N-(3-Iodobenzyl)-adenosine-5 -N-methylcarboxamide Confers Cardioprotection at Reperfusion by Inhibiting Mitochondrial Permeability Transition Pore Opening via Glycogen Synthase Kinase 3

Although the adenosine A3 receptor agonist N -(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 . IBMECA (1 M) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A3 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 M), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidantinduced loss of mitochondrial membrane potential ( m), which was reversed by MRS1334. IB-MECA also reduced Ca -induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3 (Ser) upon reperfusion, and the GSK-3 inhibitor 3-(2,4dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) (3 M) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of m. In addition, the effect of IB-MECA on GSK-3 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 phosphorylation by IB-MECA, and IB-MECA did not alter phosphorylation of either mTOR (Ser) or 70s6K (Thr). Taken together, these data suggest that IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3 at reperfusion. IB-MECA-induced GSK-3 inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway. It is well known that activation of adenosine A3 receptors by selective agonists applied before ischemia can trigger pharmacological preconditioning to protect the heart against ischemia/reperfusion injury (Tracey et al., 1997; Takano et al., 2001; Zhao and Kukreja, 2002). However, because pretreatments are seldom possible in the clinical setting of acute myocardial infarction, it is important to determine whether A3 receptor activation after the onset of ischemia or during reperfusion can also confer cardioprotection. In this regard, Vinten-Johansen’s group has reported that the selective A3 receptor agonist 2-CI-IB-MECA given at reperfusion protects isolated rabbit hearts by decreasing polymorphonuclear neutrophil-endothelial cell interactions (Jordan et al., 1997). Likewise, several recent studies have also shown a cardioprotective effect of A3 receptor activation with IB-MECA or 2-CI-IB-MECA upon reperfusion in rat (Maddock et al., 2002), guinea pig (Maddock et al., 2003), and dog (Auchampach et al., 2003) hearts. Nevertheless, curiously little is known about the cellular and molecular mechanisms that mediate the cardioprotection induced by A3 receptor activation at reperfusion. This work was partially supported by American Heart Association Grants 0365534U and 0555430U. S.-S.P. and H.Z. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.101477. ABBREVIATIONS: 2-CI-IB-MECA, 2-chloro-N-(3-iodobenzyl)-adenosine-5 -N-methylcarboxamide; IB-MECA, N-(3-iodobenzyl)-adenosine-5 N-methyluronamide; mPTP, mitochondrial permeability transition pore; GSK, glycogen synthase kinase; PI3-kinase, phosphatidylinositol 3-kinase; mTOR, molecular target of rapamycin; p70s6K, 70-kDa ribosomal protein S6 kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MRS1334, 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl]ester; SB216763, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; m, mitochondrial membrane potential; TMRE, tetramethylrhodamine ethyl ester; MOPS, 4-morpholinepropanesulfonic acid; MRS1191, 3-ethyl-5-benzyl-2-methyl-6-phenyl-4phenylethynyl-1,4-( )-dihydropyridine-3,5-dicarboxylate; NIM811, N-methyl-4-isoleucine-cyclosporin. 0022-3565/06/3181-124–131$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 101477/3121301 JPET 318:124–131, 2006 Printed in U.S.A. 124 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from Suleiman et al. (2001) and Weiss et al. (2003) have proposed that the opening of the mitochondrial transition pore (mPTP) plays an essential role in myocardial ischemia/reperfusion injury and that blockade of the pore opening is cardioprotective. Interestingly, it has been demonstrated that the mPTP remains closed during ischemia but opens at the onset of reperfusion (Griffiths and Halestrap, 1995) and that inhibition of mPTP opening at early reperfusion can protect the heart from reperfusion injury (Hausenloy et al., 2003; Halestrap et al., 2004). Because adenosine A3 receptor activation at reperfusion can exert cardioprotection against reperfusion injury, it is possible that IB-MECA applied at reperfusion may induce cardioprotection by modulating the opening of the mPTP. If this is the case, it is necessary to investigate the signaling mechanisms underlying the inhibition of the mPTP opening by IB-MECA. Inhibition of GSK-3 has been shown to contribute to opioid-induced cardioprotection at reperfusion (Gross et al., 2004) and serves as one important mechanism by which pharmacological preconditioning prevents the mPTP opening in cardiomyocytes (Juhaszova et al., 2004). GSK-3 has high basal activity and is activated by phosphorylation of its Tyr residue, whereas phosphorylation at Ser decreases its activity (Cohen and Frame, 2001). Some intracellular signals such as PI3-kinase/ Akt, mTOR/p70s6K, and mitogen-activated protein kinases have been proposed to decrease GSK-3 activity by phosphorylating it (Cohen and Frame, 2001; Murphy, 2004). Because these signals may play an important role in cardioprotection against reperfusion injury (Hausenloy and Yellon, 2004) and stimulation of adenosine A3 receptors can activate Akt (Gao et al., 2001) and ERK (Schulte and Fredholm, 2003), it is possible that IB-MECA-induced cardioprotection at reperfusion may also be due to suppression of GSK-3 activity. In this study, we examined whether IB-MECA given at reperfusion protects the heart by modulating mPTP opening via GSK-3 and to dissect the signaling mechanisms that mediate the protective effect of IB-MECA. Materials and Methods All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. Chemicals and Antibodies. IB-MECA, MRS1334, SB216763, wortmannin, and rapamycin were purchased from Tocris Cookson Inc. (Ellisville, MO) and were dissolved in a final concentration of 0.01% dimethyl sulfoxide. Atractyloside was obtained from Sigma Chemical (St. Louis, MO) and was directly dissolved in Krebs-Henseleit bicarbonate solution. All antibodies were purchased from Cell Signaling Technology Inc (Beverly, MA). Perfusion of Isolated Rat Heart. Rat hearts were isolated and perfused as described previously (Xu et al., 2001). Male Wistar rats (300–350 g) were anesthetized with thiobutabarbital sodium (100 mg/kg i.p.). The hearts were removed rapidly and mounted on a Langendorff apparatus. The hearts were perfused with KrebsHenseleit buffer containing 118.5 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, 24.8 mM NaHCO3, 1.2 mM KH2PO4, and 10 mM glucose, which was heated to 37°C and gassed with 95% O2-5% CO2. A latex balloon connected to a pressure transducer was inserted into the left ventricle through the left atrium. The left ventricular pressure and heart rate were continuously recorded with a PowerLab system (ADInstruments, Mountain View, CA). A 5-0 silk suture was placed around the left coronary artery, and the ends of the suture were passed through a small piece of soft vinyl tubing to form a snare. All hearts were allowed to stabilize for at least 20 min. Ischemia was induced by pulling the snare and then fixing it by clamping the tubing with a small hemostat. Total coronary artery flow was measured by timed collection of the perfusate dripping from the heart into a graduated cylinder. Measurement of Infarct Size. At the end of the experiments, the coronary artery was reoccluded, and fluorescent polymer microspheres (2–9 M diameter; Duke Scientific Corporation, Palo Alto, CA) were infused to demarcate the risk zone as the tissue without fluorescence. The hearts were weighed, frozen, and cut into 1-mm slices. The slices were incubated in 1% triphenyltetrazolium chloride in sodium phosphate buffer at 37°C for 20 min. The slices were immersed in 10% formalin to enhance the contrast between stained (viable) and unstained (necrotic) tissue and then squeezed between glass plates spaced exactly 1 mm apart. The myocardium at risk was identified by illuminating the slices with UV light. The infarcted and risk zone regions were traced on a clear acetate sheet and quantified with ImageTool. The areas were converted into volumes by multiplying the areas by slice thickness. Infarct size is expressed as a percentage of the risk zone. Isolation of Adult Rat Cardiomyocytes. Rat cardiomyocytes were isolated enzymatically (Xu et al., 2005). Male Wistar rats weighing 250 to 350 g were anesthetized with thiobutabarbital sodium (100 mg/kg i.p.). A midline thoracotomy was performed, and the heart was removed and rapidly mounted on a Langendorff apparatus. The heart was perfused in a nonrecirculating mode with Krebs-Henseleit buffer (37°C) containing 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.25 mM CaCl2, and 10 mM glucose for 5 min to wash out blood. The buffer was bubbled with 95% O2-5% CO2. Then the heart was perfused with a calcium-free buffer that contained all of the above components except CaCl2. After 5 min of perfusion, collagenase (type II) was added to the buffer (0.1%), and the heart was perfused in a recirculating mode for 15 min. The heart was removed from the apparatus, and the ventricles were placed into a beaker containing the calcium-free buffer. The ventricles were agitated in a shaking bath (37°C) at a rate of 50 cycles/min until individual cells were released. The released cells were suspended in an incubation buffer containing all of the components of the calcium-free buffer, 1% bovine serum albumin, 30 mM HEPES, 60 mM taurine, 20 mM creatine, and amino acid supplements at 37°C. Calcium was gradually added to the buffer containing the cells to a final concentration of 1.2 mM. The cells were filtered through nylon mesh and centrifuged briefly. Finally the cells were suspended in culture medium M199 for 4 h before