Abstract 5137: Visualization of Accumulating Reactive Oxygen Species During Myocardial Ischemia by Real-time Two-photon Imaging of Perfused Rat Hearts
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 (ΔΨm), 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 ΔΨm, respectively. Under the two-photon excitation, spatio-temporal changes of ROS and ΔΨm 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 ΔΨm. Importantly, the rate of ROS accumulation during the early phase of ischemia was variable from cell to cell, and irreversible ΔΨm 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 ΔΨm 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 ΔΨm loss (r=−0.89). Ischemic preconditioning delayed the increase of ROS during ischemia and protected against ΔΨm 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 ΔΨm loss. A free radical scavenger N-(2-mercaptopropionyl)-glycine attenuated the ROS accumulation, but it did not protect against ΔΨm 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.