Sugiura S, Kobayakawa N, Fujita H, Momomura S, Omata M
Second Department of Internal Medicine, School of Medicine, University of Tokyo, Japan.
Heart Vessels. 1997;Suppl 12:97-9.
Distinct crossbridge kinetics among cardiac myosin isoforms have been proposed as the basis of differences in energetic characteristics. However, direct evidence for this hypothesis is lacking because of experimental difficulty. As a preliminary approach to this problem, we applied an in-vitro force measurement technique to directly observe force impulse generated by a single cardiac myosin molecule. The force measurement system was constructed with an inverted microscope coupled with a laser optical trap. With the feedback of the position signal to the driving circuit for a galvanomirror that steers the laser beam, trap stiffness was increased; thus, isometric force measurement was made possible. We measured the force generated by the cardiac myosin V3 isoform purified from hypothyroid rat ventricular muscle. With very low myosin density and low adenosine triphosphate (ATP) concentration of the assay buffer, we successfully observed a single force impulse similar in shape to that of skeletal muscle myosin. With this approach, we will be able to gain a clear view of the molecular basis of cardiac mechanoenergetics.
不同心肌肌球蛋白亚型之间独特的横桥动力学被认为是能量特征差异的基础。然而，由于实验难度，这一假设缺乏直接证据。作为解决该问题的初步方法，我们应用体外力测量技术直接观察单个心肌肌球蛋白分子产生的力脉冲。力测量系统由一台倒置显微镜和一个激光光镊组成。通过将位置信号反馈到控制激光束的检流计镜的驱动电路，增加了光镊刚度；因此，可以进行等长力测量。我们测量了从甲状腺功能减退大鼠心室肌中纯化的心肌肌球蛋白V3亚型产生的力。在肌球蛋白密度非常低且测定缓冲液中三磷酸腺苷（ATP）浓度低的情况下，我们成功观察到了一个形状与骨骼肌肌球蛋白相似的单力脉冲。通过这种方法，我们将能够清楚地了解心脏机械能量学的分子基础。
