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Ischemia-reperfusion injury is a major concern for graft quality in heart transplantation with donation after circulatory death (DCD). Given that mitochondrial preservation is critical for recovery after cardiac ischemia-reperfusion, we aimed to investigate the time-dependent effects of ischemia on cardiac mitochondrial damage, stress and function in order to improve the timing and choice of therapeutic targets for cardioprotection.Isolated, working rat hearts underwent 0 (sham), 21, 24, 27, 30, or 33 min warm, global ischemia followed by 60 min reperfusion. Left ventricular work (LV work; heart rate-developed pressure product) and cytochrome c release into the coronary effluent (indicator of mitochondrial damage) were monitored.Another series of hearts was stopped after 10 min reperfusion for mitochondrial isolation and analysis of Ca2+ retention capacity and ROS production (both indicators of mitochondrial stress), as well as mitochondrial respiration (indicator of mitochondrial function). ATP and phosphocreatine levels after 10 min reperfusion were measured in snap-frozen ventricular tissue samples.LV work at 60 min reperfusion significantly decreased with ≥27 min ischemia compared with sham (p<0.05, n=6-8 per group). Cytochrome c release at 10 min reperfusion increased with ≥27 min ischemia vs. sham (p<0.001, n=6-8 per group), and inversely correlated with LV work at 60 min reperfusion (r=−0.824, p<0.001). At 10 min reperfusion, mitochondrial Ca2+ retention capacity was decreased with ischemia, regardless of duration. ROS production increased with 21 and 27 min ischemia vs. sham (p<0.001; n=5-7 per group), but was unchanged after 33 min ischemia. Compared with sham, complex II respiration was impaired with ≥27 min ischemia, while complex I respiration was impaired only after 33 min ischemia (p<0.001, n=5-7 per group). Levels of ATP, but not phosphocreatine, were significantly decreased with ≥21 ischemia (p<0.001, n=6 per group).Short ischemia (up to 21 min) induces mitochondrial stress, and only prolonged ischemia (≥27 min) results in impaired hemodynamics, mitochondrial damage and dysfunction. Cytochrome c release is a sensitive, early predictor of hemodynamic recovery. These findings may aid in developing mitochondria-based therapeutic reperfusion strategies aimed at facilitating the transplantation of hearts obtained with DCD.