Phosphoinositide 3-kinaseγ (PI3Kγ) is activated by G-protein coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the β-adrenergic receptor (βAR, a GPCR) complex altering G-protein coupling. PI3Kγ inhibition results in significant increase of βAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 & 93 resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of β2ARs is observed with phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in β2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.

It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.Heart failure has been classified as an epidemic of the 21st century and is now the major cause of morbidity in the elderly in the United States. End-stage heart failure is characterized by significantly perturbed neurohormonal and mechanical (hemodynamic) stimuli to the heart. The altered pathological signaling leads to remodeling of the heart with adaptive to maladaptive hypertrophy transitioning into dilated cardiomyopathy (DCM).3 DCM is the most common and well documented outcome of various deleterious stimuli the heart perceives (1). DCM is characterized clinically by left ventricular dilatation, ventricular wall thinning, and homogeneous myocardial dysfunction leading to congestive heart failure (1). The myocytes under the continuously changing conditions of biomechanical stress during this transition undergo a remodeling process through the activation of intracellular signaling pathways and transcriptional mediators (2). The pathological end-stage DCM is a result of the concomitant cross-talk between various deleterious and compensatory signaling pathways. The balance between these two dynamic pathways ultimately determines the progression of the pathology. Despite significant advances in identification of genes and signaling pathways, the overall complexity of hypertrophic remodeling suggests the involvement of additional global regulatory mechanisms...

Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.

Back ground Pro-inflammatory cytokine tumor necrosis factor α (TNFα) induces β-adrenergic receptor (βAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results Two different pro-inflammatory transgenic mouse models with cardiac overexpression of Myotrophin (a pro-hypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate βAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling βAR desensitization independent of sympathetic overdrive. TNFα-mediated βAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both the mouse models. In vitro studies in β2 AR overexpressing HEK 293 cells showed significant βAR desensitization, GRK2 upregulation and recruitment to the βAR complex following TNFα. Interestingly, inhibition of PI3K abolished GRK2-mediated βAR phosphorylation and GRK2 recruitment upon TNFα. Furthermore, TNFα-mediated βAR phosphorylation was not blocked with βAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gβγ sequestering peptide βARK-ct could not prevent βAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the βAR is Gβγ independent. siRNA knock down of GRK2 resulted in loss of TNFα-mediated βAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility showing that TNFα-induced βAR desensitization is GRK2 dependent. Conclusions TNFα-induced βAR desensitization is mediated by GRK2 and is independent of Gβγ uncovering a hitherto unknown cross-talk between TNFα and βAR function providing the underpinnings of inflammation-mediated cardiac dysfunction.