Modulation of the mitochondrial permeability transition pore complex in GSK-3beta-mediated myocardial protection.
Recently we found that the level of anti-infarct tolerance afforded by ischemic preconditioning (IPC) and erythropoietin (EPO) infusion was closely correlated with the level of Ser9-phospho-GSK-3beta upon reperfusion in the heart. To get an insight into the mechanism by which phospho-GSK-3beta protects the myocardium from ischemia/reperfusion injury, we examined the effects of IPC and EPO on interactions between GSK-3beta and subunits of the mitochondrial permeability transition pore (mPTP) in this study. Rat hearts were subjected to 25-min global ischemia and 5-min reperfusion in vitro with or without IPC plus EPO infusion (5 units/ml) before ischemia. Ventricular tissues were sampled before or after ischemia/reperfusion to separate subcellular fractions for immunoblotting and immunoprecipitation. Reperfusion increased mitochondrial GSK-3beta by 2-fold and increased phospho-GSK-3beta level in all fractions examined. Major subunits of mPTP, adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), were co-immunoprecipitated with GSK-3beta after reperfusion. Phospho-GSK-3beta was co-immunoprecipitated with ANT but not with VDAC. IPC+EPO significantly increased the levels of GSK-3beta and phospho-GSK-3beta that were co-immunoprecipitated with ANT to 145+/-8% and 143+/-16%, respectively, of baseline but did not induce phospho-GSK-3beta-VDAC binding. A PKC inhibitor and a PI3 kinase inhibitor suppressed the IPC+EPO-induced increase in the level of phospho-GSK-3beta-ANT complex. The level of cyclophilin D co-immunoprecipitated with ANT after reperfusion was significantly reduced to 39+/-10% of the control by IPC+EPO. These results suggest that reduction in affinity of ANT to cyclophilin D by increased phospho-GSK-3beta binding to ANT may be responsible for suppression of mPTP opening and myocardial protection afforded by IPC+EPO.
Nishihara
Masahiro
M
Second Department of Internal Medicine, Sapporo Medical University School of Medicine, S-1 W-16, Chuo-ku, Sapporo 060-8543, Japan.
Miura
Tetsuji
T
Miki
Takayuki
T
Tanno
Masaya
M
Yano
Toshiyuki
T
Naitoh
Kazuyuki
K
Ohori
Katsuhiko
K
Hotta
Hiroyuki
H
Terashima
Yoshiaki
Y
Shimamoto
Kazuaki
K
D000818:Animals; D004195:Disease Models, Animal; D004921:Erythropoietin / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000502:physiology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects / Q000502:physiology*; D000083162:Mitochondrial Permeability Transition Pore; D017202:Myocardial Ischemia / Q000503:physiopathology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000517:prevention & control; D016276:Ventricular Function
D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D004921:Erythropoietin; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3