Qin Yimin, Vanden Hoek Terry L, Wojcik Kim, Anderson Travis, Li Chang-Qing, Shao Zuo-Hui, Becker Lance B, Hamann Kimm J
Department of Medicine MC6076, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA.
Am J Physiol Heart Circ Physiol. 2004 Jun;286(6):H2280-6. doi: 10.1152/ajpheart.01063.2003. Epub 2004 Feb 19.
We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 +/- 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH(2)F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.
我们最近证明，在模拟缺血1小时后，再灌注能迅速诱导鸡心肌细胞发生凋亡的线粒体途径。在此，我们测试了在这种心肌细胞损伤模型中，缺血再灌注（I/R）诱导的凋亡是否可由半胱天冬酶依赖性细胞色素c释放引发。半胱天冬酶活性的荧光测定显示，仅由1小时缺血诱导的半胱天冬酶激活水平几乎没有超过基线水平（即便有也很少）。然而，这些测定揭示了半胱天冬酶-2的迅速激活，在再灌注的第1小时内其活性增加了2.95±0.52倍（相对于仅缺血时），而半胱天冬酶-3、-8和-9的活性仅从基线水平略有增加。半胱天冬酶-2的迅速且显著激活表明它可能是该模型中的一个重要起始半胱天冬酶，我们使用仅在再灌注时给予的特异性半胱天冬酶抑制剂来验证这一假设。半胱天冬酶-2抑制剂苄氧羰基-Val-Asp（Ome）-Val-Ala-Asp（Ome）-CH₂F是唯一能显著抑制细胞色素c从线粒体释放的半胱天冬酶抑制剂。该抑制剂还完全阻断了半胱天冬酶-3、-8和-9的激活。半胱天冬酶-3/7抑制剂只是短暂且部分地阻断了半胱天冬酶-2的活性，在阻断半胱天冬酶-8和-9的活性方面效果较差。半胱天冬酶-8抑制剂未能显著阻断半胱天冬酶-2或-3，而半胱天冬酶-9抑制剂仅阻断了半胱天冬酶-9。此外，半胱天冬酶-2抑制剂可保护细胞免受I/R诱导的细胞死亡，而半胱天冬酶-8抑制剂则不能。这些数据表明，活性半胱天冬酶-2在再灌注后引发细胞色素c释放，并且它对于该模型中I/R诱导的凋亡至关重要。
