The cytokine erythropoietin (EPO) protects the heart from ischemic injury, in part by preventing apoptosis. However, EPO administration can also raise the hemoglobin concentration, which, by increasing oxygen delivery, confounds assignment of cause and effect. The availability of EPO analogs that do not bind to the dimeric EPO receptor and lack erythropoietic activity, e.g., carbamylated EPO (CEPO), provides an opportunity to determine whether EPO possesses direct cardioprotective activity. In vivo, cardiomyocyte loss after experimental myocardial infarction (MI) of rats (40 min of occlusion with reperfusion) was reduced from Ϸ57% in MI-control to Ϸ45% in animals that were administered CEPO daily for 1 week (50 g͞kg of body weight s.c.) with the first dose administered intravenously 5 min before reperfusion. CEPO did not increase the hematocrit, yet it prevented increases in left ventricular (LV) end-diastolic pressure, reduced LV wall stress in systole and diastole, and improved LV response to dobutamine infusion compared with vehicle-treated animals. In agreement with the cardioprotective effect observed in vivo, staurosporineinduced apoptosis of adult rat or mouse cardiomyocytes in vitro was also significantly attenuated (Ϸ35%) by CEPO, which is comparable with the effect of EPO. These data indicate that prevention of cardiomyocyte apoptosis, in the absence of an increase in hemoglobin concentration, explains EPO's cardioprotection. Nonerythropoietic derivatives such as CEPO, devoid of the undesirable effects of EPO, e.g., thrombogenesis, could represent safer and more effective alternatives for treatment of cardiovascular diseases, such as MI and heart failure. Furthermore, these findings expand the activity spectrum of CEPO to tissues outside the nervous system. apoptosis ͉ cardioprotection ͉ cytokine ͉ tissue injury E rythropoietin (EPO) protects the brain and the spinal cord from ischemic and traumatic injury (1, 2), the peripheral nerve from diabetic damage (3), the kidney from ischemic (4, 5) or toxic insults (6), and the heart from acute ischemia, either permanent (7-9) or with reperfusion (10). Current data suggest that the observed protective effects of EPO depend on an antiapoptotic effect of this cytokine (7, 10). In the brain, EPO also greatly reduces the inflammatory response after ischemiareperfusion (11). It is notable that in several acute models, e.g., brain ischemia, a single dose of EPO that does not increase the Hb concentration nevertheless confers neuroprotection. However, in other in vivo injury models, including cardiac ischemia with reperfusion and diabetic neuropathy, injury develops gradually, and multiple doses of EPO appear superior but also increase the Hb concentration. The possibility that part of the benefit obtained with EPO in these models may depend on the increased oxygen-carrying capacity of the blood cannot be excluded. However, cardiac protection has clearly been demonstrated after only a single dose (8, 10) when evaluated 1-3 days after infarction before any increase i...