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

背景缺血性心脏病是包括日本在内的发达国家的主要死亡原因之一，了解其发病机制和开发新的治疗策略具有重要意义。线粒体在细胞死亡中起着关键作用，线粒体膜电位丧失(ΔΨm)是导致细胞死亡的最早事件。在这里，我们报道了一种新的实时成像方法，利用双光子激光扫描显微镜对大鼠心脏单个心肌细胞中的ΔΨm进行实时成像，该方法与传统的共聚焦显微镜相比具有独特的优点：更大的组织渗透率和更低的组织毒性。方法与结果在朗宁多夫灌流的大鼠心脏上负载了ΔΨm的荧光指示剂四甲基罗丹明乙酯。用810 nm的钛宝石激光激发TMRE，在距心外膜表面50μm处成功地观察到其在心肌细胞内的荧光。利用该系统，我们监控了Spat…在亚细胞水平上，ΔΨm在缺血/再灌注反应中有更多的时相变化。无血流缺血导致进行性ΔΨm丢失，再灌流时ΔΨm丢失更为显著。在缺血/再灌注期间，细胞在细胞间特定的潜伏期内保持恒定的ΔΨm，随后迅速、完全且不可逆地丢失ΔΨm，而这一过程不影响邻近细胞。在细胞内，-ΔΨm的丢失开始于线粒体的特定区域，并沿纵轴快速传播。ΔΨm的这些时空变化导致了线粒体功能的显著细胞和亚细胞异质性。缺血预适应可减少ΔΨm丢失的细胞数，而环孢素A可部分抑制ΔΨm丢失。结论研究细胞在自然环境中的反应将增加对缺血/再灌注损伤的认识，并为以线粒体为靶点的抗缺血/再灌注治疗提供更深入的认识。较少

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