Promiscuous actions of small molecule inhibitors of the protein kinase D-class IIa HDAC axis in striated muscle.

PKD-mediated phosphorylation of class IIa HDACs frees the MEF2 transcription factor to activate genes that govern muscle differentiation and growth. Studies of the regulation and function of this signaling axis have involved MC1568 and Gö-6976, which are small molecule inhibitors of class IIa HDAC and PKD catalytic activity, respectively. We describe unanticipated effects of these compounds. MC1568 failed to inhibit class IIa HDAC catalytic activity in vitro, and exerted divergent effects on skeletal muscle differentiation compared to a bona fide inhibitor of these HDACs. In cardiomyocytes, Gö-6976 triggered calcium signaling and activated stress-inducible kinases. Based on these findings, caution is warranted when employing MC1568 and Gö-6976 as pharmacological tool compounds to assess functions of class IIa HDACs and PKD.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Roles of histone deacetylation and AMP kinase in regulation of cardiomyocyte PGC-1α gene expression in hypoxia.

The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a key determinant of cardiac metabolic function by regulating genes governing fatty acid oxidation and mitochondrial biogenesis. PGC-1α expression is reduced in many cardiac diseases, and gene deletion of PGC-1α results in impaired cardiomyocyte metabolism and function. Reduced fuel supply generally induces PGC-1α expression, but the specific role of oxygen deprivation is unclear, and the mechanisms governing PGC-1α gene expression in these situations are poorly understood. During hypoxia of primary rat cardiomyocytes up to 12 h, we found that PGC-1α expression was downregulated via a histone deacetylation-dependent mechanism. Conversely, extended hypoxia to 24 h concomitant with glucose depletion upregulated PGC-1α expression via an AMP-activated protein kinase (AMPK)-mediated mechanism. Our previous work demonstrated that estrogen-related receptor-α (ERRα) regulates PGC-1α expression, and we show here that overexpression of ERRα was sufficient to attenuate PGC-1α downregulation in hypoxia. We confirmed that chronic hypoxia downregulated cardiac PGC-1α expression in a hypoxic but nonischemic hypobaric rat model of pulmonary hypertension. Our data demonstrate that depletion of oxygen or fuel results in repression or induction, respectively, of PGC-1α expression via discrete mechanisms, which may contribute to cardiac energetic derangement during hypoxia, ischemia, and failure.

Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism.

RATIONALE: Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension with associated right ventricular cardiac remodeling are poorly understood. OBJECTIVE: This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. METHODS AND RESULTS: Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. CONCLUSIONS: By targeting distinct pathogenic mechanisms, isoform-selective HDAC inhibitors have potential as novel therapeutics for pulmonary hypertension that will complement vasodilator standards of care.

Emergence of fibroblasts with a proinflammatory epigenetically altered phenotype in severe hypoxic pulmonary hypertension.

Persistent accumulation of monocytes/macrophages in the pulmonary artery adventitial/perivascular areas of animals and humans with pulmonary hypertension has been documented. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. We hypothesized that perivascular inflammation is perpetuated by activated adventitial fibroblasts, which, through sustained production of proinflammatory cytokines/chemokines and adhesion molecules, induce accumulation, retention, and activation of monocytes/macrophages. We further hypothesized that this proinflammatory phenotype is the result of the abnormal activity of histone-modifying enzymes, specifically, class I histone deacetylases (HDACs). Pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (termed PH-Fibs) expressed a constitutive and persistent proinflammatory phenotype defined by high expression of IL-1ß, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4, GM-CSF, CD40, CD40L, and VCAM-1. The proinflammatory phenotype of PH-Fibs was associated with epigenetic alterations as demonstrated by increased activity of HDACs and the findings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression levels in these cells. PH-Fibs induced increased adhesion of THP-1 monocytes and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages as well as activated monocytes/macrophages to express proinflammatory cytokines and profibrogenic mediators (TIMP1 and type I collagen) at the transcriptional level. Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce monocyte migration and proinflammatory activation. The emergence of a distinct adventitial fibroblast population with an epigenetically altered proinflammatory phenotype capable of recruiting, retaining, and activating monocytes/macrophages characterizes pulmonary hypertension-associated vascular remodeling and thus could contribute significantly to chronic inflammatory processes in the pulmonary artery wall.

Cardiac HDAC6 catalytic activity is induced in response to chronic hypertension.

Small molecule histone deacetylase (HDAC) inhibitors block adverse cardiac remodeling in animal models of heart failure. The efficacious compounds target class I, class IIb and, to a lesser extent, class IIa HDACs. It is hypothesized that a selective inhibitor of a specific HDAC class (or an isoform within that class) will provide a favorable therapeutic window for the treatment of heart failure, although the optimal selectivity profile for such a compound remains unknown. Genetic studies have suggested that class I HDACs promote pathological cardiac remodeling, while class IIa HDACs are protective. In contrast, nothing is known about the function or regulation of class IIb HDACs in the heart. We developed assays to quantify catalytic activity of distinct HDAC classes in left and right ventricular cardiac tissue from animal models of hypertensive heart disease. Class I and IIa HDAC activity was elevated in some but not all diseased tissues. In contrast, catalytic activity of the class IIb HDAC, HDAC6, was consistently increased in stressed myocardium, but not in a model of physiologic hypertrophy. HDAC6 catalytic activity was also induced by diverse extracellular stimuli in cultured cardiac myocytes and fibroblasts. These findings suggest an unforeseen role for HDAC6 in the heart, and highlight the need for pre-clinical evaluation of HDAC6-selective inhibitors to determine whether this HDAC isoform is pathological or protective in the setting of cardiovascular disease.

The role of MEKK1 in hypertrophic cardiomyopathy.

MEKK1 is a ubiquitously expressed mitogen activated protein kinase that is involved in tissue remodeling in a variety of settings including carotid artery blood flow cessation, wound healing, and breast adenocarcinoma intravasation. Here, we have tested the function of MEKK1 in genetic hypertrophic cardiomyopathy (HCM). MEKK1 was genetically deleted in C57Bl6/J mice expressing a mutant alpha-myosin heavy chain (HCM-MEKK1(-/-)). The absence of MEKK1 in HCM resulted in a more pronounced hypertrophy when compared to HCM mice with the MEKK1 gene intact without further increases in atrial natriuretic factor and beta-myosin heavy chain (MyHC) expression and fibrosis. Since MEKK1 is required for the induction of several tissue proteases, we tested the hypothesis that cardiac enlargement of HCM- MEKK1(-/-) mice was due to altered expression of urokinase-type plasminogen activator (uPA), JunB, matrix-metalloproteinase (MMP), and tissue inhibitors of MMPs (TIMPs). Because of its role in preventing apoptosis, we also tested the loss of MEKK1 on apoptotic mediators Bcl-2, cytochrome C, caspase-9, and caspase-3. uPA expression was decreased while JunB, MMP-9, caspase-9, and caspase-3 activities were elevated in HCM- MEKK1(-/-) hearts when compared to MEKK1(-/-), wild-type (WT), and HCM mice. Bcl-2 and Cyt C expression was elevated only in HCM mice. We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis.