The A3 Adenosine Receptor Agonist CP-532,903 [N6-(2,5-Dichlorobenzyl)-3′-aminoadenosine-5′-N-methylcarboxamide] Protects against Myocardial Ischemia/Reperfusion Injury via the Sarcolemmal ATP-Sensitive Potassium Channel

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Abstract
We examined the cardioprotective profile of the new A3 adenosine receptor (AR) agonist CP-532,903 [N6-(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 μg/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 ATP-sensitive 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.