Abstract 36: Disrupting the Delta Protein Kinase C Interaction with the “d” Subunit of F1Fo ATP Synthase Protects Against Ischemia-Reperfusion Injury in Perfused Rat Hearts

Abstract

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces >90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p<0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p<0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p < 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.