Abstractβ-adrenergic receptor (βAR) blockade is standard therapy for cardiac failure and ischemia. G-protein coupled receptor kinases (GRKs) desensitize βAR, suggesting that genetic GRK variants might modify outcomes in these syndromes. Re-sequencing of GRK2 and GRK5 revealed a nonsynonymous polymorphism of GRK5, common in African Americans (AA), substituting leucine (L) for glutamine (Q) at position 41. GRK5-L41 more effectively uncoupled isoproterenol-stimulated responses than GRK5-Q41 in transfected cells and transgenic mice, and like pharmacological βAR blockade, GRK5-L41 protected against experimental catecholamine-induced cardiomyopathy. Human association studies showed a pharmacogenomic interaction between GRK5-L41 and β-blocker treatment on mortality outcome in independent cohorts of AA cardiac failure (P=0.036) and ischemia (P=0.023). In 375 prospectively followed AA heart failure subjects, GRK5-L41 was protective against death/cardiac transplant (single allele: RR=0.28, 95% CI=0.12-0.66; two alleles: RR=0.08, 95% CI=0.04-0.19; P=0.004). The gain-of-function GRK5-L41 polymorphism facilitates βAR desensitization during catecholamine excess, imparting "genetic β-blockade" and improving survival in heart failure.

G protein-coupled receptor (GPCR) kinases (GRKs) are critical regulators of cellular signaling and function. In cardiomyocytes, GRK2 and GRK5 are two GRKs important for myocardial regulation, and both have been shown to be up-regulated in the dysfunctional heart. We report that increased levels and activity of GRK5 in failing myocardium may have unique significance due to its nuclear localization, a property not shared by GRK2. We find that transgenic mice with elevated cardiac GRK5 levels have exaggerated hypertrophy and early heart failure compared with control mice after pressure overload. This pathology is not present in cardiac GRK2-overexpressing mice or in mice with overexpression of a mutant GRK5 that is excluded from the nucleus. Nuclear accumulation of GRK5 is enhanced in myocytes after aortic banding in vivo and in vitro in myocytes after increased Gαq activity, the trigger for pressure-overload hypertrophy. GRK5 enhances activation of MEF2 in concert with Gq signals, demonstrating that nuclear localized GRK5 regulates gene transcription via a pathway critically linked to myocardial hypertrophy. Mechanistically, we show that this is due to GRK5 acting, in a non-GPCR manner, as a class II histone deacetylase (HDAC) kinase because it can associate with and phosphorylate the myocyte enhancer factor-2 repressor, HDAC5. Moreover, significant HDAC activity can be found with GRK5 in the heart. Our data show that GRK5 is a nuclear HDAC kinase that plays a key role in maladaptive cardiac hypertrophy apparently independent of any action directly on GPCRs.

Cardiac-restricted overexpression of the Ca 2+ -binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca 2+ transients and sarcoplasmic reticulum (SR) Ca 2+ load mechanistically due to increased SR Ca 2+ uptake and reduced SR Ca 2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na + concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca 2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.

Background-A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein ␣ subunit, G␣ i2 . It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G␣ subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. Methods and Results-We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of G␣ i2 that interacts specifically with G protein-coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a "functional knockout" of cardiac G␣ i2 signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9Ϯ2.5% to 50.9Ϯ4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Conclusions-Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of G␣ i2 is an adaptive protective response after ischemia to shield myocytes from apoptosis.

G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5’s newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca2+/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.

Background-Both the A 1 -and A 3 -adenosine receptors (ARs) have been implicated in mediating the cardioprotective effects of adenosine. Paradoxically, overexpression of both A 1 -AR and A 3 -AR is associated with changes in the cardiac phenotype.To evaluate the temporal relationship between AR signaling and cardiac remodeling, we studied the effects of controlled overexpression of the A 1 -AR using a cardiac-specific and tetracycline-transactivating factor-regulated promoter. Methods and Results-Constitutive A 1 -AR overexpression caused the development of cardiac dilatation and death within 6 to 12 weeks. These mice developed diminished ventricular function and decreased heart rate. In contrast, when A 1 -AR expression was delayed until 3 weeks of age, mice remained phenotypically normal at 6 weeks, and Ͼ90% of the mice survived at 30 weeks. However, late induction of A 1 -AR still caused mild cardiomyopathy at older ages (20 weeks) and accelerated cardiac hypertrophy and the development of dilatation after pressure overload. These changes were accompanied by gene expression changes associated with cardiomyopathy and fibrosis and by decreased Akt phosphorylation. Discontinuation of A 1 -AR induction mitigated cardiac dysfunction and significantly improved survival rate. Conclusions-These data suggest that robust constitutive myocardial A 1 -AR overexpression induces a dilated cardiomyopathy, whereas delaying A 1 -AR expression until adulthood ameliorated but did not eliminate the development of cardiac pathology. Thus, the inducible A 1 -AR transgenic mouse model provides novel insights into the role of adenosine signaling in heart failure and illustrates the potentially deleterious consequences of selective versus nonselective activation of adenosine-signaling pathways in the heart. (Circulation. 2006;114:2240-2250.)

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