Vanden Hoek T L, Shao Z, Li C, Schumacker P T, Becker L B
Department of Medicine, The University of Chicago, Chicago, Illinois, 60637, USA.
J Mol Cell Cardiol. 1997 Sep;29(9):2441-50. doi: 10.1006/jmcc.1997.0481.
Ischemia/reperfusion causes oxidant injury in isolated cardiomyocytes without neutrophils or xanthine oxidase. Since these cells contain mitochondria, we hypothesized that the mitochondrial electron transport chain (ETC) causes this injury. To test this, we altered two conditions known to change within the mitochondria during ischemia/reperfusion-the extent of ETC redox-reduction and oxygen levels-and measured the resulting oxidant generation and injury. Specifically, we exposed cardiomyocytes for 1 h to the mitochondrial ETC inhibitors cyanide, antimycin, and rotenone and measured oxidant generation, using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH, sensitive to H2O2 and hydroxyl radicals). Inhibitors causing more extensive redox-reduction of the ETC (cyanide or antimycin) generated more oxidants than did partial ETC reduction with the inhibitor rotenone (10-fold v five-fold increases in DCFH oxidation). In addition, the DCFH oxidation caused by cyanide could be completely attenuated by the antioxidants 2-mercaptopropionylglycine (MPG) and 1,10 phenanthroline (PHEN). Finally, we tested the relevance of this oxidant generation on cell survival and contraction, with and without antioxidant interventions. Cell viability and contraction after 3-h recovery from cyanide exposure was significantly improved by either the addition of antioxidants, or by the "antioxidant" strategy of lowering O2 levels (i.e. from 150 to 3 tau) during the cyanide exposure (13.8% death with hypoxic cyanide v 48.6% cell death with normoxic cyanide). Collectively, these findings demonstrate that mitochondrial ETC carriers can cause significant oxidant injury, greatest when fully redox-reduced and exposed to oxygen, conditions known to occur in the transition from ischemia to reperfusion.
缺血/再灌注可在无中性粒细胞或黄嘌呤氧化酶的情况下，导致离体心肌细胞发生氧化损伤。由于这些细胞含有线粒体，我们推测线粒体电子传递链（ETC）会引发这种损伤。为验证这一推测，我们改变了两个已知在缺血/再灌注期间线粒体内会发生变化的条件——ETC氧化还原程度和氧水平，并测量由此产生的氧化剂生成量和损伤情况。具体而言，我们将心肌细胞暴露于线粒体ETC抑制剂氰化物、抗霉素和鱼藤酮1小时，使用细胞内荧光探针2',7'-二氯荧光素（DCFH，对过氧化氢和羟基自由基敏感）测量氧化剂生成量。导致ETC发生更广泛氧化还原的抑制剂（氰化物或抗霉素）比使ETC部分还原的抑制剂鱼藤酮产生更多的氧化剂（DCFH氧化增加10倍对5倍）。此外，氰化物引起的DCFH氧化可被抗氧化剂2-巯基丙酰甘氨酸（MPG）和1,10-菲咯啉（PHEN）完全减弱。最后，我们测试这种氧化剂生成对细胞存活和收缩的相关性，有无抗氧化剂干预。从氰化物暴露中恢复3小时后的细胞活力和收缩，通过添加抗氧化剂或在氰化物暴露期间采用降低氧水平（即从150降至3τ）的“抗氧化剂”策略得到显著改善（低氧氰化物处理后13.8%细胞死亡，常氧氰化物处理后48.6%细胞死亡）。总体而言，这些发现表明线粒体ETC载体可导致显著的氧化损伤，在完全氧化还原并暴露于氧时损伤最大，这是已知在从缺血到再灌注转变过程中会出现的情况。
