Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

Loss of mitochondrial membrane potential (ΔΨ(m)) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ(m) recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ(m) contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K(+) (mK(ATP)) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ(m) of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ(m) to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ(m) was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK(ATP) channel openers improved the ΔΨ(m) recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ(m) during AA treatment. Activation of the mK(ATP) channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK(ATP) channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ(m). These results indicate that inactivation of GSK-3β directly or indirectly by mK(ATP) channel activation facilitates recovery of ΔΨ(m) by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.