Dichlorobenzyl)-3 -aminoadenosine-5 -N-methylcarboxamide] Protects against Myocardial Ischemia/Reperfusion Injury via the Sarcolemmal ATP-Sensitive Potassium Channel

We examined the cardioprotective profile of the new A3 adenosine receptor (AR) agonist CP-532,903 [N-(2,5-dichlorobenzyl)-3 -aminoadenosine-5 -N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A3ARs (Ki 9.0 2.5 nM) and with high selectivity versus mouse A1AR (100-fold) and A2AARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 g/kg CP-532,903 reduced infarct size from 59.2 2.1% of the risk region in vehicle-treated mice to 42.5 2.3 and 39.0 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A3AR gene, A3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/ reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATPsensitive potassium (KATP) channel. Whole-cell patch-clamp recordings provided evidence that the A3AR is functionally coupled to the sarcolemmal KATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A3AR that protects against ischemia/reperfusion injury by activating sarcolemmal KATP channels. A3 adenosine receptor (AR) agonists have been shown to effectively limit infarct size and reduce contractile dysfunction in several different animal models of ischemia/reperfusion injury (Auchampach et al., 1997b, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999; Ge et al., 2004, 2006). A3AR agonists are attractive as cardioprotective agents because they do not alter systemic hemodynamic parameters in nonrodent species and are effective if administered before the ischemic event or only during reperfusion (Auchampach et al., 1997b, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999; Ge et al., 2004, 2006). The most widely available A3AR agonists that have been tested in experimental animal models of ischemia/reperfusion injury include the N-benzyladenosine5 -N-methylcarboxamide derivative IB-MECA and its This work was supported by the National Institutes of Health (Grants R01 HL60051, R01 HL07707, and T32 HL73643) and by the American Heart Association (Research Fellowship Grants 0320019Z and 0225454Z). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.127480. ABBREVIATIONS: AR, AR, adenosine receptor; IB-MECA, N-(3-iodobenzyl)adenosine-5 -N-methyluronamide; Cl-IB-MECA, 2-chloro-N-(3iodobenzyl)adenosine-5 -N-methylcarboxamide; CP-532,903, N-(2,5-dichlorobenzyl)-3 -aminoadenosine-5 -N-methylcarboxamide; KATP, ATPsensitive potassium; KO, knockout; HEK, human embryonic kidney; [I]AB-MECA, N-(4-amino-3-[I]iodobenzyl)adenosine-5 -N-methylcarboxamide; Ro-20,1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; dP/dt, maximal rate of contraction/relaxation; IPC, ischemic preconditioning; ECG, electrocardiogram; LAD, left anterior descending; CPX, 1,3-dipropyl-8-cyclopentylxanthine; ZM 241385, 4-[2-[7-amino-2(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol; CCPA, 2-chloro-N-cyclopentyladenosine; CP-608,039, N-[2-(3-methylisoxazol-5-ylmethoxy)-5-chloro]benzyl-3 -aminoadenosine-5 -N-methylcarboxamide; DP, developed pressure; MRS 1754, 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine. 0022-3565/08/3241-234–243$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 1 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 127480/3282986 JPET 324:234–243, 2008 Printed in U.S.A. 234 at A PE T Jornals on A ril 7, 2017 jpet.asjournals.org D ow nladed from 2-chloro derivative Cl-IB-MECA (Fig. 1). Although IB-MECA and Cl-IB-MECA are potent A3AR agonists, they exhibit moderate selectivity. In radioligand binding assays, the selectivity of IB-MECA and Cl-IB-MECA has been reported to range from 6 to 2500-fold versus the A1AR and 4 to 1400-fold versus the A2AAR, depending on the species and the assay conditions (Gallo-Rodriguez et al., 1994; Kim et al., 1994; Hill et al., 1997; Takano et al., 2001; Murphree et al., 2002). In the present investigation, we characterized the cardioprotective profile of the A3AR agonist CP-532,903 in an isolated mouse heart model of global ischemia and reperfusion and an in vivo mouse model of infarction. The goal of this work was to examine the cardioprotective effectiveness of CP-532,903 and to confirm whether it mediates cardioprotection via the A3AR. A second goal of this investigation was to determine whether A3AR activation provides ischemic protection by facilitating opening of the sarcolemmal isoform of the ATP-sensitive potassium (KATP) channel. This second question was addressed using Kir6.2 gene knockout (KO) mice lacking the pore-forming subunit of the sarcolemmal KATP channel (Suzuki et al., 2002). Materials and Methods