Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion

Background— Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (ΔΨ m ) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of ΔΨ m in individual cardiomyocytes within perfused rat hearts using 2-photon laser-scanning microscopy, which has unique advantages over conventional confocal microscopy: greater tissue penetration and lower tissue toxicity. Methods and Results— The Langendorff-perfused rat heart was loaded with a fluorescent indicator of ΔΨ m , tetramethylrhodamine ethyl ester. Tetramethylrhodamine ethyl ester was excited with an 810-nm line of a Ti:sapphire laser, and its fluorescence in the heart cells was successfully visualized up to ≈50 μm from the epicardial surface. Taking advantage of this system, we monitored the spatiotemporal changes of ΔΨ m in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive ΔΨ m loss and a more prominent ΔΨ m loss on reperfusion. During ischemia/reperfusion, cells maintained a constant ΔΨ m for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible ΔΨ m loss, and this process did not affect the neighboring cells. Within a cell, ΔΨ m loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in ΔΨ m resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing ΔΨ m loss, whereas cyclosporin A partially inhibited ΔΨ m loss in each cell. Conclusions— Investigation of cellular responses in the natural environment will increase knowledge of ischemia/reperfusion injury and provide deeper insights into antiischemia/reperfusion therapy that targets mitochondria.