Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia

Aim: Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66(Shc), a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66(Shc) protects against IR injury, but it is unknown if and when p66(Shc) is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKC beta alters p66(Shc) activation during IR is associated with cardioprotection. Methods: Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKC beta inhibitor. Phosphorylation of p66(Shc) at serine 36 and levels of p66(Shc) in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion. Results: Phosphorylation of p66(Shc) and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66(Shc) levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66(Shc) and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66(Shc) by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion. Conclusion: Our results show that IR activates p66(Shc) and that reversible blockade of electron transfer from complex I, or inhibition of PKC beta activation, decreases p66(Shc) activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.