Non-canonical regulation of GRK2 by TNFalpha impairs BetaAR function

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 DESCRIPTION (provided by applicant): Tumor necrosis factor alpha (TNFα) is a major pro-inflammatory cytokine that is significantly elevated in Type 2 diabetes and obesity which are known co-morbid cardiovascular risk factors. It is known that TNFα exposure leads to cardio-depressant negative inotropic effects. Despite this observation, little is understood about the underlying mechanisms. We have recently shown that TNFα causes β-adrenergic receptor (βAR) dysfunction through G-protein coupled receptor kinase 2 (GRK2) which may underlie cardio-depressant negative inotropic effects of TNFα. βARs are one of the most powerful regulators of cardiac function and βAR desensitization (i.e., diminished catecholamine) response is a hallmark of heart failure. Reduced βAR response to catecholamines is due to phosphorylation of βARs that is predominantly mediated by GRK2 which is markedly elevated in cardiac stress. Interestingly, our studies show that TNFα up-regulates GRK2 mediating βAR desensitization as siRNA knock down of GRK2 normalizes βAR function despite TNFα. TNFα pre-treatment of murine cardiomyocytes inhibits contractility to βAR agonist isoproterenol (ISO) that is remarkably preserved in GRK2 null myocytes indicating a role for GRK2 in TNFα mediated βAR function. Since GRK2 recruitment is mediated by Gβγ subunits of G-proteins, we determined whether TNFα mediated βAR function could be rescued by expression of Gβγ sequestering peptide GRK2-ct (βARK-ct). Although GRK2-ct preserved βAR function to ISO, it could not preserve βAR function to TNFα. Furthermore, TNFα administration in GRK2-ct transgenic mice with cardiac expression of GRK2-ct resulted in cardiac dysfunction associated with βAR desensitization despite no changes in catecholamines. Surprisingly, TNFα treatment resulted in marked β2AR phosphorylation even in the presence of βAR antagonist propranolol. These data suggest that TNFα mediates βAR desensitization by GRK2 in an agonist- and Gβγ-independent manner contrary to the current paradigm of βAR desensitization. In addition to elevation in TNFα, mouse models of obesity (mice on high fat-diet or lipolysis deficient adipose triglyceride lipase null mice (ATGL-/-)) are characterized by increased cardiac β2AR phosphorylation and upregulation of GRK2. Based on these exciting observations, we hypothesize that TNFα contributes to cardiac βAR desensitization by non-traditional GRK2 recruitment to the βAR complex suggesting a yet, unidentified cross-talk between TNFα and βAR signaling. Thus, to mechanistically understand TNFα- induced βAR desensitization, we have designed the following studies: Specific Aim 1: To determine whether TNFα-TNFR1/2-TRAF2 axis facilitates Gβγ-independent GRK2-mediated βAR desensitization. Specific Aim 2: To identify the mechanism of TRAF2-dependent GRK2 recruitment to βAR complex. Specific Aim 3: To demonstrate whether conditional cardiomyocyte GRK2 ablation (GKR2 del) in mice will ameliorate cardiac dysfunction/hypertrophy in mouse models of obesity. Understanding this pathway may provide novel therapeutic targets/strategies and importantly could also be a universal phenomenon of desensitizing other GPCRs in response to TNFα. 1