Maulik N, Kagan V E, Tyurin V A, Das D K
Department of Surgery, University of Connecticut School of Medicine, Farmington 06030, USA.
Am J Physiol. 1998 Jan;274(1):H242-8. doi: 10.1152/ajpheart.1998.274.1.H242.
Although cardiomyocyte death and infarction associated with ischemia-reperfusion are traditionally believed to be induced via necrosis, recent studies implicated apoptotic cell death in ischemic reperfused tissue. To examine whether myocardial ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15 and 30 min of ischemia as well as 15 min of ischemia followed by 30, 90, or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from cardiomyocytes to 1.8% agarose gel electrophoresis and photographed under ultraviolet illumination. In addition, high-performance thin-layer chromatography (HPTLC) of aminophospholipids labeled with 2,4,6-trinitrobenzenesulfonate was performed to evaluate phospholipid topography in cardiomyocytes. The results of our study revealed apoptotic cells only in the 90- and 120-min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscope. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation that showed increased ladders of DNA bands in the 120-min reperfused hearts, representing integer multiples of the internucleosomal DNA length (approximately 180 bp). Two-dimensional HPTLC of the phospholipids obtained from the cardiomyocytes and transbilayer organization of the phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the myocytes indicated translocation of both PE and PS from the inner leaflet to the outer leaflet of the membrane as early as after 20 min of ischemia. These results demonstrate that the redistribution of PS and PE precedes the apototic cell death and DNA fragmentation associated with the reperfusion of ischemic myocardium, suggesting that ischemia may trigger the signal for apoptosis although it becomes evident during reperfusion.
虽然传统上认为与缺血再灌注相关的心肌细胞死亡和梗死是由坏死引起的，但最近的研究表明凋亡性细胞死亡参与了缺血再灌注组织的过程。为了研究心肌缺血再灌注损伤是否由凋亡性细胞死亡介导，对离体灌注的大鼠心脏进行15分钟和30分钟的缺血处理，以及15分钟缺血后再灌注30分钟、90分钟或120分钟。在每个实验结束时，对心脏进行处理以评估凋亡和DNA梯状条带。通过使用APOPTAG原位凋亡检测试剂盒对洋地黄毒苷标记的基因组DNA进行直接荧光检测来可视化凋亡心肌细胞，从而研究凋亡。通过将从心肌细胞获得的DNA进行1.8%琼脂糖凝胶电泳并在紫外光下拍照来评估DNA梯状条带。此外，进行了用2,4,6-三硝基苯磺酸标记的氨基磷脂的高效薄层色谱（HPTLC），以评估心肌细胞中的磷脂拓扑结构。我们的研究结果显示，只有在再灌注90分钟和120分钟的心脏中观察到凋亡细胞，荧光显微镜下观察到免疫染色的洋地黄毒苷标记的基因组DNA发出强烈荧光即可证明。缺血心脏均未显示任何凋亡迹象。这些结果与DNA片段化的结果一致，后者显示再灌注120分钟的心脏中DNA条带的梯状条带增加，代表核小体间DNA长度（约180 bp）的整数倍。从心肌细胞获得的磷脂的二维HPTLC以及心肌细胞中磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）的跨膜组织表明，早在缺血20分钟后，PE和PS就从膜的内小叶转移到了外小叶。这些结果表明，PS和PE的重新分布先于与缺血心肌再灌注相关的凋亡性细胞死亡和DNA片段化，这表明缺血可能触发凋亡信号，尽管它在再灌注期间才变得明显。
