| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Background
Ischemic heart disease is one of the leading causes of death in developed countries including Japan, and it is of crucial importance to understand the pathogenesis and develop a novel therapeutic strategy. Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (ΔΨm) is the earliest event which commits the cell to death. Here, we report a novel real-time imaging of ΔΨm in individual cardiomyocytes within perfused rat hearts using two-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 (TMRE). TMRE was excited with 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 spat
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io-temporal changes of ΔΨm in response to ischemia/reperfusion at subcellular level. No-flow ischemia caused progressive ΔΨm loss, as well as a more prominent ΔΨm loss upon 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 spatio-temporal 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
Investigating cellular responses in the natural environment will increase knowledge of ischemia/reperfusion injury, and provide deeper insights into anti-ischemia/reperfusion therapy targeting mitochondria. Less
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