Background. Adenosine monophosphate-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and -consuming pathways, and protects the heart against ischemic injury and apoptosis. Recent progress shed light on various factors, including adiponectin, MIF, H11K, and metformin in the activation of AMPK. It is uncertain whether the activation of AMPK is contributed to cardioprotection of opioids. Here we show that morphine, an exogenous non-peptide opioid receptor agonist, can modulate the activation of the cardioprotective AMPK pathway during ischemia and exert anti-apoptotic effects through AMPK. Methods. Isolated rat hearts were perfused on a constant pressure Langendorff system and subjected to 30min of global ischemia followed by 60min of reperfusion. The hearts received vehicles, morphine, a combination of morphine and compound C, a combination of morphine and STO609, a combination of morphine and BAPTA-AM at the onset of ischemia. Hemodynamics parameters, infarct size, release of intracellular creatine kinase, expression of AMPK, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining were analyzed. Results. Morphine significantly increased phosphorylation level of Thr172 site on AMPK, left ventricular function, and reduced infarct size as a percentage of the area at risk (IS/AAR from 63% +/- 7% to 40% +/- 5%), release of intracellular creatine kinase ( from 319 +/- 46 to 156 +/- 42IU/60min/gdw), apoptosis ratio (from 16% +/- 2% to 5% +/- 1.4%) during reperfusion in comparison with the control group. A inhibitor of AMPK, compound C abrogated phosphorylation of AMPK induced by morphine, the improvement in myocardial function, and the reduction of IS/AAR (58% +/- 6%), release of intracellular creatine kinase (270 +/- 40IU/60min/gdw), apoptosis ratio (13% +/- 1.5%). A Ca(2+)/calmodulin-dependent protein kinase kinase inhibitor STO609 and a chelator of intracellular Ca(2+) stores BAPTA-AM also abolished the cardioprotection of morphine. Conclusions. Morphine can ameliorate myocardial contractile dysfunction and limit infarct size following ischemia and reperfusion by a mechanism involving activation of AMPK, and activate AMPK by Ca(2+)-CaMKK beta-dependent phosphorylation. (C) 2011 Elsevier Inc. All rights reserved.