Abstract 1285: GSK-3β Signaling Module in Mitochondria Leading to Myocardial Protection Against Infarction
Objective Recent studies suggest that phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser-9 increases the threshold for opening mitochondrial permeability transition pores (mPTPs), resulting in suppression of lethal myocardial ischemia/reperfusion injury. However, the mechanism by which phospho-GSK-3β modulates the mPTP threshold is unclear. The aim of this study was to clarify intracellular translocation of GSK-3β in response to ischemia/reperfusion and its modulation by cardioprotective signals.
Methods Isolated buffer-perfused rat hearts underwent 25-min global ischemia/5-min reperfusion with or without ischemic preconditioning (IPC) or pre-ischemic infusion of erythropoietin (Epo, 5 units/ml) for 15 min. IPC was performed with 2 cycles of 5-min ischemia/5-min reperfusion. These protocols of IPC and Epo have been confirmed to afford significant infarct size limitation in our previous studies. Ventricular tissues were sampled for cell fractionation, immunopre-cipitation and immunoblotting for protein kinases and two components of mPTP (adenine nucleotide translocase [ANT] and voltage-dependent anion channel [VDAC]).
Results Under baseline conditions, 70% of GSK-3β was localized in the cytosol of the myocardium. Ischemia/reperfusion induced translocation of GSK-3β to the mitochondria. Furthermore, in the reperfused myocardium, GSK-3β was co-immunoprecipitated with ANT, VDAC and protein kinase C-κ (PKC-κ) but not with Akt. IPC and Epo increased phosphorylation of GSK-3β co-immunoprecipitated with ANT and VDAC, though the total amount of GSK-3β bound to these mPTP components was not changed. Phosphorylation of GSK-3β by IPC and Epo was attenuated by chelerythrine, a PKC inhibitor, and by wortmannin, a PI3 kinase inhibitor.
Conclusion The results indicate that GSK-3β forms a protein complex with PKC-κand components of mPTP, including ANT and VDAC, after ischemia/reperfusion. Direct modulation of mPTPs by GSK-3β that is phosphorylated by cytosolic Akt and mitochondrial PKC-κ may contribute to myocyte protection afforded by IPC and Epo.