Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes
Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes
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DOI：
10.1016/j.yexcr.2007.03.002
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发表时间：
2007-05-15
影响因子：
3.7
通讯作者：
Kanzaki, Makoto
中科院分区：
文献类型：
--
作者：
Fujita, Hideaki;Nedachi, Taku;Kanzaki, Makoto
肌节是横纹肌中最小的收缩单位，其组装是一个复杂且高度协调的过程，其依赖于肌节蛋白的时空组织，该过程需要自发的Ca 2+瞬变。为了研究C2 C12肌管中Ca 2+瞬变和肌节组装之间的关系，我们采用电脉冲刺激（EPS），其允许操纵Ca 2+瞬变的频率。我们监测收缩活动的一种手段，评估功能肌节建立差分图像减法（DIS）的方法。由于缺乏肌节结构，C2 C12肌管最初没有显示出与EPS的收缩性。然而，C2 C12肌管表现出显着的收缩活动与EPS诱导的重复Ca 2+瞬变（1 Hz）仅在2 h内。这种活动与肌节结构的发育是同步的。重要的是，所需的时间为收购的收缩活动，以响应激励依赖于钙振荡的频率，但持续增加细胞内钙（不振荡）的高频EPS（10赫兹）是无法赋予收缩或肌节组装的肌管。EPS促进的从头功能肌节组装似乎需要钙蛋白酶介导的蛋白水解。此外，通过加入IV型胶原或RGD肽调节整合素信号，显著影响了EPS诱导的收缩性的发展。两者合计，这些观察结果表明，Ca 2+振荡的频率决定了建立功能活性肌节组装所需的时间，也表明Ca 2+振荡信号可以通过钙蛋白酶介导的蛋白水解整合素-细胞骨架蛋白复合物的重组来解码。(c)2007爱思唯尔公司All rights reserved.
The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca2+ transients. To investigate the relationship between Ca2+ transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca2+ transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca2+ transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca2+ oscillations, but a sustained increase in intracellular Ca2+ (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca2+ oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca2+ oscillatory signal may be decoded through reorganization of the integrin-cytoskeletal protein complex via calpain-mediated proteolysis. (c) 2007 Elsevier Inc. All rights reserved.