Lundblad A, Gonzalez-Serratos H, Inesi G, Swanson J, Paolini P
J Gen Physiol. 1986 Jun;87(6):885-905. doi: 10.1085/jgp.87.6.885.
Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77-159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.
为了在技术上改进对肌节动力学的测量，并评估肌浆网在原位收缩激活方面的作用，通过用胶原酶灌注大鼠心脏来制备功能去膜和电化学分流的心肌细胞。在存在微摩尔钙的情况下，心肌细胞表现出从一端开始并沿心肌细胞传播的阶段性收缩波。通过对从连接到配备有温度控制载物台的显微镜的相机以170帧/秒记录的16毫米胶片进行手动或自动分析，获得了跳动频率、激活波的传播速度以及单个肌节的缩短和松弛速度。在平行实验中，通过直接同位素示踪法测量了原位心肌细胞肌浆网的钙积累。自发收缩的频率（10 - 38次/分钟）、肌节缩短的速度（1.9 - 7.4微米·秒⁻¹）和肌节松弛的速度（1.7 - 6.8微米·秒⁻¹）表现出相同的温度依赖性（Q10 = 2.2），这与肌浆网钙泵的温度依赖性相似，并且与酶催化机制对所有这些参数施加的速率限制一致。另一方面，连续肌节激活的速度（77 - 159微米·秒⁻¹）表现出较低的温度依赖性（Q10 = 1.5），这与钙从一个肌节向另一个肌节的扩散限制和自传播释放一致。在原位肌浆网钙积累被证明最佳进行的条件下，解离的心肌细胞的阶段性收缩活动被10⁻⁸ - 10⁻⁶ M的ryanodine（而不是肌膜钙阻滞剂）抑制。ryanodine的作用归因于该药物与肌管结构的相互作用，导致肌浆网钙释放受到抑制。由此导致的肌节激活缺失强调了肌浆网摄取和释放在电化学分流心肌细胞的阶段性收缩激活中的作用。
