Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion
Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion
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DOI：
10.1152/ajpheart.00683.2005
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发表时间：
2006-05-01
影响因子：
4.8
通讯作者：
Lemasters, JJ
中科院分区：
文献类型：
--
作者：
Kim, JS;Jin, YG;Lemasters, JJ
我们研究了pH、活性氧（ROS）、Ca ~（2+）和线粒体通透性转换（MPT）在成年大鼠心肌细胞pH依赖性缺血-再灌注损伤中的作用。将肌细胞在pH 6.2的缺氧Krebs-Ringer-HEPES缓冲液中孵育3小时以模拟缺血。为了模拟再灌注，将肌细胞在pH 6.2或7.4下再氧合2小时。用MPT阻断剂（环孢菌素A和N-甲基-4-异亮氨酸环孢菌素）和抗氧化剂（去铁醛、二苯基苯二胺和2-巯基丙酰甘氨酸）处理一些心肌细胞。线粒体膜电位，内膜透化，和ROS的形成分别与四甲基罗丹明甲酯，钙黄绿素，和氯甲基二氯荧光素二乙酸酯成像。对于Ca 2+成像，用rhod-2和fluo-4对肌细胞进行共染色，以分别评价线粒体和胞质Ca 2+。在pH 7.4下再灌注10分钟后，钙黄绿素重新分布在线粒体内膜上，这是一个由线粒体ROS形成并伴随着过度挛缩、线粒体去极化然后细胞死亡的事件。酸性再灌注、抗氧化剂和MPT阻断剂各自阻止MPT、去极化、过度收缩和细胞杀伤。抗氧化剂，但既不是MPT阻断剂，也不是酸再灌注，抑制再灌注后ROS的形成。此外，在pH 7.4的缺氧再灌注防止细胞死亡。线粒体和胞浆Ca 2+在缺血期间增加，但在再灌注的第一分钟恢复。再灌注后，线粒体和胞浆Ca 2+超载再次发生。MPT抑制可阻断这种晚期钙超载。通过BAPTA冷负荷/热孵育进行的线粒体内Ca 2+螯合并不能阻止再灌注后的细胞死亡。总之，线粒体活性氧，连同正常化的pH值，促进MPT发病和随后的心肌细胞死亡后再灌注。相反，Ca 2+超载似乎是MPT后生物能量衰竭的结果，而不是促进MPT发作的因素。
We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.