Stuyvers B D, Miura M, Jin J P, ter Keurs H E
University of Calgary, Alberta, Canada.
Prog Biophys Mol Biol. 1998;69(2-3):425-43. doi: 10.1016/s0079-6107(98)00018-2.
The stiffness of the sarcomeres was studied during the diastolic interval of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; temperature = 25 degrees C). Sarcomere length (SL) and force (F) were measured using, respectively, laser diffraction techniques (resolution: 4 nm) and a silicon strain gauge (resolution: 0.63 microN). Sinusoidal perturbations (frequency = 500 Hz) were imposed to the length of the preparation. The stiffness was evaluated from the corresponding F and SL sinusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinusoidal perturbations was repeated at 5 predetermined times during diastole providing 5 measurements of stiffness during the time interval separating two twitches. These measurements revealed that stiffness increases by approximately 30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements of the fluorescence of fura-2 under the same conditions revealed a continuous exponential decline of [Ca2+]i from 210 to 90 nM (constant of time approximately 300 ms) during diastole. In order to test the possibility that the increase of sarcomere stiffness and the decline of [Ca2+]i were coupled during diastole of intact trabeculae, we studied the effect of different free Ca(2+)-concentrations ([Ca2+]) between 1 and 430 nM on sarcomere stiffness in rat cardiac trabeculae skinned by saponin (n = 17). Stiffness was studied using 500 Hz sinusoidal perturbations of muscle length (ML). We found that, below 70 nM, the stiffness was independent of [Ca2+]; between 70 and 200 nM, the stiffness declined with increase of [Ca2+]; above 200 nM, the stiffness increased steeply with [Ca2+]. The data fitted accurately to the sum of two sigmoids (Hill functions): (1) at [Ca2+] < 200 nM the stiffness decreased with [Ca2+] (EC50 = 160 +/- 13 nM; n = -2.6 +/- 0.7) and (2) at [Ca2+] > 200 nM, stiffness increased with [Ca2+] (EC50 = 3.4 +/- 0.3 microM; n = 2.1 +/- 0.2) due to attachment of cross-bridges. From these results, it was possible to reproduce accurately the time course of diastolic stiffness observed in intact trabeculae and to predict the effect on stiffness of a spontaneous elevation of the diastolic [Ca2+]. Identical stiffness measurements were performed in 4 skinned preparations exposed to a cloned fragment of titin (Ti I-II) which has been shown to exhibit a strong interaction with F-actin in vitro. It was anticipated that Ti I-II would compete with endogenous titin for the same binding site on actin in the I-band. Below 200 nM, Ti I-II (2 microM) eliminated the Ca(2+)-dependence of stiffness. These results are consistent with the hypothesis that the Ca(2+)-sensitivity of the sarcomeres at [Ca2+] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, involves an association between titin molecules and the thin filament.
在大鼠的18条受刺激（0.5赫兹）的心脏小梁舒张期（pH 7.4；温度=25摄氏度）研究肌节的刚度。分别使用激光衍射技术（分辨率：4纳米）和硅应变片（分辨率：0.63微牛）测量肌节长度（SL）和力（F）。对标本的长度施加正弦扰动（频率=500赫兹）。通过在时域或频域中对相应的F和SL正弦波同时进行分析来评估刚度。在舒张期的5个预定时间重复短脉冲（持续时间=30毫秒）的正弦扰动，在两个收缩期之间的时间间隔内提供5次刚度测量。这些测量结果显示，舒张期刚度增加约30%，同时检测到肌节有同步扩展（幅度=10 - 60纳米）。在相同条件下对fura - 2荧光的测量显示，舒张期[Ca2+]i从210纳米持续指数下降至90纳米（时间常数约300毫秒）。为了测试完整小梁舒张期肌节刚度增加与[Ca2+]i下降是否相关，我们研究了1至430纳米之间不同游离Ca(2+)浓度（[Ca2+]）对经皂角苷处理的大鼠心脏小梁肌节刚度的影响（n = 17）。使用500赫兹的肌肉长度（ML）正弦扰动研究刚度。我们发现，低于70纳米时，刚度与[Ca2+]无关；在70至200纳米之间，刚度随[Ca2+]增加而下降；高于200纳米时，刚度随[Ca++]急剧增加。数据精确拟合为两个S形曲线（希尔函数）之和：（1）在[Ca2+]<200纳米时，刚度随[Ca2+]下降（半数有效浓度EC50 = 160±13纳米；n = -2.6±0.7），（2）在[Ca2+]>200纳米时，由于横桥附着，刚度随[Ca2+]增加（EC50 = 3.4±0.3微摩尔；n = 2.1±0.2）。根据这些结果，可以准确再现完整小梁中观察到的舒张期刚度的时间进程，并预测舒张期[Ca2+]自发升高对刚度的影响。在4个暴露于肌联蛋白克隆片段（Ti I-II）的去膜标本中进行了相同的刚度测量，该片段在体外已显示与F-肌动蛋白有强烈相互作用。预计Ti I-II会与内源性肌联蛋白竞争I带中肌动蛋白上的相同结合位点。在200纳米以下，Ti I-II（2微摩尔）消除了刚度的Ca(2+)依赖性。这些结果与以下假设一致：在[Ca2+]<200纳米时，即完整肌肉中的心肌细胞在舒张期的状态下，肌节的Ca(2+)敏感性涉及肌联蛋白分子与细肌丝之间的结合。
