Modulation of the mitochondrial permeability transition pore complex in GSK-3β-mediated myocardial protection

Recently we found that the level of anti-infarct tolerance afforded by ischemic preconditioning (IPC) and erythropoietin (EPO) infusion was closely correlated with the level of Ser9-phospho-GSK-3 beta upon reperfusion in the heart. To get an insight into the mechanism by which phospho-GSK-3 beta protects the myocardium from ischemia/reperfusion injury, we examined the effects of IPC and EPO on interactions between GSK-3 beta and subunits of the mitochondrial permeability transition pore (mPTP) in this study. Rat hearts were subjected to 25-min global ischemia and 5-min reperfusion in vitro with or without IPC plus EPO infusion (5 units/ml) before ischemia. Ventricular tissues were sampled before or after ischemia/reperfusion to separate subcellular fractions for immunoblotting and immunoprecipitation. Reperfusion increased mitochondrial GSK-3 beta[ by 2-fold and increased phospho-GSK-3 beta level in all fractions examined. Major subunits of mPTP, adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), were co-immunoprecipitated with GSK-3 beta after reperfusion. Phospho-GSK-3 beta was co-immunoprecipitated with ANT but not with VDAC. IPC + EPO significantly increased the levels of GSK-3 beta and phospho-GSK-3 beta that were co-immunoprecipitated with ANT to 145 +/- 8% and 143 +/- 16%, respectively, of baseline but did not induce phospho-GSK-3 beta-VDAC binding. A PKC inhibitor and a PI3 kinase inhibitor suppressed the IPC + EPO-induced increase in the level of phospho-GSK-3 beta-ANT complex. The level of cyclophilin D co-immunoprecipitated with ANT after reperfusion was significantly reduced to 39 +/- 10% of the control by IPC + EPO. These results suggest that reduction in affinity of ANT to cyclophilin D by increased phospho-GSK-3 beta binding to ANT may be responsible for suppression of mPTP opening and myocardial protection afforded by IPC+EPO. (c) 2007 Elsevier Inc. All rights reserved.