Shepherd M, Bruening M, Auld A M, Barritt G J
Department of Biochemistry and Chemical Pathology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, South Australia.
Biochem Med Metab Biol. 1987 Oct;38(2):195-204. doi: 10.1016/0885-4505(87)90080-6.
The effect of energy deprivation and H2O2 on the contraction, shape, and intracellular free Ca2+ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mM extracellular Ca2+, CCCP (0.25 microM) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca2+ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mM extracellular K+, CCCP caused a marked increase in the myoplasmic free Ca2+ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K+-induced depolarization. Exposure of myocytes to H2O2 (0.5 mM) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H2O2 were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca2+, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H2O2, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca2+ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.
利用新鲜分离的、电刺激的大鼠心室肌细胞悬液，研究了能量剥夺和过氧化氢对心肌细胞收缩、形态及细胞内游离钙离子浓度的影响。线粒体解偶联剂羰基氰化物间氯苯腙（CCCP）用于降低ATP合成速率。在细胞外钙离子浓度为0.9 mM时，CCCP（0.25 μM）在5分钟后使收缩细胞数量减少50%，10分钟后使杆状细胞数量减少40%。CCCP的作用与测得的细胞ATP浓度大幅降低有关。细胞外钙离子浓度升高可增强心肌细胞暴露于CCCP长达5分钟的有害作用，但在无电刺激时明显减弱。维拉帕米在最初5分钟可保护心肌细胞免受CCCP的有害作用，但在之后则不能。在细胞外钾离子浓度为46 mM时，CCCP使肌浆游离钙离子浓度（用喹啉2测量）显著升高。该作用被维拉帕米抑制，且在无钾离子诱导的去极化时未观察到。心肌细胞暴露于过氧化氢（0.5 mM）导致正常端端同步收缩的细胞数量以及部分或完全收缩的细胞总数大幅减少。过氧化氢浓度越高、暴露时间越长、细胞外钙离子浓度越高，过氧化氢的作用越明显，且二甲基亚砜可部分逆转其作用。结果表明，ATP剥夺和过氧化氢可能通过自由基的产生，对心肌细胞造成严重且快速的损伤，并诱导额外的钙离子穿过肌膜进入肌浆。在ATP剥夺的情况下，这最初是通过电压门控通道发生的。
