Abstract 13978: Facilitated Re-Closure of the Mitochondrial Permeability Transition Pore Underlies Protection of Cardiomyocytes from ROS-Induced Necrosis by Activation of the Mitochondrial ATP-Sensitive K+ Channel
Background Compelling evidence indicates that opening of the mitochondrial permeability transition pore (mPTP) is inhibited by phosphorylation of glycogen synthase kinase-3β (GSK-3β) or by activation of the mitochondrial ATP-sensitive K+ channel (mKATP channel). However, the effect of mKATP channel activation on the time course of mPTP opening and re-closure remains unclear. Here, we examined this issue by use of nicorandil (NC), an activator of mKATP channels.
Methods and results H9c2 cardiomyocytes were exposed to antimycin A (AA, 40 microM), a reactive oxygen species (ROS)-producing agent, or to a vehicle for 60 min with or without pretreatment with NC (300 microM, 60 min) and then incubated in normal culture medium for 120 min. NC significantly reduced AA-induced cell death as determined by LDH compared with the vehicle (15.7±0.3% vs. 23.2±0.1%, P<0.01). Mitochondrial membrane potential (ΔΨm), which was monitored by TMRE fluorescence, markedly decreased after exposure to AA (23.8±2.6% of baseline), suggesting opening of the mPTP, and then gradually recovered during 120 min of DMEM incubation (51.2±2.2% of baseline). NC did not affect the reduction of ΔΨm by AA (26.9±2.7%) but significantly improved recovery of ΔΨm at 120 min after incubation (68.5±3.1%, P<0.01 vs. AA). Immunoblotting revealed that NC significantly increased the level of phospho-Ser9 GSK-3β by 34% compared with the vehicle at 5 min after exposure to AA. On the other hand, the protein levels of mPTP subunit proteins (i.e., adenine nucleotide translocase, voltage-dependent anion channel and inorganic phosphate carrier) were not altered by exposure to AA with or without NC. Pharmacological inhibition of cyclophilin D (CyPD) by cyclosporine A or siRNA-mediated knockdown of CyPD neither suppressed cell death nor affected ΔΨm after exposure to AA.
Conclusion Transient generation of ROS induces mPTP-mediated necrosis of cardiomyocytes in a CyPD-independent manner. Activation of the mKATP channel protects cardiomyocytes from the ROS-induced cell necrosis by promoting re-closure of the mPTP, in which Ser9 phosphorylation of GSK-3β may play a role.
- © 2012 by American Heart Association, Inc.