Abstract 1012: The Delta Protein Kinase C Isozyme Inhibits F1Fo ATPase Activity Following Prolonged Oxygen Deprivation in Rat Cardiac Myocytes (CMs)
Under physiological conditions the F1Fo ATP synthase produces ~95 % of cardiac ATP. However, during prolonged cardiac ischemia the electrochemical gradient across the inner mitochondrial membrane is dissipated and the F1Fo ATP synthase is inhibited and ultimately operates in reverse to destroy ATP. Recovery of oxidative phosphorylation following ischemia / reperfusion (I/R) injury is crucial as the heart cannot sustain itself using anaerobic glycolysis. Our preliminary proteomic analyses suggest that the “d” subunit of the F1Fo ATP synthase (dF1Fo) may be phosphorylated following exposure of CMs to protein kinase C (PKC) stimuli. We therefore, evaluated PKC isozyme modulation of F1Fo ATP synthase / ATPase in CMs. In gradient-purified mitochondria isolated from CMs exposed to 100 nM PMA, δPKC (but notα, ϵor ζPKC) co-immunoprecipitated with antisera directed against dF1Fo (p<0.001, n=3) and correlated with a 55 ± 9 % inhibition of F1Fo ATPase activity (p<0.01, n=5). AδPKC-selective translocation inhibitor (δV1–1) chemically coupled to the HIV-Tat protein transduction domain antagonized the effects of PMA (p<0.01, n=5). In 2-D electrophoresis studies PMA treatment of CMs induced Pro-Q Diamond (a phospho-protein sensing dye) staining of mitochondrial proteins, many of which were attenuated by δV1–1. Incubation of CMs for 4 hrs in an anaerobic chamber also induced a δPKC-selective co-immunoprecipitation with dF1Fo antisera which correlated with an 85 ± 4 % inhibition of F1Fo ATPase activity (p<0.001, n=4). This effect was attenuated by δV1–1 (p<0.05, n=3). In contrast, an ϵPKC-selective translocation inhibitor (ϵV1–2) markedly enhanced the inhibition of F1Fo ATPase activity (p<0.01, n=3) suggesting opposing roles for the δ and ϵPKC isozymes. δPKC has been previously implicated in both cardiac preconditioning (PC) and I/R injury but few molecular targets of δ PKC in either response have been described. This is the first report of a specific PKC isozyme contributing F1Fo ATPase inhibition. Our future work will identify the sites of interaction between PKC isozymes and F1Fo ATPase subunits to form the basis for cell permeant and mitochondrial-selective peptidomimetics that may preserve ATP and improve clinical outcomes following cardiac I/R injury.