), 1 angiotensin II, and ␣ 1 -adrenergic agonists (2). At the genetic level, activation of a program of immediate early genes, such as c-fos, c-jun, and c-myc, is the first detectable response to hypertrophic stimuli. This is followed by alterations in contractile protein compositions, including reactivation of ␤-myosin heavy chain, skeletal ␣-actin, and myosin light chain-2 genes (3, 4). Hypertrophy also results in induction of noncontractile protein genes such as atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which are known members of the mammalian cardiac natriuretic peptide system (5-7). ANP and BNP defend against increased hemodynamic load by decreasing blood pressure, regulating fluid homeostasis by increasing salt and water excretion, and regulating several hormones, such as angiotensin II, ET-1, and vasopressin (5, 8). In the normal adult heart, ANP is mainly synthesized in the atria, whereas BNP is abundant in cardiac atria and ventricles where its gene expression is rapidly up-regulated in response to cardiac wall stretch. Indeed, the induction of BNP gene expression is o...

Terminally differentiated cardiac myocytes adapt to mechanical and neurohumoral stress via morphological changes of individual cells accompanied by reactivation of fetal pattern of gene expression. Endothelin-1, a powerful paracrine mediator of myocyte growth, induces similar changes in cultured cardiac myocytes as those seen in hypertrophied heart in vivo. By using rat B-type natriuretic peptide promoter, we identified a novel ETS binding sequence, on which nuclear protein binding is activated in endothelin-1-treated cultured cardiac myocytes. This sequence binds ETS-like gene-1 transcription factor and mediates endothelin-1-specific activation of transcription, but not responses to increased calcium signaling via L-type calcium channels, angiotensin II treatment, or mechanical stretch of myocytes. Interestingly, endothelin-1 activated signaling converges via p38 mitogen-activated protein kinase-dependent mechanism on ETS binding site, whereas this element inhibits extracellular signal-regulated kinase activated transcription. In conclusion, given the fundamental role of the interaction of mitogen-activated protein kinases and ETS factors in regulation of eukaryotic cell differentiation, growth, and oncogenesis, these results provide the unique evidence of a endothelin-1-and mitogenactivated protein kinase-regulated ETS factor pathway for cardiac myocytes.Terminally differentiated cardiac myocytes, due to their inability to divide, adapt to increased mechanical load and the activation of the neurohumoral system by hypertrophy. Initiation of hypertrophic growth is accompanied by a rapid and transient expression of immediate early genes (e.g. c-jun, c-fos, and Egr-1) followed by activation of a pattern of cardiac genes, including atrial and B-type natriuretic peptide (ANP 1 and BNP)

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.

Volume excess and mechanical load lead to the induction of the endocrine activity of the heart. The increased production and secretion of A- and B-type natriuretic peptides (ANP and BNP), in turn, unload the heart due to their physiological effects. To find out the mechanisms of cardiac-specific expression and sensitivity to mechanical stimuli of the natriuretic peptide genes, we have used salmon (Salmo salar) as our model organism, because osmoregulating fish have a particularly well developed defense mechanism against volume excess. We have previously cloned a complementary DNA from salmon heart encoding a novel vasorelaxant cardiac hormone, salmon cardiac peptide (sCP). Its production is restricted to the heart, and its release is very sensitive to mechanical load. We have now cloned the gene encoding sCP. The structure of the gene suggests that sCP may represent an ancestral form of the mammalian natriuretic peptides. Remarkably, despite the large phylogenetic distance, the sCP promoter is as effective as mammalian ANP promoters in cultured neonatal rat atrial cardiomyocytes. Therefore, structural and functional comparisons of the promoters of sCP and ANP provide an excellent means of identifying the elements and transcription factors required for atrial-specific gene expression and the regulation of the endocrine function of the heart. Isolation of the protein product of sCP gene from salmon atrium demonstrated that the storage form of sCP is the prohormone of 126 amino acids. The final processing of the prohormone appears to take place during exocytosis of the secretory granules, as the released and circulating form is the biologically active 29-amino acid sCP.