A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression.

Stress signaling in the myocardium results in enhanced expression of fetal ß-myosin heavy chain (ß-MyHC) and reduced expression of adult α-myosin heavy chain (α-MyHC), with the net outcome of diminished myofibrillar ATPase activity and impaired contractility. Pharmacological approaches aimed at preventing this myosin isoform "switch" could provide therapeutic benefit to patients with heart failure. Myosin isoform protein expression is typically quantified using gel electrophoresis methods, which are time-consuming and prone to variability. Here we describe a facile, reversed-phase high-performance liquid chromatography (HPLC) method for rapidly determining the relative amounts of full-length α- and ß-MyHC in rat hearts. The assay was validated using cardiac tissues from rats in which a key transcriptional regulator of MyHC expression, the thyroid hormone receptor, was pharmacologically manipulated. This novel assay should facilitate drug discovery efforts focused on the MyHC axis.

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1.

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling.

The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart.

Regulation of cardiac stress signaling by protein kinase d1.

In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.

Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5.

A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation- and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.