Attenuating the Interaction Between Delta Protein Kinase C and the

Abstract

Cardiac ischemia / reperfusion (IR) injury most often results from the thrombotic blockade of the coronary arteries and is the most frequent cause of death in humans. Despite the significant role energy deprivation plays in cardiac IR injury, few studies have targeted the IR-induced impairment of the mitochondrial F1Fo ATP synthase. We have previously demonstrated delta protein kinase C (δPKC) involvement in cardiac myocyte energy deprivation via its interaction with the “d” subunit of F1Fo ATP synthase (dF1Fo) and have developed a peptide inhibitor [NH2YGRKKRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of this interaction. It targets to the mitochondrial matrix / inner membrane. The inhibitor peptide contains a FLAG epitope which allowed confirmation of its uptake into cardiac mitochondria. Our early studies in neonatal cardiac myocytes (NCMs) led us to the hypothesis that PKC inhibits ATP production in vivo via an interaction with dF1Fo to exacerbate cardiac IR injury. To directly test our hypothesis, we first utilized the Langendorff isolated heart model to show that PKC co-immunoprecipitates (co-IPs) with antisera to dF1Fo in myocardium exposed to simulated IR injury. Administration of the inhibitor peptide to the isolated rat hearts prior to cardiac IR attenuated the co-IP of ����PKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced myocardial infarct size (as assessed by 2, 3, 5 triphenyltetrazolium chloride (TTC) staining) following simulated IR exposures. Additionally, this peptide enhanced ATP levels 2.1 fold, improved ADP-stimulated mitochondrial respiration, and attenuated Ca++-induced mitochondrial swelling in ischemic myocardium. We next evaluated the inhibitor peptide in an in situ rat coronary ligation model for its ability to protect live rats from cardiac IR injury. A 10 min coronary ligation increased the PKC-dF1Fo co-IP in the region at risk (RAR) by 5-fold which was attenuated by 71% with intravenous infusion of the inhibitor peptide. This response correlated with an enhancement of ATP levels, a 2-fold reduction in oxidative stress markers, improvement in systolic cardiac function, and a reduction in TTC monitored myocardial infarct size in the RAR. These results support further development of this peptide as a first-in-class-translational therapeutic for the treatment of cardiac IR injury.