Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z discs.
Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z discs.
复制标题
DOI：
10.1085/jgp.201311118
复制
发表时间：
2014-04
期刊：
影响因子：
--
通讯作者：
Fukuda N
中科院分区：
文献类型：
--
作者：
Shintani SA;Oyama K;Kobirumaki-Shimozawa F;Ohki T;Ishiwata S;Fukuda N
培养心肌细胞的纳米级成像可以定量评估收缩事件期间单个肌节长度的变化。纳米测量学广泛应用于生物科学中，用于分析细胞和体内分子或分子组装体的运动。在心肌中，肌节长度 (SL) 仅约 100 nm 的变化就会引起收缩力的显着变化，这表明需要以高空间和时间分辨率同时测量心肌细胞中的 SL 和细胞内 Ca2+ 浓度 ([Ca2+]i)。为了在兴奋-收缩耦合过程中以纳米精度准确分析单个肌节的运动，我们将纳米技术应用于原代培养的大鼠新生心肌细胞。首先，我们开发了一个实验系统，通过 Z 盘中 AcGFP 的表达，对单个肌节动力学和 [Ca2+]i 变化进行同步纳米级分析。我们发现，当今心肌研究中普遍使用的一种方法是沿肌细胞的肌节长度平均，由于顺序连接的肌节之间不同延长时间的叠加，导致肌节延长速度明显低估。然后，我们发现用离子霉素处理后，新生肌细胞在部分激活时表现出自发性肌节振荡（cell-SPOC），并阻断肌浆网功能，并且波形特性与电场刺激中获得的波形特性没有区别。肌球蛋白激活剂 omecamtiv mecarbil 显着增强了细胞 SPOC 期间的 Z 盘位移。最后，我们在 SPOC 数学模型的框架中解释了目前的实验结果。本实验系统具有广泛的应用可能性，可揭示各种设置下心肌细胞兴奋-收缩耦合过程中的单个肌节动力学。
Nanoscale imaging of cultured cardiomyocytes allows the quantitative assessment of changes in the length of single sarcomeres during contractile events. 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 Ca2+ concentration ([Ca2+]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 [Ca2+]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.
登录
查看更多内容
DOI：
10.1073/pnas.1210718109
发表时间：
2012-08-07
影响因子：
11.1
作者：
Drago, Ilaria;De Stefani, Diego;Pozzan, Tullio
通讯作者：
Pozzan, Tullio
影响因子：
5.6
作者：
Luther, Pradeep K.;Bennett, Pauline M.;Moss, Richard L.
通讯作者：
Moss, Richard L.
影响因子：
3.6
作者：
Boulesteix, T;Beaurepaire, E;Schanne-Klein, MC
通讯作者：
Schanne-Klein, MC
影响因子：
6.1
作者：
Kojima, K;Moriguchi, H;Yasuda, K
通讯作者：
Yasuda, K
影响因子：
5.2
作者：
Jacot JG;Kita-Matsuo H;Wei KA;Chen HS;Omens JH;Mercola M;McCulloch AD
通讯作者：
McCulloch AD