The Impact of Reperfusion Duration on the Activation of Apoptosis, Ferroptosis, Autophagy, and Pyroptosis in Ischemia-Reperfusion Injury in Rat Hearts

Reperfusion injury is a paradoxical event characterized by mitochondrial dysfunction and the activation of cell death, which results in diminished heart function after blood flow is restored following myocardial infarction. Several cell death mechanisms have been linked to this process, but the specific contributions of these pathways to the development of reperfusion injury and cardiac dysfunction at different stages of reperfusion remain unclear. In this study, we assessed apoptosis, ferroptosis, autophagy, and pyroptosis at various time points during reperfusion in rat hearts. Isolated hearts from adult male Sprague Dawley rats (200-250 g) were perfused using the Langendorff-mode retrograde method with normal Krebs-Henseleit solution. Hearts were subjected to 20 min equilibration period, and 25 min of global ischemia followed by short (5 min), moderate (30 min) or long (60 min) reperfusion. At the end of each experiment, mitochondria were isolated and used to analyze Ca 2+ -induced swelling and ROS production. Protein levels of death markers for apoptosis, ferroptosis, autophagy, and pyroptosis were measured using SDS-PAGE and western blotting. Our results showed a 77% (p<0.001) decrease in cardiac function, measured by LVDP (difference between LVSP and LVEDP) during reperfusion compared to pre-ischemic values. Mitochondrial Ca 2+ -induced swelling showed a significant time-dependent increase (p<0.01) during reperfusion, while ROS production showed a 4.7-fold increase (p<0.01) at moderate reperfusion and a 3.6-fold increase (p<0.005) at long reperfusion. Our western blot data revealed a significant increase (p<0.005) in Caspase-3 cleavage, an apoptosis marker, during ischemia and all reperfusion time points. Glutathione peroxidase 4, an endogenous ferroptosis inhibitor, showed decreased expression in ischemia (p<0.05), short (p<0.005) and moderate (p<0.01) reperfusion compared to control. Autophagy marker p62, showed a time-dependent significant increase in expression (p<0.01) compared to the control group. Interestingly, GSDMD cleavage, the endpoint of the pyroptosis pathway that leads to the cell's death, showed a significant decrease during short reperfusion (p<0.05). Our data demonstrates an association between cardiac and mitochondrial dysfunction, and the distinct activation of four cell death pathways depending on the reperfusion time. This suggests that certain mechanisms may prevail over others at different stages of reperfusion, indicating a potential time-sensitive interaction between these pathways that could influence future therapeutic strategies for the treatment of MI patients. National Institutes of Health NIGMS G-RISE T32GM148406, Grant R16GM145390, National Science Foundation Award 2006477. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.