Abstract 12526: AKAP150-Mobilized PKC Activation Mediates Myocardial Dysfunction in Diabetes

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Background: Chronic activation of conventional PKC (cPKC) is a key signaling that directs the cardiac toxicity of hyperglycemia. AKAP150, a scaffold protein of the A-kinase anchoring proteins (AKAPs) family, is less defined regarding its capability to anchor and regulate cPKC signaling in cardiomyocytes. This study was aimed to investigate the role of AKAP150 in cPKC-mediated cardiac injury in diabetes. Methods and Results: In cardiac tissues from streptozotocin-induced diabetic rats and high-glucose treated neonatal rat cardiomyocytes, expression of AKAP150 increased significantly at both mRNA and protein levels, with a marked elevation in expression and activity of cPKC and phosphorylation levels of p65NF-κB and p47phox. AKAP150 knockdown was established via intramyocardial injection in vivo and transfection in vitro of adenovirus carrying AKAP150 targeted shRNA. Downregulation of AKAP150 reversed diabetes-induced diastolic dysfunction, as evidenced by decreased left ventricular end-diastolic diameter and early/late mitral diastolic wave ratio. AKAP150 inhibition also abrogated high-glucose induced cardiomyocyte apoptosis (TUNEL staining and annexin V/PI flow cytometry) and oxidative stress (ROS production, NADPH oxidase activity and lipid peroxidation). More importantly, reduced AKAP150 expression significantly reversed high-glucose induced membrane translocation and activation of cPKC, and inhibited phosphorylation of p65NF-κB and p47phox. Immunofluorescent co-expression and immunoprecipitation analysis indicated enhanced anchoring of AKAP150 with cPKC within the plasma membrane in both diabetic myocardium and high-glucose treated cardiomyocytes. Further studies using antibodies and specific inhibitors against different cPKC isoforms revealed that AKAP150 preferentially co-localized and functionally bound with PKC α and β, which are the major isoforms responsible for cardiac glucotoxicity. Conclusions: Cardiac AKAP150 positively responds to hyperglycemic stimuli and functions to enhance the efficiency of glucotoxicity signaling through a cPKC/p47phox/ROS pathway that induces myocardial dysfunction, cardiomyocyte apoptosis and oxidative stress.