N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide confers cardioprotection at reperfusion by inhibiting mitochondrial permeability transition pore opening via glycogen synthase kinase 3β

Although the adenosine A 3 receptor agonist N-6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase (GSK) 3 beta. IB-MECA (1 mu M) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A 3 receptor antagonist 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl] ester (MRS1334) (100 nM). The effect of IB-MECA was abrogated by the mPTP opener atractyloside (20 mu M), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential (Delta Psi(m)), which was reversed by MRS1334. IB-MECA also reduced Ca2+-induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3 beta (Ser(9)) upon reperfusion, and the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) (3 mu M) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of Delta Psi(m). In addition, the effect of IB-MECA on GSK-3 beta was reversed by wortmannin (100 nM), and IB-MECA was shown to enhance Akt phosphorylation upon reperfusion. In contrast, rapamycin (2 nM) failed to affect GSK-3 beta phosphorylation by IB-MECA, and IB-MECA did not alter phosphorylation of either mTOR (Ser(2448)) or 70s6K (Thr(389)). Taken together, these data suggest that IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3 beta at reperfusion. IB-MECA-induced GSK-3 beta inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.