title: 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 (delta psi(m)) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of delta psi(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.
  
  
  RESULTS
  The Langendorff-perfused rat heart was loaded with a fluorescent indicator of delta psi(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 approximately 50 microm from the epicardial surface. Taking advantage of this system, we monitored the spatiotemporal changes of delta psi(m) in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive delta psi(m) loss and a more prominent delta psi(m) loss on reperfusion. During ischemia/reperfusion, cells maintained a constant delta psi(m) for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible delta psi(m) loss, and this process did not affect the neighboring cells. Within a cell, delta psi(m) loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in delta psi(m) resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing delta psi(m) loss, whereas cyclosporin A partially inhibited delta psi(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.
authors.affiliation: Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Matsumoto-Ida
authors.forename: Masaharu
authors.initials: M
authors.lastname: Akao
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Takeda
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Toru
authors.initials: T
authors.lastname: Kita
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008564:Membrane Potentials; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008929:Mitochondria, Heart / Q000378:metabolism / Q000473:pathology* / Q000502:physiology*; D051336:Mitochondrial Membranes / Q000502:physiology; D017202:Myocardial Ischemia / Q000473:pathology; D015428:Myocardial Reperfusion Injury; D032383:Myocytes, Cardiac / Q000473:pathology / Q000648:ultrastructure; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D005456:Fluorescent Dyes