Ethanol Prevents Oxidant-Induced Mitochondrial Permeability Transition Pore Opening in Cardiac Cells

Aims: The purpose of this study was to determine if ethanolprevents the mitochondrial permeability transition pore (mPTP)opening via glycogen synthase kinase 3β (GSK-3β).Methods: Cardiac H9c2 cells were exposed to ethanol (10–1000µM) for 20 min. GSK-3β activity was determined bymeasuring its phosphorylation at Ser⁹. Mitochondrial membranepotential (_m) was assessed by imaging (confocal microscopy)H9c2 cells loaded with tetramethylrhodamine ethyl ester (TMRE).To activate GSK-3β, cells were transfected with constitutivelyactive GSK-3β (GSK-3β-S9A-HA) mutant plasmid. Results:Treatment of cardiac cells with low doses of ethanol (10–500µM) significantly enhanced GSK-3β phosphorylation,indicating that ethanol can inactivate GSK-3β in H9c2 cells.The effect of ethanol on GSK-3β activity was reversed bythe phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002and ethanol could enhance Akt phosphorylation, implying thatthe PI3K/Akt pathway accounts for the action of ethanol. Ethanolprevented oxidant (H₂O₂)-induced loss _m, an effect that wasreversed by LY294002, indicating that ethanol can modulate themPTP opening caused by oxidant stress through the PI3K/Akt pathway.Ethanol failed to preserve _m in cells transfected with the constitutivelyactive GSK-3β (GSK-3β-S9A-HA) mutant, suggesting thatethanol prevents the mPTP opening by inactivating GSK-3β.Conclusions: These data suggest that ethanol prevents the mPTPopening through inactivation of GSK-3β. The PI3K/Akt signalingpathway is responsible for inactivation of GSK-3β by ethanol.