Real-time 2-photon imaging of mitochondrial function in perfused rat hearts subjected to ischemia/reperfusion

Abstract

BACKGROUND - Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (ΔΨm) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of ΔΨm 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 ΔΨm, 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 ΔΨm in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive ΔΨm loss and a more prominent ΔΨm loss on reperfusion. During ischemia/reperfusion, cells maintained a constant ΔΨm for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible ΔΨm loss, and this process did not affect the neighboring cells. Within a cell, ΔΨm loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in ΔΨm resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing ΔΨm loss, whereas cyclosporin A partially inhibited ΔΨm 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. © 2006 American Heart Association, Inc.