Abstract 221: Inhibition Of Wild Type P66shca In Cardiac Myocytes Prevents Hyperglycemia Induced Oxidative Stress And Promotes The Survival Phenotype
Hyperglycemia and diabetes mellitus (DM) are associated with an exponential increase in reactive oxygen species (ROS) at the cellular level. Phosphorylation of wild type (WT) p66ShcA protein at a critical Ser36 residue plays a pivotal role in the generation of hyperglycemia induced ROS. We hypothesized that gene based strategies targeting WT p66 ShcA, will attenuate or prevent ROS induced phenotype of apoptosis in adult rat ventricular myocytes (ARVM), maintained at high glucose (HG). To test this hypothesis, ARVM were cultured, plated and infected with adenovirus vector expressing dominant negative (DN) mutant 36p66ShcA construct. WT and DNp66ShcA ARVM were cultured in SFM containing 5mM and 25mM glucose for 16 hours. The phosphorylation status of Ser36 was determined by probing ARVM cell lysates with phospho-anti-ShcA (Ser36) antibody. At HG, phosphorylation at Ser36 was inhibited in DNp66ShcA ARVM. To explore whether DNp66ShcA ARVM exhibit an oxidant resistant phenotype, we examined oxidative DNA damage (apoptosis) and mitochondrial transmembrane potential (ΔΨm). DNp66ShcA ARVM were resistant to ROS-induced apoptosis at HG, whereas WT ARVM showed a 30% increase (p ≤ 0.05; n = 7); ΔΨm was preserved in DNp66ShcA at HG while collapse of ΔΨm was detected in WT ARVM. Similarly, catalase expression, a surrogate marker of oxidative stress was increased indicative of increased ROS production. We next examined the phosphorylation status of FOXO3a, a potent stress response regulator and downstream target of WT p66ShcA. The phosphorylation status of FOXO3a is a critical determinant of subcellular localization. At HG, DNp66ShcA ARVM exhibit decreased phosphorylation of AKT/PKB and FOXO3a, consistent with nuclear localization of FOXO3a. We conclude, silencing WT p66ShcA confers an oxidant resistant phenotype and promotes ARVM survival at HG. This may represent a parallel mechanism for PKCε induced cardioprotection in DM.