Loor Gabriel, Kondapalli Jyothisri, Iwase Hirotaro, Chandel Navdeep S, Waypa Gregory B, Guzy Robert D, Vanden Hoek Terry L, Schumacker Paul T
Department of Surgery, University of Chicago, Chicago, IL 60637, USA.
Biochim Biophys Acta. 2011 Jul;1813(7):1382-94. doi: 10.1016/j.bbamcr.2010.12.008. Epub 2010 Dec 23.
To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O(2) during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X(L) in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X(L), failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.
为了阐明缺血再灌注（I/R）过程中活性氧（ROS）与细胞死亡之间的关系，我们在I/R细胞模型中研究了细胞死亡机制。使用比率氧化还原传感器线粒体roGFP并通过Mito-Sox Red氧化，在模拟缺血期间检测线粒体基质中的氧化应激。再灌注诱导的死亡通过锰超氧化物歧化酶（Mn-SOD）或线粒体磷脂氢过氧化物谷胱甘肽过氧化物酶（mito-PHGPx）的过表达而减弱，但过氧化氢酶、线粒体靶向过氧化氢酶或铜锌超氧化物歧化酶则不能。化学性质不同的抗氧化化合物也具有保护作用，NAC或在缺血期间清除残余O₂（缺氧缺血）可减弱mito-roGFP氧化。通过线粒体钙黄绿素荧光的实时成像监测线粒体通透性转换孔（mPTP）的振荡/开放。氧化应激在缺血期间导致钙黄绿素释放到细胞质中，这种反应受到化学性质不同的抗氧化剂、缺氧或Mn-SOD或mito-PHGPx过表达的抑制。这些发现表明，线粒体氧化应激在再灌注之前导致mPTP振荡。直到再灌注后才检测到细胞色素c从线粒体释放到细胞质中，并且在缺血期间通过缺氧缺血或抗氧化剂给药受到抑制。尽管在I/R后检测到DNA片段化，但未检测到Bax激活的证据。抗凋亡蛋白Bcl-X(L)在心肌细胞中的过表达并未赋予对I/R诱导的细胞死亡的保护作用。此外，Bax和Bak基因缺失或Bcl-X(L)过表达的小鼠胚胎成纤维细胞未能显示出对I/R的保护作用。这些发现表明，缺血期间的线粒体ROS通过不依赖Bax/Bak的细胞死亡途径触发mPTP激活、线粒体去极化和再灌注期间的细胞死亡。因此，线粒体凋亡似乎代表了这种模拟I/R模型中的一种冗余死亡途径。本文是名为：线粒体与心脏保护的特刊的一部分。
