Background.The preconditioning effect of diadenosine tetraphosphate (AP4A) was reported in ischemia/reperfused hearts, but its effect in heart preservation was unknown. According to the possible role of mitochondrial ATP-sensitive potassium channel (mKATPchannel) in the effect of ischemic preconditioning, the contribution of mKATPchannel to the effect of AP4A was tested.Methods.Isolated rat hearts were arrested and preserved by Eurocollin’s (EC) solution at 4°C for 8 hr. AP4A (80 &mgr;M) or AP4A with the 5-hydroxydecanoic acid (100 &mgr;M), a selective inhibitor of the mKATPchannel, was added into the EC solution. The preischemic and postischemic cardiac functions were evaluated on a buffer-perfused Langendorff apparatus before storage and after 20 min of reperfusion.Results.AP4A administration improved the recovery of poststorage cardiac functions (the rate-pressure production, left ventricular systolic pressure, heart rate, coronary flow rate, and derivative of left ventricular systolic pressure;P<0.05) and reduced the leakage of lactate dehydrate and creatine kinase during reperfusion, compared with EC alone. Those effects of AP4A were completely reversed by 5-hydroxydecanoic acid administration in combination subjects.Conclusion.AP4A administration protects the heart through opening of the mKATPchannel during hypothermic preservation. Thus, addition of AP4A into cardioplegia may be a novel method of ischemic preconditioning in the transplantation context.