Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, Rootstown, OH 44272, United States of America.
Campus Chemical Instrument Center, Proteomics and Mass Spectrometry Facility, The Ohio State University, Columbus, OH 43210, United States of America.
J Mol Cell Cardiol. 2021 Dec;161:23-38. doi: 10.1016/j.yjmcc.2021.07.008. Epub 2021 Jul 29.
A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSOH) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C/C of cytochrome c via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSOH levels at the C ligand of the [2Fe2S] cluster of the Rieske iron‑sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSOH of the hinge protein at C and of cytochrome c at C and C, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSOH of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSOH detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSOH. In conclusion, reperfusion-mediated heme damage with increased PrSOH controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.
心肌缺血再灌注损伤（I/R）的一个严重后果是氧化损伤，这会导致线粒体功能障碍。级联反应的 ROS 可以传播，并可能导致线粒体电子传递链中的血红素漂白和蛋白质半胱氨酸磺酸化（PrSOH）。在这里，我们研究了在体内经历 30 分钟缺血和 24 小时再灌注的大鼠心脏线粒体中，I/R 介导的复合物 III 不可逆氧化损伤的机制。在 I/R 区域，复合物 III 的催化活性显著受损。光谱分析表明，I/R 介导了线粒体中血红素 b 和 c+c 的破坏，支持了 I/R 介导的复合物 III 损伤。然而，在仅经历 30 分钟缺血的大鼠心脏风险区域的线粒体中，没有观察到复合物 III 活性和血红素损伤的显著损伤，尽管状态 3 呼吸减少。在 I/R 线粒体中，通过烷基化复合物 III 并下调 HCCS，检测到通过碳甲酰化使细胞色素 c 的 C/C 与半胱氨酸形成硫醚缺陷，这支持了 I/R 介导的血红素 c 的硫醚缺陷。LC-MS/MS 分析显示，I/R 线粒体中 Rieske 铁-硫蛋白（uqcrfs1）[2Fe2S]簇的 C 配体上的复合物 III PrSOH 水平显著增加，从而损害电子传递活性。MS 分析还表明，在跨膜空间中受限的铰链蛋白的 C 和 C 以及细胞色素 c 的 C 和 C 上的 PrSOH 增加。MS 分析还表明，I/R 广泛增强了基质腔中核心 1（uqcrc1）和核心 2（uqcrc2）亚基的 PrSOH，从而支持了复合物 III 在再灌注期间向内膜两侧释放 ROS 的结论。对缺血线粒体的分析表明，与 sham 对照组心脏线粒体中检测到的基础复合物 III PrSOH 相比，有适度降低，这表明再灌注期间生理超氧和 ROS 过度产生介导了复合物 III PrSOH 的增强。总之，再灌注介导的血红素损伤伴随着增加的 PrSOH 控制了复合物 III 的氧化损伤，并加重了缺血后心脏的线粒体功能障碍。
