Chen Qun, Moghaddas Shadi, Hoppel Charles L, Lesnefsky Edward J
Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Am J Physiol Cell Physiol. 2008 Feb;294(2):C460-6. doi: 10.1152/ajpcell.00211.2007. Epub 2007 Dec 12.
Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.
心肌缺血会降低肌膜下线粒体（SSM）和肌原纤维间线粒体（IFM）中细胞色素氧化酶的复合体III活性、细胞色素c含量及呼吸作用。缺血期间用异戊巴比妥对电子传递进行可逆性阻断可保护线粒体呼吸作用，并减少再灌注期间的心肌损伤。这些发现支持线粒体损伤在缺血期间发生，并导致再灌注期间的心肌损伤。当前研究探讨了电子传递链（ETC）的缺血性损伤是否会增加线粒体活性氧（ROS）的净生成量。从6月龄Fisher 344大鼠心脏中分离出SSM和IFM，一组进行25分钟全心缺血，另一组仅灌注40分钟作为对照。使用安吖啶红检测法测量SSM和IFM中过氧化氢（H₂O₂）的释放量。与对照组（n = 8）相比，缺血后以谷氨酸作为复合体I底物时，SSM和IFM中H₂O₂的净生成量分别增加了178±14%和179±17%（n = 9）。在存在鱼藤酮的情况下以琥珀酸作为底物时，缺血后SSM和IFM中H₂O₂分别增加了272±22%和171±21%。使用电子传递抑制剂来评估最大ROS生成量。缺血后用鱼藤酮抑制复合体I，SSM和IFM中H₂O₂的生成量分别增加了179±24%和155±14%。缺血还增加了抗霉素A刺激的复合体III产生H₂O₂的量。因此，ETC的缺血性损伤增加了复合体I和复合体III产生H₂O₂的能力及净生成量，并为再灌注期间ROS生成增加作为心脏损伤机制奠定了基础。
