Local delivery of PKCε-activating peptide mimics ischemic preconditioning in aged hearts through GSK-3β but not F1-ATPase inactivation

In adult heart, selective PKC epsilon activation limits ischemia (I)-reperfusion (R) damage and mimics the protection associated with ischemic preconditioning. We sought to determine whether local delivery of PKCe activator peptide Psi epsilon-receptor for activated C-kinase (Psi epsilon-RACK) is sufficient to produce a similarly protected phenotype in aged hearts. Langendorff-perfused hearts isolated from adult (5 mo; n = 9) and aged (24 mo; n = 9) male Fisher 344 rats were perfused with Psi epsilon-RACK conjugated to Tat (500 nM) or Tat only (500 nM) for 10 min before global 31-min ischemia. Western blotting was used to measure mitochondrial targeting of PKC epsilon, PKC delta, phospho (p)-GSK-3 beta (Ser(9)) and GSK-3 beta in hearts snap-frozen during I. Recovery of left ventricular developed pressure was significantly improved by Psi epsilon-RACK (P < 0.01) and infarct size reduced in 24-mo rats vs. age-matched controls (60% vs. 34%; P < 0.01). Mitochondrial PKCe levels were 30% greater during I with Psi epsilon-RACK in aged vs. control rats (P < 0.01). Interestingly, mitochondrial GSK-3 beta levels were threefold greater in aged vs. adult rats during I, and Psi epsilon-RACK prevented this increase (P < 0.01). Mitochondrial p-GSK-3 beta levels were also greater in aged rats after Psi epsilon-RACK (P < 0.01), and subsequent inhibition of GSK-3 beta with SB-216763 (3 mu M) before I/R elicited protection similar to that of Psi epsilon-RACK (n = 3/group). Mitochondrial proteomic analysis further identified group differences in the F-1-ATPase beta-subunit, and coimmunoprecipitation studies revealed a novel interaction with PKCe. F-1-ATPase-PKC epsilon association was affected by Psi epsilon-RACK in adult but not aged rats. Our results provide evidence, for the first time, for PKC epsilon-mediated protection in aged rat heart after I/R and suggest a central role for mitochondrial GSK-3 beta but not F-1-ATPase as a potential target of PKCe to limit I/R damage with aging.