Diacylglycerol kinase-epsilon restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Galpha(q) signaling cascade

Galpha(q) protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKepsilon acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKepsilon prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKepsilon (DGKepsilon-TG) using an alpha-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKepsilon-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKepsilon-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKepsilon-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKepsilon-TG mice, and the survival rate after TAC was higher in DGKepsilon-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKepsilon-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKepsilon-TG mice. In conclusion, these results demonstrate the first evidence that DGKepsilon restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKepsilon may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.     

Transgenic replacement of type V adenylyl cyclase identifies a critical mechanism of beta-adrenergic receptor dysfunction in the G alpha q overexpressing mouse.

Chronic activation of Gq coupled receptors, or overexpression of G alpha q, in cardiomyocytes results in hypertrophy, enhanced expression of fetal genes, decreased basal and beta-adrenergic receptor (beta AR) stimulated adenylyl cyclase (AC) activities, and depressed cardiac contractility in vivo. Among several abnormalities of the beta AR-Gs-AC pathway that occur in G alpha q overexpressing transgenic mice, we have investigated whether the observed approximately 45% decrease in type V AC expression and function compared to non-transgenic (NTG) is the basis of the above phenotype. Transgenic mice were generated that overexpressed by approximately 50% the rat type V AC in the heart using the alpha-myosin heavy chain promoter. These mice were mated with the G alpha q transgenics resulting in animals (ACV/G alpha q) that had restored levels of forskolin stimulated AC activities in cardiac membranes. In addition, basal cardiac AC activities were normalized in the ACV/G alpha q mice (NTG=23+/-4.4, G alpha q=14+/-3.6, ACV/G alpha q=29+/-5.3 pmol/min/mg) as were maximal isoproterenol stimulated activities (59+/-8.9, 34+/-4.6, 52+/-6.7 pmol/min/mg respectively). Cardiac contractility was also improved by ACV replacement, with increased fractional shortening (51+/-2%, 36+/-6%, 46+/-3% respectively). In contrast, hypertrophy and expression of hypertrophy associated fetal genes were not affected. Thus the observed decrease in type V AC that accompanies the development of the cardiac phenotype in the G alpha q model is the dominant mechanism of dysfunctional beta AR signalling and contractility. In contrast, the decrease in type V AC or beta AR signalling to cAMP is not the basis of the hypertrophic response.     

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.     

Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy

Loss of cardiomyocytes through programmed cell death is a key event in the development of heart failure, but the inciting molecular mechanisms are largely unknown. We used microarray analysis to identify a genetic program for myocardial apoptosis in Gq-mediated and pressure-overload cardiac hypertrophy. A critical component of this apoptotic program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. A previously undescribed truncated Nix isoform, termed sNix, was not targeted to mitochondria but heterodimerized with Nix and protected against Nix-mediated apoptosis. Forced in vivo myocardial expression of Nix resulted in apoptotic cardiomyopathy and rapid death. Conversely, sNix protected against apoptotic peripartum cardiomyopathy in G(alpha)q-overexpressors. Thus, Nix/Bnip3L is upregulated in myocardial hypertrophy, and is both necessary and sufficient for Gq-mediated apoptosis of cardiomyocytes and resulting hypertrophy decompensation.     

Changes in ionic currents and beta-adrenergic receptor signaling in hypertrophied myocytes overexpressing G alpha(q)

Transgenic overexpression of G alpha(q) causes cardiac hypertrophy and depressed contractile responses to beta-adrenergic receptor agonists. The electrophysiological basis of the altered myocardial function was examined in left ventricular myocytes isolated from transgenic (G alpha(q)) mice. Action potential duration was significantly prolonged in G alpha(q) compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K(+) currents, transient outward K(+) currents (I(to)), and Na(+)/Ca(2+) exchange currents were reduced in G alpha(q) myocytes. Consistent with functional measurements, Na(+)/Ca(2+) exchanger gene expression was reduced in G alpha(q) hearts. Kinetics or sensitivity of I(to) to 4-aminopyridine was unchanged, but 4-aminopyridine prolonged the action potential more in G alpha(q) myocytes. Isoproterenol increased L-type Ca(2+) currents (I(Ca)) in both groups, with a similar EC(50), but the maximal response in G alpha(q) myocytes was approximately 24% of that in NTG myocytes. In NTG myocytes, the maximal increase of I(Ca) with isoproterenol or forskolin was similar. In G alpha(q) myocytes, forskolin was more effective and enhanced I(Ca) up to approximately 55% of that in NTG myocytes. These results indicate that the changes in ionic currents and multiple defects in the beta-adrenergic receptor/Ca(2+) channel signaling pathway contribute to altered ventricular function in this model of cardiac hypertrophy.     

PKCepsilon is essential for gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells

Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects. In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces gelsolin mRNA independently of the de novo protein synthesis. Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression. Furthermore, inhibition of PKCepsilon by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCepsilon is the major isoform for this process. In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity. Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin.