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.

The zinc finger-containing transcription factors GATA4 and GATA6 are important regulators of basal and inducible gene expression in cardiac and smooth muscle cell types. Here we demonstrate a direct functional role for GATA4 and GATA6 as regulators of cardiomyocyte hypertrophic growth and gene expression. To model the increase in endogenous GATA4 and GATA6 transcriptional activity that occurs in response to hypertrophic stimulation, each factor was overexpressed in cardiomyocytes using recombinant adenovirus. Overexpression of either GATA4 or GATA6 was sufficient to induce cardiomyocyte hypertrophy characterized by enhanced sarcomeric organization, a greater than 2-fold increase in cell surface area, and a significant increase in total protein accumulation. In vivo, transgenic mice with 2.5-fold overexpression of GATA4 within the adult heart demonstrated a slowly progressing increase in heart to body weight ratio, histological features of cardiomyopathy, and activation of hypertrophy-associated genes, suggesting that GATA factors are sufficient regulators of cardiomyocyte hypertrophy in vitro and in vivo. To evaluate the requirement of GATA factors as downstream transcriptional mediators of hypertrophy, a dominant negative GATA4-engrailed repressor fusionencoding adenovirus was generated. Expression of GATA4-engrailed blocked GATA4-and GATA6-directed transcriptional responses and agonist-induced cardiomyocyte hypertrophy, demonstrating that cardiac-expressed GATA factors are necessary mediators of this process.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonistinduced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.Increasing evidence suggests that multiple trans-acting factors and cis-acting elements cooperatively regulate the expression of cardiac muscle-specific genes (reviewed in references 28 and 36), unlike skeletal muscle myogenesis where myogenic basic helix-loop-helix factors can activate the entire myogenic program (reviewed by Olson and Klein [37a]). For example, the cardiac ␣-myosin heavy chain gene (␣-MHC) is synergistically activated by myocyte-specific enhancer factor 2 (MEF2) and thyroid hormone receptor, and this activation depends on the binding of each factor to the DNA target sequences (27). Multiple transcription factors, such as E-box and CArG-box binding factors and Sp1, are required for the muscle-specific expression of the cardiac ␣-actin gene (37b). Cardiac myosin light chain 2v (MLC2v) gene expression appears to depend on several factors, including YB-1 and CARP (44,45).Homeobox genes have been studied extensively in many animal species, where they play fundamental roles in specifying cell fate and positional identity in embryos. The nk-4/msh-2 Drosophila gene, tinman, has been ...

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

Basal and aortic constriction-stimulated transcription of the beta-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within beta-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. Here we determined that GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activated expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the ␣-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box siteindependent. A mechanism for the transcriptional synergy was suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND was associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacted with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. Taken together with previous observations, these results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type 1a receptor (AT 1a R)-dependent mechanism. To understand how the level of AT 1a Rs is altered in this pathological state, we studied the expression of an injected AT 1a R promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the ␣-skeletal actin promoter 1.7-fold whereas the ␣-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT 1a R promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT 1a R promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT 1a R promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.Pathological conditions resulting in increased cardiac workload generally are associated with activation of systemic and local renin-angiotensin systems and increased levels of circulating angiotensin II (AngII) (1, 2). However, little is understood about how AngII type 1a receptors (AT 1a R) are modulated under these same pathological conditions. AngII is a potent growth factor that mediates the hypertrophic growth of cardiac muscle cells and is a chemical mediator of stretchinduced cardiomyocyte hypertrophy (3-7). The interaction of AngII with AT 1a R activates a signal transduction cascade that effects the phosphorylation of serum response factor and p62 TCF by pp90 RSK and mitogen-activated protein kinase, respectively, resulting in increased c-fos gene expression (5-7). Hypertrophic stimuli also increase the level of AT1 a R mRNA in cardiomyocytes. A 3-fold increase in AT 1a R mRNA and a 2-fold increase in AT 1a R densities have been reported in spontaneously hypertensive and two kidney one-clip renovascular hypertensive rats with established cardiac hypertrophy (8). It is not known whether this increase in AT 1a R mRNA is mediated by a transcriptional or posttranscriptional mechanism.In this study, we use direct injection of DNA into the heart in conjunction with aortic coarctation (CoA) to study the activity of the AT 1a R promoter in the normal and pressureoverloaded rat heart. The AT 1a R promoter was found to be active in normal adult cardiac muscle, whereas gene expression was increased in response to an acute pressure overload (PO). The induced expression was blocked by mutation of either an AP-1 or a GATA binding site, however, these mutations had no effect on basal expression. Administration of the angiotensin-...