A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia-reperfusion (MI-R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI-R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H9C2 cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H2O2). Cells treated with HNG in the presence of H2O2 demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H2O2. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo. (C) 2013 Elsevier Inc. All rights reserved.