Accelerated recovery of mitochondrial membrane potential by GSK-3β inactivation affords cardiomyocytes protection from oxidant-induced necrosis.
Loss of mitochondrial membrane potential (ΔΨm) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨm recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨm contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K+ (mKATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨm 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 ΔΨm to 15 ± 1% of the baseline and induced calcein leak from mitochondria. ΔΨm 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. mKATP channel openers improved the ΔΨm recovery and mitochondrial calcein to 73 ± 2% and 30 ± 7%, respectively, without change in ΔΨm during AA treatment. Activation of the mKATP 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 mKATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨm. These results indicate that inactivation of GSK-3β directly or indirectly by mKATP channel activation facilitates recovery of ΔΨm by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Daisuke
D
Sunaga
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Masaya
M
Tanno
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Atsushi
A
Kuno
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Satoko
S
Ishikawa
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Makoto
M
Ogasawara
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Toshiyuki
T
Yano
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Takayuki
T
Miki
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Tetsuji
T
Miura
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D017209:Apoptosis; D002460:Cell Line; D005452:Fluoresceins / Q000378:metabolism; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D051379:Mice; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced / Q000201:enzymology / Q000235:genetics / Q000517:prevention & control*; D009942:Organometallic Compounds / Q000737:chemistry; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation; D015221:Potassium Channels / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction
D005452:Fluoresceins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009942:Organometallic Compounds; D016877:Oxidants; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C117780:mitochondrial K(ATP) channel; C110932:tetramethyl rhodamine ethyl ester; D000968:Antimycin A; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; C007740:fluorexon