Abstract 5137: Visualization of Accumulating Reactive Oxygen Species During Myocardial Ischemia by Real-time Two-photon Imaging of Perfused Rat Hearts

Abstract

Background: During myocardial ischemia/reperfusion injury, a burst of reactive oxygen species (ROS) is considered to occur at the onset of reperfusion and cause cardiomyocyte death through the loss of mitochondrial membrane potential (ΔΨ m ), but there are evidences against this concept. We aimed to visualize the dynamic changes of ROS during ischemia/reperfusion in intact rat hearts using two-photon laser scanning microscopy. Methods and Results: Langendorff-perfused rat hearts were loaded with 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl-ester and tetramethylrhodamine ethyl-ester, fluorescent indicators of ROS and ΔΨ m , respectively. Under the two-photon excitation, spatio-temporal changes of ROS and ΔΨ m in response to ischemia/reperfusion were simultaneously monitored at cellular level. As soon as ischemia started, ROS level of each cell began to increase, despite maintained ΔΨ m . Importantly, the rate of ROS accumulation during the early phase of ischemia was variable from cell to cell, and irreversible ΔΨ m loss occurred in an all-or-none manner depending on cellular ROS level, with a clear cut-off value. Upon reperfusion, we were unable to detect a burst of ROS, but there were more cells undergoing similar ΔΨ m loss after the cell-to-cell specific period of latency. Importantly, there was a significant inverse correlation between the level of ROS accumulation during ischemia and the latency until which the cell underwent ΔΨ m loss (r=−0.89). Ischemic preconditioning delayed the increase of ROS during ischemia and protected against ΔΨ m loss. An iron chelator deferoxamine, which reduces the generation of hydroxyl radical through inhibiting the iron-mediated Fenton reaction, neither attenuated the ROS generation nor protected against ΔΨ m loss. A free radical scavenger N-(2-mercaptopropionyl)-glycine attenuated the ROS accumulation, but it did not protect against ΔΨ m loss. Conclusion: In myocardial ischemia/reperfusion, ROS are mainly generated during ischemia, but not after reperfusion, and play a critical role in mitochondrial collapse and cardiomyocyte death. However, reducing the ROS accumulation during ischemia may not suffice to prevent cell death.