Attenuating complex I activity decreases p66 shc phosphorylation and translocation to mitochondria during cardiac ischemia reperfusion injury

Mitochondrial reactive oxygen species (mROS), produced via complexes I and III electron leak, play a critical role in cardiac ischemia (I) and reperfusion (R) injury. p66 shc , a splice variant of the ShcA adaptor protein family, enhances mROS by oxidizing reduced cyt c to yield H 2 O 2 . p66 shc ablation protects against IR injury by decreasing mROS. It is not known if blocking complex I electron transfer impacts p66 shc activation during IR. To test this, isolated guinea pig hearts were buffer perfused and subjected to 5, 10, 20 or 35 min I and 20 min R ± amobarbital (AMB), a complex I blocker. Levels of p66 shc in mitochondria (mitop66 shc ) and phosphorylation of p66 shc at serine 36 (phoSer 36 ) were measured by Western blot and immunoprecipitation. We found that phoSer 36 and mitop66 shc increased during reperfusion after I 20, 35, but not during ischemia. Moreover, I 20 or I 35+R 20, but not I 5 or I 10+R 20 increased phoSer 36 and mitop66 shc . AMB given 1 min before I reduced phoSer 36 and mitop66 shc induced by I 20 or I 30+R 20, but not I 35+R 20. Low levels of phoSer 36 and mitop66 shc were associated with better recovery during R. Our results show that p66 shc was activated by IR. Shorter I time or attenuating complex I decreased activation of p66 shc . AMB reduces electron transfer during IR and thereby decrease reduction of cyt c , a p66 shc substrate mediated mROS production, as a possible mechanism to improve function during reperfusion. (NIH,VA)