The heart responds to stress signals by hypertrophic growth, which is accompanied by activation of the MEF2 transcription factor and reprogramming of cardiac gene expression. We show here that class II histone deacetylases (HDACs), which repress MEF2 activity, are substrates for a stress-responsive kinase specific for conserved serines that regulate MEF2-HDAC interactions. Signal-resistant HDAC mutants lacking these phosphorylation sites are refractory to hypertrophic signaling and inhibit cardiomyocyte hypertrophy. Conversely, mutant mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals and exhibit stress-dependent cardiomegaly. Thus, class II HDACs act as signal-responsive suppressors of the transcriptional program governing cardiac hypertrophy and heart failure.

The adult heart responds to stress signals by hypertrophic growth, which is often accompanied by activation of a fetal cardiac gene program and eventual cardiac demise. We showed previously that histone deacetylase 9 (HDAC9) acts as a suppressor of cardiac hypertrophy and that mice lacking HDAC9 are sensitized to cardiac stress signals. Here we report that mice lacking HDAC5 display a similar cardiac phenotype and develop profoundly enlarged hearts in response to pressure overload resulting from aortic constriction or constitutive cardiac activation of calcineurin, a transducer of cardiac stress signals. In contrast, mice lacking either HDAC5 or HDAC9 show a hypertrophic response to chronic ␤-adrenergic stimulation identical to that of wild-type littermates, suggesting that these HDACs modulate a specific subset of cardiac stress response pathways. We also show that compound mutant mice lacking both HDAC5 and HDAC9 show a propensity for lethal ventricular septal defects and thin-walled myocardium. These findings reveal central roles for HDACs 5 and 9 in the suppression of a subset of cardiac stress signals as well as redundant functions in the control of cardiac development.Postnatal growth of the heart occurs primarily through hypertrophy, in which cardiac myocytes increase in size but not in number (reviewed in reference 34). Hypertrophy can occur in response to physiological stimuli, such as exercise, or pathological stimuli, such as myocardial infarction, hypertension, aortic stenosis, or valve dysfunction. While stress-induced hypertrophy serves initially to normalize ventricular wall stress, this form of hypertrophy, when prolonged, can progress to dilated cardiomyopathy and sudden death. Pathological cardiac hypertrophy is a major predictor of human morbidity and mortality and a major cause of heart failure (17,18,25).Numerous intracellular signaling pathways have been implicated in the transduction of hypertrophic signals from the cardiomyocyte cell surface to the nucleus (reviewed in references 2, 8, 22, and 34). Many hypertrophic agonists acting through cell surface receptors coupled to G␣q mobilize intracellular calcium, which activates downstream kinases and the calcium-and calmodulin-dependent phosphatase calcineurin. Activation of these effectors is sufficient and, in many cases, necessary for hypertrophic growth of the heart (14, 31, 35, 49). Elevation of cyclic AMP in response to ␤-adrenergic agonists also stimulates cardiac hypertrophy via protein kinase A and other downstream effectors (36). The identification of nodal points in hypertrophic signaling pathways and the mechanisms that link signaling in the cytoplasm with changes in gene expression that contribute to maladaptive growth of the heart represent major challenges in the field.Pathological cardiac hypertrophy is coupled to the activation of a fetal cardiac gene program, which results in the expression of fetal proteins involved in contractility, metabolism, and calcium handling that are incompatible with sustained function of the adult...

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Development and homeostasis of the cardiovascular system require intimate interactions between endothelial and smooth muscle cells, which form a seamless circulatory network. We show that histone deacetylase 7 (HDAC7) is specifically expressed in the vascular endothelium during early embryogenesis, where it maintains vascular integrity by repressing the expression of matrix metalloproteinase (MMP) 10, a secreted endoproteinase that degrades the extracellular matrix. Disruption of the HDAC7 gene in mice results in embryonic lethality due to a failure in endothelial cell-cell adhesion and consequent dilatation and rupture of blood vessels. HDAC7 represses MMP10 gene transcription by associating with myocyte enhancer factor-2 (MEF2), a direct activator of MMP10 transcription and essential regulator of blood vessel development. These findings reveal an unexpected and specific role for HDAC7 in the maintenance of vascular integrity and have important implications for understanding the processes of angiogenesis and vascular remodeling during cardiovascular development and disease.

The ␦ B and ␦ C splice variants of Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII), which differ by the presence of a nuclear localization sequence, are both expressed in cardiomyocytes. We used transgenic (TG) mice and CaMKII expression in cardiomyocytes to test the hypothesis that the CaMKII␦ C isoform regulates cytosolic Ca 2؉ handling and the ␦ B isoform, which localizes to the nucleus, regulates gene transcription. Phosphorylation of CaMKII sites on the ryanodine receptor (RyR) and on phospholamban (PLB) were increased in CaMKII␦ C TG. This was associated with markedly enhanced sarcoplasmic reticulum (SR) Ca 2؉ spark frequency and decreased SR Ca 2؉ content in cardiomyocytes. None of these parameters were altered in TG mice expressing the nuclear-targeted CaMKII␦ B . In contrast, cardiac expression of either CaMKII␦ B or ␦ C induced transactivation of myocyte enhancer factor 2 (MEF2) gene expression and up-regulated hypertrophic marker genes. Studies using rat ventricular cardiomyocytes confirmed that CaMKII␦ B and ␦ C both regulate MEF2-luciferase gene expression, increase histone deacetylase 4 (HDAC4) association with 14-3-3, and induce HDAC4 translocation from nucleus to cytoplasm, indicating that either isoform can stimulate HDAC4 phosphorylation. Finally, HDAC4 kinase activity was shown to be increased in cardiac homogenates from either CaMKII␦ B or ␦ C TG mice. Thus CaMKII␦ isoforms have similar effects on hypertrophic gene expression but disparate effects on Ca 2؉ handling, suggesting distinct roles for CaMKII␦ isoform activation in the pathogenesis of cardiac hypertrophy versus heart failure.is the predominant isoform of CaMKII in the heart. Splice variants differing in the presence of a nuclear localization sequence (NLS) show distinct subcellular targeting to either cytoplasmic or nuclear compartments (1-3). The CaMKII␦ B isoform contains an 11 amino acid NLS that is absent from ␦ C . Thus CaMKII heteromers comprised predominantly of ␦ B subunits localize to the nucleus while those with predominantly ␦ C localize to the cytoplasm (1-3). We recently demonstrated that both ␦ B and ␦ C CaMKII are activated in response to pressure overload induced by thoracic aortic banding but that expression of these isoforms is differentially regulated (4). The possibility that there are discrete roles for these two isoforms in regulating Ca 2ϩ homeostasis and gene transcription has not yet been explored.CaMKII has long been implicated as a regulator of Ca 2ϩ homeostasis and excitation-contraction (E-C) coupling in ventricular myocytes. This enzyme has been shown to phosphorylate proteins involved in sarcoplasmic reticulum (SR) Ca 2ϩ handling including the cardiac ryanodine receptors (RyR2) and phospholamban (PLB) (4 -10). Phosphorylation of the RyR2 appears to alter its channel open probability (9 -11) while phosphorylation of PLB by CaMKII can regulate SR Ca 2ϩ uptake (10, 12). Altered intracellular Ca 2ϩ handling plays an important role in the pathogenesis of heart failure with changes in Ca 2ϩ cycling pr...

The mammary gland consists of a branched ductal system comprised of milk-producing epithelial cells that form ductile tubules surrounded by a myoepithelial cell layer that provides contractility required for milk ejection. Myoepithelial cells bear a striking resemblance to smooth muscle cells, but they are derived from a different embryonic cell lineage, and little is known of the mechanisms that control their differentiation. Members of the myocardin family of transcriptional coactivators cooperate with serum response factor to activate smooth muscle gene expression. We show that female mice homozygous for a loss-of-function mutation of the myocardin-related transcription factor A (MRTF-A) gene are unable to effectively nurse their offspring due to a failure in maintenance of the differentiated state of mammary myoepithelial cells during lactation, resulting in apoptosis of this cell population, a consequent inability to release milk, and premature involution. The phenotype of MRTF-A mutant mice reveals a specific and essential role for MRTF-A in mammary myoepithelial cell differentiation and points to commonalities in the transcriptional mechanisms that control differentiation of smooth muscle and myoepithelial cells.

Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.

Class II histone deacetylases (HDACs) repress transcription by associating with a variety of transcription factors and corepressors. Phosphorylation of a set of conserved serine residues in the N-terminal extensions of class II HDACs creates binding sites for 14-3-3 chaperone proteins, which trigger nuclear export of these HDACs, thereby derepressing specific target genes in a signaldependent manner. To identify intracellular signaling pathways that control phosphorylation of HDAC5, a class II HDAC, we designed a eukaryotic cDNA expression screen in which a GAL4-dependent luciferase reporter was expressed with the DNAbinding domain of GAL4 fused to the N-terminal extension of HDAC5 and the VP16 transcription activation domain fused to 14-3-3. The transfection of COS cells with cDNA expression libraries results in activation of luciferase expression by cDNAs encoding HDAC5 kinases or modulators of such kinases that enable phosphorylated GAL4 -HDAC5 to recruit 14-3-3-VP16 with consequent reconstitution of a functional transcriptional complex. Our results reveal a remarkable variety of signaling pathways that converge on the signal-responsive phosphorylation sites in HDAC5, thereby enabling HDAC5 to connect extracellular signals to the genome.endothelial differentiation genes ͉ lysophosphatidic acid receptors ͉ Rho signaling ͉ sphingosine-1 phosphate C hanges in histone acetylation represent a key mechanism for the modulation of gene transcription (1). Acetylation of nucleosomal histones by histone acetyltransferases enhances transcription by relaxing the condensed structure of the nucleosome, whereas deacetylation of histones by histone deacetylase (HDAC) activity reverses this process and promotes chromatin condensation and transcriptional repression. The recruitment of histone acetyltransferases and HDACs by specific transcription factors enables these chromatin-modifying enzymes to regulate specific sets of target genes.