Canton Marcella, Neverova Irina, Menabò Roberta, Van Eyk Jennifer, Di Lisa Fabio
Dipartimento di Chimica Biologica, Viale G. Colombo, 3, 35121 Padova, Italy.
Am J Physiol Heart Circ Physiol. 2004 Mar;286(3):H870-7. doi: 10.1152/ajpheart.00714.2003.
Although the contribution of reactive oxygen species to myocardial ischemia is well recognized, the possible intracellular targets, especially at the level of myofibrillar proteins (MP), are not yet fully characterized. To assess the maximal extent of oxidative degradation of proteins, isolated rat hearts were perfused with 1 mM H(2)O(2). Subsequently, the MP maximally oxidative damage was compared with the effects produced by 1) 30 min of no-flow ischemia (I) followed in other hearts by 3 min of reperfusion (I/R); and 2) I/R in the presence of a potent antioxidant N-(2-mercaptopropionyl)glycine (MPG). Samples from the H(2)O(2) group electrophoresed under nonreducing conditions and probed with actin, desmin, or tropomyosin monoclonal antibodies showed high-molecular mass complexes indicative of disulfide cross-bridges along with splitting and thickening of tropomyosin and actin bands, respectively. Only these latter changes could be detected in I/R samples and were prevented by MPG. Carbonyl groups generated by oxidative stress on MP were detected by Western blot analysis (oxyblot) under optimized conditions. The analyses showed one major band corresponding to oxidized actin, the density of which increased 1.2-, 2.8-, and 6.8-fold in I, I/R, and H(2)O(2) groups, respectively. The I/R-induced increase was significantly reduced by MPG. In conclusion, oxidative damage of MP occurs on reperfusion, although at a lower extent than in H(2)O(2) perfused hearts, whereas oxidative modifications could not be detected in ischemic hearts. Furthermore, the inhibition of MP oxidation by MPG might underlie the protective efficacy of antioxidants.
尽管活性氧对心肌缺血的作用已得到充分认识，但其可能的细胞内靶点，尤其是在肌原纤维蛋白（MP）水平，尚未完全明确。为评估蛋白质氧化降解的最大程度，用1 mM过氧化氢灌注离体大鼠心脏。随后，将MP的最大氧化损伤与以下情况产生的影响进行比较：1）30分钟无血流缺血（I），然后在其他心脏中进行3分钟再灌注（I/R）；2）在强效抗氧化剂N-（2-巯基丙酰基）甘氨酸（MPG）存在下的I/R。在非还原条件下对过氧化氢组的样品进行电泳，并用肌动蛋白、结蛋白或原肌球蛋白单克隆抗体进行检测，结果显示高分子量复合物，表明存在二硫键交联，同时原肌球蛋白和肌动蛋白条带分别出现分裂和变厚。只有后一种变化能在I/R样品中检测到，且被MPG阻止。在优化条件下，通过蛋白质免疫印迹分析（氧化印迹法）检测MP上由氧化应激产生的羰基。分析显示一条对应氧化型肌动蛋白的主要条带，其密度在I组、I/R组和过氧化氢组中分别增加了1.2倍、2.8倍和6.8倍。MPG显著降低了I/R诱导的增加。总之，MP的氧化损伤在再灌注时发生，尽管程度低于过氧化氢灌注的心脏，而在缺血心脏中未检测到氧化修饰。此外，MPG对MP氧化的抑制作用可能是抗氧化剂保护作用的基础。
