Reversible blockade of complex I or inhibition of PKCβ reduces activation and mitochondria translocation of p66Shc to preserve cardiac function after ischemia.

Meiying Yang,David F Stowe,James S Heisner,Amadou K S Camara,Kenechukwu B Udoh

doi:10.1371/journal.pone.0113534

Abstract

AimExcess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection.MethodsIsolated 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 PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc 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.ResultsPhosphorylation of p66Shc 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 p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion.ConclusionOur results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.

Highlights

Mitochondria are proximal effectors and determinants of cell fate during ischemia and reperfusion (IR)-mediated oxidative stress
Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of protein kinase C b (PKCb) activation, decreases p66Shc activation and translocation and reduces IR damage
We first examined if and when p66Shc was activated during cardiac IR by determining the level of phosphorylation of p66Shc at serine 36 (Ser36), an indication of p66Shc activation [12, 13]

Summary

Introduction

Mitochondria are proximal effectors and determinants of cell fate during ischemia and reperfusion (IR)-mediated oxidative stress. They are potential therapeutic targets to ameliorate oxidative damage [1]. P66Shc gene ablation (p66Shc2/2) has been shown to reduce hypoxia/reoxygenation-induced damage to hepatocytes [9] and to decrease necrosis and apoptosis of myofibrils after hind limb ischemia compared to the wild type [10]. Pinton et al [13] reported that in MEF, inhibition of PKCb with hispidin inhibited H2O2 -induced p66Shc phosphorylation; overexpression of PKCb mediated H2O2-induced mitochondrial dysfunction in wild type MEFs, but not in p66Shc2/2 MEFs. It was reported that activation of PKCbII in ventricular tissue increased after IR and that gene deletion or pharmacological blockade of PKCbII was associated with protection against ischemia [14]

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