Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR ؊/؊ ) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1␣ (HIF-1␣) under normoxic conditions. We further show that S-nitrosylated HIF-1␣ binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.angiogenesis ͉ HIF-1␣ ͉ myocardial infarction ͉ nitric oxide ͉ S-nitrosylation

beta-Adrenergic receptor (betaAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in betaAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac betaARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for betaAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated betaARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/betaAR colocalization is required to preserve betaAR signaling, since deletion of a single PI3K isoform (PI3Kgamma knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent betaAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of betaAR turnover and show that abnormalities in betaAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of betaAR-localized PI3K activity represents a novel therapeutic approach to restore normal betaAR signaling and preserve cardiac function in the pressure overloaded failing heart.

. Jeffrey J. Nienaber and Hideo Tachibana contributed equally to this work. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: β-adrenergic receptor (βAR); G protein-coupled receptors (GPCRs); phosphatidylinositol (PtdIns); phosphoinositide kinase domain (PIK); phosphatidylinositol-3,4,5-tri-phosphate (PtdIns-3,4,5-P3); inactive mutant of PI3Kγ (PI3Kγinact); PI3Kγ knockout (PI3Kγ-KO); hemagglutinin (HA); transverse aortic constriction (TAC); left ventricle (LV); PtdIns-3,4,5-tri-phosphate (PIP3); phosphoprotein kinase B (pPKB); phospho-glycogen synthase kinase (pGSK); extracellular signal-regulated kinase (ERK); LV weight/body weight (LVW/BW); isoproterenol (ISO); immunoblotting (IB); c-terminal region of β-adrenergic receptor kinase-1 (βARK1-ct).

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Background-Downregulation of ␤-adrenergic receptors (␤ARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)-␥ and redistribution of ␤ARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3K␥ activity and intracellular distribution of ␤ARs. Additionally, we tested whether displacement of PI3K␥ from activated ␤ARs would restore agonist responsiveness in failing human cardiomyocytes. Methods and Results-To test the role of PI3K on ␤AR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in ␤AR kinase 1 (␤ARK1)-associated PI3K activity that was attributed exclusively to enhanced activity of the PI3K␥ isoform. Increased ␤ARK1-coupled PI3K activity in the failing hearts was associated with downregulation of ␤ARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3K␥ activation, normalized the levels of agonist-responsive ␤ARs at the plasma membrane, and depleted the ␤ARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the ␤AR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts ␤ARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to ␤-agonist. Conclusions-These results suggest a novel paradigm in which human ␤ARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3K␥ and ␤ARK1-associated PI3K activation. Furthermore, displacement of active PI3K from ␤ARK1 restores ␤AR responsiveness in failing myocytes.