Borutaite V, Budriunaite A, Morkuniene R, Brown G C
Department of Biochemistry, University of Cambridge, UK.
Biochim Biophys Acta. 2001 Sep 28;1537(2):101-9. doi: 10.1016/s0925-4439(01)00062-x.
It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.
先前的研究表明，缺血/再灌注心脏中的细胞凋亡会增加。然而，对于缺血期间心肌细胞凋亡的诱导机制知之甚少。我们研究了长时间心肌缺血是否会导致半胱天冬酶的激活，以及这种激活是否与缺血期间细胞色素c从线粒体释放到细胞质中有关。使用心脏缺血的体外模型，我们发现60分钟的缺血会导致细胞质中细胞色素c的显著积累以及线粒体中细胞色素c含量的减少，但细胞色素a含量未减少。细胞色素c从线粒体的释放伴随着caspase-3样蛋白酶的激活（通过荧光肽底物DEVD-amc的切割来测量）以及DNA链断裂细胞数量的大幅增加（通过TUNEL染色来测量）。缺血期间caspase-1样蛋白酶（通过YVAD-amc切割来测量）未被激活。向从对照心脏制备的细胞质提取物中添加14微摩尔的细胞色素c会诱导caspase-3样蛋白酶活性的ATP依赖性激活。我们的数据表明，延长的心脏缺血可导致由细胞色素c从线粒体释放以及随后caspase-3的激活介导的细胞凋亡。
