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Parenteral administration of recombinant human erythropoietin (rhEPO) to rats induces protection against myocardial ischemia/reperfusion injury 24 hours later. However, the mechanisms by which rhEPO mediates protection have not been determined.rhEPO was perfused into isolated rat hearts over 15 minutes immediately before 30 minutes of no-flow ischemia and 45 minutes of reperfusion. Compared with saline-perfused control hearts, recovery of left ventricular developed pressure was increased in rhEPO-perfused hearts. rhEPO also increased AKT activity and decreased apoptosis. All of these effects were blocked when the phosphatidylinositol-3-kinase inhibitor wortmannin was infused with rhEPO.rhEPO provides immediate protection against ischemia/reperfusion injury in the isolated perfused rat heart that is mediated by the phosphatidylinositol-3-kinase pathway.