Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Abstract

Background: The generation of reactive oxygen species (ROS) during myocardial ischemia (I)/reperfusion (R) contributes to postreperfusion cardiac injury. The increase in ROS is attributed in part to the uncoupling of endothelial nitric oxide synthase (eNOS) that produces ROS instead of nitric oxide (NO) and mitochondrial derived ROS which in part is derived from opening of mitochondrial ATPsensitive K + channels (mK ATP ). Both the activity of uncoupled eNOS and the opening of mK ATP channels in mitochondria are stimulated by protein kinase C epsilon (PKCε) that is activated during reperfusion. We hypothesize that a cell permeable PKCε peptide inhibitor, (N-myristic acid-EAVSLKPT, Myr-PKCε-) given at reperfusion will improve postreperfused cardiac function and attenuate infarct size compared to untreated isolated perfused rat hearts subjected to ischemic reperfusion injury. Methods:Male Sprague-Dawley rats (275-325 g) were anesthetized with sodium pentobarbital (60 mg/kg) and anticoagulated with heparin 1000 units given intraperitoneally. The hearts were excised and attached to a Langendorff perfusion system. All hearts were subjected to 30 min of global ischemia, followed by 90 min of reperfusion. Myr-PKCε-was dissolved in Krebs' buffer and infused during the first 10 min of reperfusion at final concentrations of 5, 10 and 20 µM.Results: Myr-PKCε-treated hearts (10 and 20 μM) exhibited significant improvement in post-reperfused cardiac function at 90 min compared to untreated controls. The maximal rate of left ventricular developed pressure generation (+dP/dt max ) of Myr-PKCε-treated hearts showed significant recovery to 56±4% (10 μM, p<0.05, n=8) and 50±3%; (20 μM, p<0.05, n=8) of initial baseline values. By contrast, the +dP/ dt max of both untreated control hearts (n=9) and those treated with the lowest concentration of Myr-PKCε-(5 μM, n=8) recovered to only 30±4% and 33±4% of initial baseline values, respectively. Interestingly, all Myr-PKCε-treated hearts (5-20 μM) showed significant reduction in infarct size to 28±2% compared to untreated control hearts, which averaged 38±3% (p<0.05, n=9). Conclusion:The results suggest that Myr-PKCε-given at reperfusion effectively reduces infarct size, improves cardiac function and is a putative treatment that could aid in clinical myocardial infarction/ organ transplantation patient recovery. [3][4][5][6][7]. The necessity for effective pharmacological intervention still exists, despite numerous basic studies firmly establishing that I/R injury can be delayed or even reduced by the introduction of cardioprotective agents or strategies prior to a prolonged ischemic insult (preconditioning) or at the start of reperfusion [1,8]. Such interventions have been shown to reduce infarct size, attenuate the frequency and severity of reperfusion-induced arrhythmias, and prevent endothelial dysfunction [9][10][11][12][13].Preconditioning of the myocardium can be induced by several rounds of brief ischemia (i.e. less than 4 min) followed by several rounds of brief reperfus...