Real-time imaging of myocardial ischemia-reperfusion using multi-photon laser microscopy

在包括日本在内的发达国家，非化学性心脏病是导致死亡的主要原因之一，了解其发病机制和制定新的治疗策略至关重要。线粒体在细胞死亡中起关键作用；线粒体膜电位的丧失（ΔΨm）是导致细胞死亡的最早事件。在这里，我们报告了一种使用双光子激光扫描显微镜在灌注大鼠心脏内单个心肌细胞中ΔΨm的新型实时成像，它比传统的共聚焦显微镜具有独特的优势：更大的组织穿透性和更低的组织毒性。方法与结果langendorff灌注大鼠心脏负荷荧光指示剂ΔΨm，四甲基罗丹明乙酯（TMRE）。用Ti: Sapphire激光810 nm谱线激发TMRE，在距心外膜表面50 μm范围内成功地观察到其在心脏细胞内的荧光。利用该系统，我们在亚细胞水平上监测了ΔΨm对缺血/再灌注的反应。无血流缺血导致进行性ΔΨm损失，再灌注时ΔΨm损失更为突出。在缺血/再灌注过程中，细胞在细胞间特异性潜伏期内保持恒定ΔΨm，随后迅速、完全、不可逆地ΔΨm丧失，这一过程不影响邻近细胞。在细胞内，□ΔΨm损失始于线粒体的特定区域，并沿纵轴迅速传播。ΔΨm的这些时空变化导致线粒体功能的细胞和亚细胞异质性。缺血预处理减少了ΔΨm丢失的细胞数量，而环孢素A部分抑制了每个细胞的ΔΨm丢失。结论研究自然环境下的细胞反应将增加对缺血/再灌注损伤的认识，并为线粒体抗缺血/再灌注治疗提供更深入的见解。少

BackgroundIschemic 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 ResultsThe 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 … More 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.ConclusionsInvestigating 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