Regulation of cytochrome oxidase and F1Fo ATP synthase by protein kinase C isozymes: Implications for cardiac preconditioning and ischemia/reperfusion injury.

摘要

Despite decades of intense research, heart disease associated with myocardial ischemia/reperfusion (IR) injury remains the most frequent cause of lethality worldwide. It has been known for over 2 decades that the mammalian heart can be protected from IR injury if exposed to a paradoxical response known as cardiac ischemic preconditioning (PC). Of interest, mitochondrial protein kinase C (PKC) isozymes have emerged as central players in both PC and IR mechanisms. Mitochondrial oxidative phosphorylation (OXPHOS) complexes are responsible for greater than 90% of myocardial ATP synthesis, and ATP levels decline substantially during myocardial IR injury. Therefore, we determined if direct protein-protein interactions occurred between individual PKC isozyme and specific subunits of each of these complexes during cardioprotective and cardiac IR responses. First, we have utilized an in situ rat coronary ligation model to establish an epsilonPKC-cytochrome oxidase subunit IV (COIV) coimmunoprecipitation (co-IP) in myocardium exposed to PC. This co-IP correlated with a 2.8-fold increase in mitochondrial epsilonPKC autophosphorylation (activation) and a 2-fold enhancement of cytochrome- c oxidase activity. In a second line of study, we demonstrated that following prolonged 4-beta phorbol 12-myristate-13-acetate (PMA) and hypoxia (Hx), deltaPKC interacts with the "d" subunit of F1Fo ATP synthase (dF1Fo) to inhibit F1Fo) activity in neonatal cardiac myocytes (NCMs). We next developed cell-permeable, mitochondrial-targeted peptide modulators (derived from the amino acid sequence of dF1Fo) based on the deltaPKC-dF1Fo interaction. In vitro binding assays and co-IP experiments using NCMs revealed a facilitator [NH 2-YGRKKRRQRRMLATRALSLIGKRAISTSVCRVREYEKQLEKIKNMIDYKDDDK-COOH ] and an inhibitor peptide [NH2-YGRKKRRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of the deltaPKC-dF1Fo interaction. The inhibitor peptide reduced PMA/Hx-induced inhibition of F1Fo activity or PMA-Hx-induced deltaPKC-dF 1Fo co-IP in NCMs while the facilitator peptide has opposite effects. Administration of the inhibitor peptide to isolated rat hearts immediately after a 20 min global ischemia exposure, and just prior to a 90 min reperfusion, decreased infarct size and released of cardiac troponin I compared to rat hearts receiving a scrambled-sequence (inactive) peptide prior to IR exposures alone. Collectively our studies have revealed two key mitochondrial OXPHOS regulatory events involving epsilonPKC-enhancement and protection of cytochrome oxidase by ischemic PC and deltaPKC suppression of F1Fo ATP synthase during IR injury. Further, our work suggests that the deltaPKC-dF1 Fo inhibitor peptide may have potential as a therapeutic compound targeting myocardial ischemia in humans.;INDEX WORDS: Protein Kinase C, oxidative phosphorylation, preconditioning, ischemia reperfusion injury, cytochrome oxidase, F1 Fo ATPase, protein transduction and mitochondrial-targeting domains