Abstract-Diverse cardiac diseases induce cardiac hypertrophy, which leads to dilatation and heart failure. We previously reported that hypertrophy can be blocked by class I histone deacetylase (HDAC) inhibitor, which prompted us to investigate the regulatory mechanism of class I HDACs. Cardiac hypertrophy was introduced by aortic banding, by infusion of isoproterenol or angiotensin II, or by swimming. Hypertrophic stimuli transiently elevated the activity of histone deacetylase-2 (Hdac2), a class I HDAC. In cardiomyocytes, forced expression of Hdac2 simulated hypertrophy in an Akt-dependent manner, whereas enzymatically inert Hdac2 H141A failed to do so. Hypertrophic stimuli induced the expression of heat shock protein (Hsp)70. The induced Hsp70 physically associated with and activated Hdac2. Hsp70 overexpression produced a hypertrophic phenotype, which was blocked either by siHdac2 or by a dominant negative Hsp70⌬ABD. In Hsp70.1 Ϫ/Ϫ mice, cardiac hypertrophy and Hdac2 activation were significantly blunted. Heat shock either to cardiomyocytes or to mice activated Hdac2 and induced hypertrophy. However, heat shock-induced Hdac2 activation was blunted in the cardiomyocytes isolated from Hsp70.1 Ϫ/Ϫ mice. These results suggest that the induction of Hsp70 in response to diverse hypertrophic stresses and the ensuing activation of HDAC2 trigger cardiac hypertrophy, emphasizing HSP70/HDAC2 as a novel mechanism regulating hypertrophy. Key Words: cardiac hypertrophy Ⅲ class I histone deacetylases Ⅲ histone deacetylase 2 Ⅲ heat shock protein 70 Ⅲ Hsp70.1 Ϫ/Ϫ mice C ardiac hypertrophy is a response, either adaptive or maladaptive, to pressure or volume overload, mutations, or loss of contractile mass. Hypertrophic growth accompanies many forms of heart disease, including ischemic diseases, myocardial infarction, hypertension, aortic stenosis, and valvular dysfunctions. Although the initial hypertrophic responses seem to be an adaptation to those stimuli, the sustained stress may lead to cardiomyopathy and heart failure, a major cause of human morbidity and mortality. However, few interventions have proven effective in blocking the hypertrophy or in preventing the transition to congestive heart failure.Cardiomyocyte hypertrophy is characterized by an increase in individual myocyte size, enhanced protein synthesis, and heightened organization of the sarcomere, 1 which are regulated by activation of heart-specific transcription factors such as GATA4, MEF2, and immediate early genes like c-jun and c-fos. 2 The subsequent reactivation of the fetal gene program and repression of adult cardiac genes are closely related to the deterioration of heart function in hypertrophy.Recently, modulation of gene transcription by altering chromatin structure, especially by adding or removing acetyl groups to histone tails, has been implicated in diverse human pathologies, including cardiac hypertrophy. 3 Histone deacetylases (HDACs), which remove the acetyl group, repress downstream gene expression. Although HDACs are divided into 4 familie...

Background-Cardiac hypertrophy is characterized by transcriptional reprogramming of fetal gene expression, and histone deacetylases (HDACs) are tightly linked to the regulation of those genes. We previously demonstrated that activation of HDAC2, 1 of the class I HDACs, mediates hypertrophy. Here, we show that casein kinase-2␣1 (CK2␣1)-dependent phosphorylation of HDAC2 S394 is required for the development of cardiac hypertrophy. Methods and Results-Hypertrophic stimuli phosphorylated HDAC2 S394, which was necessary for its enzymatic activation, and therefore the development of hypertrophic phenotypes in rat neonatal cardiomyocytes or in isoproterenoladministered mice hearts. Transgenic mice overexpressing HDAC2 wild type exhibited cardiac hypertrophy, whereas those expressing phosphorylation-resistant HDAC2 S394A did not. Compared with that in age-matched normal human hearts, phosphorylation of HDAC2 S394 was dramatically increased in patients with hypertrophic cardiomyopathy. Hypertrophy-induced phosphorylation of HDAC2 S394 and its enzymatic activity were completely blocked either by CK2 blockers or by CK2␣1 short interfering RNA. Hypertrophic stimuli led CK2␣1 to be activated, and its chemical inhibitors blocked hypertrophy in both phenylephrine-treated cardiomyocytes and isoproterenol-administered mice. CK2␣1-transgenic mice developed hypertrophy, which was attenuated by administration of trichostatin A, an HDAC inhibitor. Overexpression of CK2␣1 caused hypertrophy in cardiomyocytes, whereas chemical inhibitors of both CK2 and HDAC as well as HDAC2 S394A blunted it. Hypertrophy in CK2␣1-transgenic mice was exaggerated by crossing these mice with wild-type-HDAC2-overexpressing mice. By contrast, however, it was blocked when CK2␣1-transgenic mice were crossed with HDAC2 S394A-transgenic mice. Conclusions-We have demonstrated a novel mechanism in the development of cardiac hypertrophy by which CK2 activates HDAC2 via phosphorylating HDAC2 S394. (Circulation. 2011;123:2392-2403.)Key Words: hypertrophy Ⅲ casein kinase 2 Ⅲ histone deacetylase 2 S394 Ⅲ phosphorylation Ⅲ transgenic mice C ardiac hypertrophy, an increase in the size of cardiomyocytes, is often caused by diverse pathological conditions such as myocardial infarction, hypertension, aortic stenosis, and valvular dysfunction. Although cardiac hypertrophy itself is an initial adaptive process, uncorrected continuous stimuli often lead the heart to heart failure. Because heart failure is a main cause of human mortality, many researchers are eager to develop interventions to reverse cardiac hypertrophy or to prevent the transition to congestive heart failure. Editorial see p 2341 Clinical Perspective on p 2403Posttranslational modifications of histones are closely involved in diverse biological processes through the regulation of transcription of downstream target genes. 1,2 Among these modifications, the acetylation status of the chromatin mediates the epigenetic regulation of gene expression. Two opposing groups of enzymes, histone acetyltransferase and hi...

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

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Skeletal muscle differentiation is well regulated by a series of transcription factors. We reported previously that enhancer of polycomb1 (Epc1), a chromatin protein, can modulate skeletal muscle differentiation, although the mechanisms of this action have yet to be defined. Here we report that Epc1 recruits both serum response factor (SRF) and p300 to induce skeletal muscle differentiation. Epc1 interacted physically with SRF. Transfection of Epc1 to myoblast cells potentiated the SRF-induced expression of skeletal muscle-specific genes as well as multinucleation. Proximal CArG box in the skeletal ␣-actin promoter was responsible for the synergistic activation of the promoter-luciferase. Epc1 knockdown caused a decrease in the acetylation of histones associated with serum response element (SRE) of the skeletal ␣-actin promoter. The Epc1⅐SRF complex bound to the SRE, and the knockdown of Epc1 resulted in a decrease in SRF binding to the skeletal ␣-actin promoter. Epc1 recruited histone acetyltransferase activity, which was potentiated by cotransfection with p300 but abolished by si-p300. Epc1 directly bound to p300 in myoblast cells. Epc1 ؉/؊ mice showed distortion of skeletal ␣-actin, and the isolated myoblasts from the mice had impaired muscle differentiation. These results suggest that Epc1 is required for skeletal muscle differentiation by recruiting both SRF and p300 to the SRE of muscle-specific gene promoters.

Cardiac hypertrophy occurs in response to increased hemodynamic demand and can progress to heart failure. Identifying the key regulators of this process is clinically important. Though it is thought that the phosphorylation of histone deacetylase (HDAC) 2 plays a crucial role in the development of pathological cardiac hypertrophy, the detailed mechanism by which this occurs remains unclear. Here, we performed immunoprecipitation and peptide pull-down assays to characterize the functional complex of HDAC2. Protein phosphatase (PP) 2 A was confirmed as a binding partner of HDAC2. PPP2CA, the catalytic subunit of PP2A, bound to HDAC2 and prevented its phosphorylation. Transient overexpression of PPP2CA specifically regulated both the phosphorylation of HDAC2 S394 and hypertrophy-associated HDAC2 activation. HDAC2 S394 phosphorylation was increased in a dose-dependent manner by PP2A inhibitors. Hypertrophic stresses, such as phenylephrine in vitro or pressure overload in vivo, caused PPP2CA to dissociate from HDAC2. Forced expression of PPP2CA negatively regulated the hypertrophic response, but PP2A inhibitors provoked hypertrophy. Adenoviral delivery of a phosphomimic HDAC2 mutant, adenovirus HDAC2 S394E, successfully blocked the anti-hypertrophic effect of adenovirus-PPP2CA, implicating HDAC2 S394 phosphorylation as a critical event for the anti-hypertrophic response. PPP2CA transgenic mice were protected against isoproterenol-induced cardiac hypertrophy and subsequent cardiac fibrosis, whereas simultaneous expression of HDAC2 S394E in the heart did induce hypertrophy. Taken together, our results suggest that PP2A is a critical regulator of HDAC2 activity and pathological cardiac hypertrophy and is a promising target for future therapeutic interventions.

Aims Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70). Methods and results Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2. Conclusion These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p ( miR-27a-3p ) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D 3 -administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p -induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3.