Piot C A, Martini J F, Bui S K, Wolfe C L
Department of Medicine, University of California, San Francisco, USA.
Cardiovasc Res. 1999 Dec;44(3):536-42. doi: 10.1016/s0008-6363(99)00227-8.
Recently, we have demonstrated that ischemic preconditioning (IP) both limits infarct size and decreases internucleosomal DNA fragmentation in rat hearts in vivo, and that there was a direct correlation between myocardial infarct size and DNA fragmentation even after IP. In this study, we examined the ability of IP to attenuate processing and activation of caspase-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), after prolonged ischemia and reperfusion using the same in vivo animal model. Rats that underwent IP and controls (Ctrl) were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion. IP was accomplished by five 5-min cycles of ischemia, each followed by 5 min of reperfusion. The amount of soluble nucleosomes was measured by enzyme-linked immunosorbent assay. Cleavage of caspases-1 and -3, and of one of their substrates PARP, was analyzed by Western blotting. Nucleosomal DNA fragmentation was significantly reduced in ischemic left ventricular (LV) tissue obtained from IP compared with Ctrl animals. The proforms of caspases-1 and -3, and the active form of PARP were not cleaved in the nonischemic LV region of both IP and Ctrl hearts. In contrast, the proform of caspase-3 and the active form of PARP were cleaved in the ischemic LV region of Ctrl hearts, while processing of caspase-1 was increased. Cleavages of caspases-1 and -3, and inactivation of PARP were prevented by IP. The results of this study indicate that IP attenuates both internucleosomal DNA fragmentation and caspases processing, and suggest that the prevention of caspases activation by IP may be important steps in protecting the heart against ischemia/reperfusion injury in vivo.
最近，我们已经证明，缺血预处理（IP）在体内可限制大鼠心脏的梗死面积并减少核小体间DNA片段化，并且即使在进行缺血预处理后，心肌梗死面积与DNA片段化之间也存在直接相关性。在本研究中，我们使用相同的体内动物模型，研究了缺血预处理在长时间缺血和再灌注后减弱半胱天冬酶-1和半胱天冬酶-3的加工和激活以及聚（ADP-核糖）聚合酶（PARP）裂解的能力。接受缺血预处理的大鼠和对照组（Ctrl）经历30分钟的左冠状动脉闭塞，随后再灌注180分钟。缺血预处理通过五个5分钟的缺血周期完成，每个周期后再灌注5分钟。通过酶联免疫吸附测定法测量可溶性核小体的量。通过蛋白质印迹分析半胱天冬酶-1和-3及其底物之一PARP的裂解情况。与对照组动物相比，从接受缺血预处理的大鼠获得的缺血左心室（LV）组织中的核小体DNA片段化明显减少。在缺血预处理组和对照组心脏的非缺血LV区域中，半胱天冬酶-1和-3的前体形式以及PARP的活性形式均未被裂解。相反，在对照组心脏的缺血LV区域中，半胱天冬酶-3的前体形式和PARP的活性形式被裂解，而半胱天冬酶-1的加工增加。缺血预处理可防止半胱天冬酶-1和-3的裂解以及PARP的失活。本研究结果表明，缺血预处理可减弱核小体间DNA片段化和半胱天冬酶的加工，并表明缺血预处理对半胱天冬酶激活的预防可能是体内保护心脏免受缺血/再灌注损伤的重要步骤。
