Increased Activity of NADPH Oxidase Contributes to Enhanced LV Myocyte Contraction in nNOS−/− Mice

Abstract

It is believed, but not without dispute, that activation of PKB is essential to obtain cardioprotection by ischemic preconditioning (IP). Here we have investigated the role of PKB activity in ischemic myocardial injury and IP using novel specific PKB inhibitors, examined whether any effect is species-dependent and determined its location in the transduction pathway. The specific PKB inhibitors VIII (0.05, 0.5 and 5mM) and XI (0.1, 1 and 10mM) were co-incubated with rat ventricular myocardium for 20min prior to 90min ischemia/120min reoxygenation at 37 C (n¼6/group). CK release and cell necrosis and apoptosis (% of nuclei) were significantly decreased by more than 60% at all concentrations of both inhibitors. Similar protection was obtained with IP, results that were unaffected by PKB inhibitors. The PI-3K inhibitors LY294002 (10mM) and wortmanin (0.1mM) administered for 20min prior to ischemia induced identical results to those seen with PKB inhibitors. The protection afforded by PKB inhibitor XI was unaffected by the presumed mitoK ATP channel blocker 5-HD (10mM) but was abrogated by the p38MAPK inhibitor SB203580 (10mM). Western Blot and Proteome Profiler studies confirmed a decrease in PKB phosphorylation in myocardium exposed to IP, wortmanin and PKB inhibitor XI. Studies using human myocardium also showed that both PKB inhibitor XI (1mM) and PI-3K inhibitor wortmanin (0.1mM) equally reduced CK release and cell necrosis and apoptosis. The diabetic myocardium, that could not be protected by IP or diazoxide (100mM), was however protected by PKB inhibitor XI and wortmanin, further suggesting that PKB is located beyond the mitochondria. In conclusion, inhibition of PKB activity is protective against ischemic injury of the rat and human myocardium and is as potent as IP. Importantly, PKB is downstream of the 5-HD target but upstream of p38MAPK.