Background--Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (_m) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of _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 _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 _m in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive _m loss and a more prominent _m loss on reperfusion. During ischemia/reperfusion, cells maintained a constant _m for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible _m loss, and this process did not affect the neighboring cells. Within a cell, _m loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in _m resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing _m loss, whereas cyclosporin A partially inhibited _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.