Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes
Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes
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DOI：
10.1006/jmcc.2001.1364
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发表时间：
2001-05-01
影响因子：
5
通讯作者：
Schaper, J
中科院分区：
文献类型：
--
作者：
Suzuki, K;Kostin, S;Schaper, J
由于成年心肌细胞凋亡级联反应的时间进程在很大程度上是未知的，因此对患病心脏中凋亡速率的解释受到阻碍。因此。我们建立了一个与心力衰竭的体内情况相关的标准化体外系统，使用成年去分化和再分化心肌细胞来确定凋亡级联反应的不同步骤发生所需的时间间隔。在培养10天的成年大鼠心肌细胞中，0.1 mmol/l H_2O_2诱导Mras凋亡。剂量> 0.5 mmol/l H2 O2会产生坏死。线粒体膜电位（Δ Psim）的破坏是细胞凋亡的最早迹象，发生在H2 O2暴露后2小时。膜联蛋白V（磷脂酰丝氨酸的易位）和PhiPhiLux（半胱天冬酶-3的活化）阳性细胞的数量在4小时后显著增加，此后保持恒定。Bcl-2水平下降。九点。Bar表达显著升高，导致Bcl-2/Bar比率降低。TUNEL和ssDNA检测到的DNA片段化在14 h达到高峰。与凋亡超微结构变化的出现平行。虽然zVAD-favorites抑制DNA片段化。Ac-DEVD-CHO。zLEVD-f抑制剂，这些半胱天冬酶抑制剂不能抑制膜的破坏，并增加坏死细胞的数量。过氧化氢酶抑制细胞凋亡和坏死。我们的研究结果表明，凋亡级联反应的不同步骤的发生是时间依赖性的，并受到严格的调控。半胱天冬酶抑制剂减少细胞凋亡，但增加坏死率。表明细胞注定在胱天蛋白酶步骤的上游死亡，即通过线粒体损伤。这些数据提供了关键的评估和解释的发生在衰竭的心脏细胞凋亡的基础。(C)北京：科学出版社.
Interpretation of the rate of apoptosis in diseased hearts is hampered by the fact that the time course of the apoptotic cascade in adult cardiomyocytes is largely; unknown. Therefore. we established a standardized in vitro system, relevant to the in vivo situation of heart failure, using adult de- and redifferentiating cardiomyocytes to determine the time intervals necessary for the different steps of the apoptotic cascade to occur. Apoptosis Mras induced with 0.1 mmol/l H2O2 in adult rat cardiomyocytes 10 days in culture. Dosages > 0.5 mmol/l H2O2 produced necrosis. Disruption of the mitochondrial membrane potential (Delta Psim) was the earliest sign of apoptosis and occurred at 2 h after H2O2 exposure. The number of annexin V (translocation of phosphatidylserine) and PhiPhiLux (activation of caspase-3) positive cells significantly increased after 4 h and remained constant thereafter. Bcl-2 levels decreased. At 9 h. Bar expression was significantly elevated resulting in a reduced Bcl-2/Bar ratio. DNA fragmentation detected by TUNEL and ssDNA peaked at 14 h. parallel to the appearance of apoptotic ultrastructural changes. Although DNA fragmentation was inhibited by zVAD-fmk. Ac-DEVD-CHO. zLEVD-fmk, these caspase inhibitors failed to inhibit disruption of film and increased the number of necrotic cells. Catalase inhibited both apoptosis and necrosis. Our results indicate that the occurrence of the different steps of the apoptotic cascade is time-dependent and tightly regulated. Caspase inhibitors reduce apoptosis but increase the rate of necrosis. suggesting that the cells are destined to die upstream of the caspase step, i.e. by mitochondrial damage. These data provide the basis for the critical evaluation and interpretation of the occurrence of apoptosis in failing hearts. (C) 2001 Academic Press.