Shintani Seine A, Oyama Kotaro, Kobirumaki-Shimozawa Fuyu, Ohki Takashi, Ishiwata Shin'ichi, Fukuda Norio
Department of Pure and Applied Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
J Gen Physiol. 2014 Apr;143(4):513-24. doi: 10.1085/jgp.201311118. Epub 2014 Mar 17.
Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca(2+) concentration ([Ca(2+)]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca(2+)]i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today's myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.
纳米测量技术在生物科学中被广泛应用，用于分析细胞内和体内分子或分子组装体的运动。在心肌中，肌节长度（SL）仅发生约100纳米的变化就会导致收缩性发生显著改变，这表明需要在高空间和时间分辨率下同时测量心肌细胞中的SL和细胞内钙离子浓度（[Ca(2+)]i）。为了在兴奋-收缩偶联过程中以纳米精度准确分析单个肌节的运动，我们将纳米测量技术应用于原代培养的新生大鼠心肌细胞。首先，我们通过在Z盘表达AcGFP，开发了一个用于同时进行单个肌节动力学和[Ca(2+)]i变化的纳米级分析的实验系统。我们发现，当今心肌研究中普遍使用的沿心肌细胞平均肌节长度的方法，由于顺序连接的肌节之间延长时间的叠加，导致肌节延长速度被显著低估。然后，我们发现用离子霉素处理后，新生心肌细胞在肌浆网功能受阻的部分激活状态下表现出自发性肌节振荡（细胞-SPOC），其波形特性与电场刺激下获得的波形特性无法区分。肌球蛋白激活剂omecamtiv mecarbil在细胞-SPOC期间显著增强了Z盘位移。最后，我们在SPOC的数学模型框架内解释了当前的实验结果。本实验系统在揭示各种条件下心肌细胞兴奋-收缩偶联过程中的单个肌节动力学方面具有广泛的应用可能性。
