Taimor G, Lorenz H, Hofstaetter B, Schlüter K D, Piper H M
Physiologisches Institut, Justus-Liebig-Universität Giessen, Germany.
Cardiovasc Res. 1999 Jan;41(1):147-56. doi: 10.1016/s0008-6363(98)00209-0.
Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion.
Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release.
Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation.
Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.
细胞凋亡是缺血再灌注后心肌损伤的一个特征，但其诱导原因尚不清楚。本研究旨在探讨心肌细胞中的细胞凋亡是否直接由缺血再灌注导致的亚致死性损伤引发。
在pH 6.4的无葡萄糖培养基中通过缺氧模拟成年大鼠离体心室心肌细胞的缺血状态。通过以下方法检测细胞凋亡的诱导情况：（1）基因组DNA的DNA梯状条带；（2）TUNEL阳性细胞（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）；（3）在排除碘化丙啶的情况下，膜联蛋白V-异硫氰酸荧光素（annexinV-FITC）与细胞结合。通过以下方法鉴定坏死细胞：（1）用膜联蛋白V-FITC和碘化丙啶染色；（2）非特异性DNA降解；（3）酶释放。
模拟缺血在2小时内导致高能磷酸盐损失超过75%，在pH 7.4复氧后可逆转。即使在模拟缺血18小时后，磷酸肌酸含量在1小时内恢复至对照的55.2±7.3%。紫外线照射（80 J/m2）、过氧化氢和NO供体N2-乙酰-S-亚硝基-D,L-青霉胺可诱导细胞凋亡。与此相反，模拟缺血和复氧不能诱导细胞凋亡，但随着缺血时间延长，更多细胞发生坏死。在模拟缺血18小时和复氧4小时后，41.2±10.2%的心肌细胞坏死（对照为6.3±4.4%），仅有1.7±0.5%的细胞凋亡（对照为8.7±4.6%）。坏死细胞百分比与乳酸脱氢酶释放增加相关，从常氧对照的总活性的9.9±0.6%增加到模拟缺血18小时和复氧12小时后的37.9±5.1%。
模拟缺血再灌注导致离体心肌细胞坏死，但不足以诱导细胞凋亡。
