White Melanie Y, Tchen Adrian S, McCarron Hugh C K, Hambly Brett D, Jeremy Richmond W, Cordwell Stuart J
Department of Medicine, The University of Sydney, Sydney, Australia.
Proteomics. 2006 Dec;6(23):6221-33. doi: 10.1002/pmic.200600219.
A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.
短暂的缺血期后及时再灌注可能导致心肌收缩功能障碍延长但可逆（心肌顿抑）。心肌损伤不仅发生在缺血期间，也发生在再灌注期间，此时会大量释放氧自由基（OFR）。我们之前利用二维电泳（2-DE）和质谱（MS）技术，在兔模型中确定了短暂缺血/再灌注（15分钟缺血，60分钟再灌注；15I/60R）损伤过程中57个蛋白质点的变化（怀特，M.Y.，科德韦尔，S.J.，麦卡伦，H.C.K.，普拉桑，A.M.等，《蛋白质组学》2005年，第5卷，1395 - 1410页），并表明其中大多数变化是由于物理和/或化学的蛋白质翻译后修饰（PTM）。在本研究中，我们在氧自由基清除剂N -（2 - 巯基丙酰基）甘氨酸（MPG）存在的情况下，对兔心肌进行15I/60R处理。在15I/60R期间发生变化的57个蛋白质点中，有37个在MPG存在（15I/60R + MPG）时保持在对照水平。收缩蛋白的变化，包括肌球蛋白轻链2（MLC - 2）和肌钙蛋白C（TnC），通过添加MPG得以预防。为了进一步研究缺血和再灌注的个体影响，我们从仅经历短暂缺血（15I/0R）的兔心肌中制备了二维电泳凝胶，并观察到33个蛋白质点的变化，其中18/20个在15I/60R处理和15I/60R + MPG处理的组织中都可见。该组织也在MPG存在的情况下进行缺血处理（15I/0R + MPG），尽管存在氧自由基清除剂，但仍有21个点的变化，代表14种蛋白质变体，保持改变。这些缺血特异性蛋白质包括参与能量代谢的蛋白质（乳酸脱氢酶和ATP合酶α）、氧化还原调节蛋白（NADH泛醌氧化还原酶51 kDa和谷胱甘肽S - 转移酶Mu）以及应激反应蛋白（热休克蛋白27和70，以及脱酰胺化的αB - 晶状体蛋白）。我们得出结论，与心肌顿抑相关的收缩功能障碍主要是由再灌注开始时的氧自由基损伤引起的，但在缺血期间也会发生不依赖氧自由基的损伤。这些缺血特异性蛋白质修饰可能表明早期心肌损伤。
