Chiesi M, Ho M M, Inesi G, Somlyo A V, Somlyo A P
J Cell Biol. 1981 Dec;91(3 Pt 1):728-42. doi: 10.1083/jcb.91.3.728.
Homogeneous populations of single myocytes showing good preservation of ultrastructure were obtained by enzymatic digestion of rabbit and rat hearts, and maintained in a relaxed state in the presence of free Ca2+ concentrations less than 10(-7) M. Ultrastructural details such as a cytoskeleton of 100-A filaments connected to the sarcolemma at the Z lines were demonstrated especially well in these preparations. In spite of seemingly normal structure, electron probe analysis of cryosections reveals similar concentrations of electrolytes in the medium and in the cytoplasm, indicating the presence of electrochemical shunting across the external membrane. The dissociated myocytes display Ca uptake and phasic contractions that are apparently dependent on mitochondrial respiration, but are not affected by mitochondrial uncouplers when ATP and phosphocreatine are added. The uptake is augmented by oxalate and, based on identification of calcium oxalate crystals by electron microscopy and electron probe analysis, is localized to the sarcoplasmic reticulum (SR). An advantageous feature of the dissociated myocytes is that they are suitable for experiments using large numbers of cells in suspension. Thereby, velocities of calcium transport were measured directly by isotopic tracer and filtration methods. It was then found that the lowest CA2+ concentrations (5 x 10(-7) M for the rabbit and 1 x 10(-7) M for the rat) sustaining Ca transport also induce phasic contractile activity in all myocytes, even though the external membrane is electrochemically shunted. A stepwise rise in the Ca2+ concentration of up to one order of magnitude, increases transport velocities in parallel with the rates of phasic contractions. Both these parameters are affected by Mg2+, temperature, cyclic-AMP, and methylxanthines, even though the Ca2+ concentration is maintained constant in the medium. Therefore, Ca transport by SR is a requirement and a rate limiting factor for the occurrence of phasic contractile activation in dissociated cardiac cells retaining an electrochemically shunted external membrane. It is suggested that transient Ca release required for phasic contractile activation is due to equilibrium oscillations across the SR membrane. The sequential pattern of sarcomere activation is consistent with a self propagating mechanism of calcium release. SR in dissociated skeletal muscle cells sustains a greater Ca transport activity than in dissociated heart cells. However, the heart cells display a much higher phasic contractile activity, indicating that cardiac SR has a greater tendency to release accumulated calcium. If free Ca2+ in the medium is raised above 10(-6) M, both cardiac and skeletal myocytes undergo contractures and degenerative phenomena, accompanied by Ca, Mg, and phosphate accumulation in cardiac mitochondria.
通过酶消化兔和大鼠心脏获得了超微结构保存良好的单一心肌细胞同质群体，并在游离钙离子浓度低于10^(-7) M的情况下保持松弛状态。在这些制备物中，超微结构细节，如在Z线处与肌膜相连的100埃细丝的细胞骨架，表现得尤为清晰。尽管结构看似正常，但对冷冻切片的电子探针分析显示，培养基和细胞质中的电解质浓度相似，表明在外膜上存在电化学分流。解离的心肌细胞表现出钙摄取和阶段性收缩，这显然依赖于线粒体呼吸，但当添加ATP和磷酸肌酸时，不受线粒体解偶联剂的影响。草酸盐可增强摄取，基于电子显微镜和电子探针分析对草酸钙晶体的鉴定，摄取定位于肌浆网（SR）。解离的心肌细胞的一个有利特征是它们适用于使用大量悬浮细胞的实验。由此，通过同位素示踪和过滤方法直接测量了钙转运速度。然后发现，维持钙转运的最低钙离子浓度（兔为5×10^(-7) M，大鼠为1×10^(-7) M）也会在所有心肌细胞中诱导阶段性收缩活动，即使外膜存在电化学分流。钙离子浓度逐步升高至一个数量级，会使转运速度与阶段性收缩速率平行增加。这两个参数都受镁离子、温度、环磷酸腺苷和甲基黄嘌呤的影响，尽管培养基中的钙离子浓度保持恒定。因此，SR的钙转运是保留电化学分流外膜的解离心脏细胞中发生阶段性收缩激活的必要条件和限速因素。有人提出，阶段性收缩激活所需的瞬时钙释放是由于SR膜上的平衡振荡。肌节激活的顺序模式与钙释放的自传播机制一致。解离的骨骼肌细胞中的SR比解离的心脏细胞维持更大的钙转运活性。然而，心脏细胞表现出更高的阶段性收缩活性，表明心脏SR有更大的倾向释放积累的钙。如果培养基中的游离钙离子升高到10^(-6) M以上，心脏和骨骼肌细胞都会发生挛缩和退行性现象，同时心脏线粒体中会积累钙、镁和磷酸盐。
