Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion
Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion
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DOI：
10.1074/jbc.m701917200
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发表时间：
2007-06-29
影响因子：
4.8
通讯作者：
Schumacker, Paul T.
中科院分区：
文献类型：
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作者：
Robin, Emmanuel;Guzy, Robert D.;Schumacker, Paul T.
缺血再灌注损伤可诱导氧化应激，缺血心肌再灌流后产生的活性氧(ROS)足以导致细胞死亡。线粒体氧化剂的产生可能在缺血再灌注之前就开始了，因为还原的等价物积累并促进了超氧化物的产生。我们利用比率氧化还原敏感蛋白传感器(热休克蛋白33荧光共振能量转移(HSP-FRET))来评估模拟缺血期间心肌细胞的氧化应激。HSP-FRET由细菌HSP-33的半胱氨酸调节结构域连接的青色和黄色荧光蛋白荧光团组成。在缺血期间，观察到ROS介导的HSP-FRET氧化，同时细胞还原型谷胱甘肽水平下降。使用氧化还原敏感的绿色荧光蛋白的测量证实了这些发现，绿色荧光蛋白是另一种蛋白质硫醇比率传感器，在模拟缺血结束时，它变得93%被氧化。然而，在缺血期间没有发生细胞死亡，这表明这种氧化应激不足以在再灌注前诱导死亡。然而，减轻缺血性氧化应激的干预措施，包括抗氧化剂或残余O-2的清除剂，在缺血期间减少/防止ROS的产生，在模拟再灌注期间消除了细胞死亡。这些发现表明，在分离的心肌细胞中，模拟缺血期间亚致死性过氧化氢的产生调节了模拟再灌流期间的细胞死亡，这是由再灌流氧化剂爆发所介导的。
Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O-2 that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H2O2 generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.