Optogenetic induction of contractile ability in immature C2C12 myotubes.
Optogenetic induction of contractile ability in immature C2C12 myotubes.
复制标题
DOI：
10.1038/srep08317
复制
发表时间：
2015-02-09
影响因子：
4.6
通讯作者：
Yawo H
中科院分区：
文献类型：
--
作者：
Asano T;Ishizuka T;Morishima K;Yawo H
成肌细胞可以分化为多核肌管，为体外骨骼肌发生提供了一个良好的、可重复的肌细胞模型。然而，在常规分化条件下，每个肌管很少表现出强大的收缩以及肌节排列。在这里，我们应用列车的光学刺激（OS）C2C12肌管，这是基因工程表达的通道视紫红质变体，通道视紫红质绿色接收器（ChRGR），以调查是否膜去极化促进成熟的肌管。我们发现，光脉冲诱导膜去极化和诱发的动作电位在ChRGR表达肌管。肌节蛋白的规则排列在OS训练后周期性地形成图案。相比之下，未经训练的控制肌管很少表现出条纹图案。OS训练和未经训练的肌管在静息电位方面也有所不同。OS训练显著增加收缩肌管的数量。硝苯地平治疗OS训练过程中显着降低收缩肌管的分数，而河豚毒素是不太有效。这些结果表明，在生肌过程中，膜电位和细胞内Ca 2+的振荡伴随OS促进了肌小节的组装和收缩性的发展。这些结果还表明，光遗传学技术可用于操纵肌源性发育过程中的活性依赖性过程。
Myoblasts can be differentiated into multinucleated myotubes, which provide a well-established and reproducible muscle cell model for skeletal myogenesis in vitro. However, under conventional differentiation conditions, each myotube rarely exhibits robust contraction as well as sarcomere arrangement. Here, we applied trains of optical stimulation (OS) to C2C12 myotubes, which were genetically engineered to express a channelrhodopsin variant, channelrhodopsin-green receiver (ChRGR), to investigate whether membrane depolarization facilitates the maturation of myotubes. We found that light pulses induced membrane depolarization and evoked action potentials in ChRGR-expressing myotubes. Regular alignments of sarcomeric proteins were patterned periodically after OS training. In contrast, untrained control myotubes rarely exhibited the striated patterns. OS-trained and untrained myotubes also differed in terms of their resting potential. OS training significantly increased the number of contractile myotubes. Treatment with nifedipine during OS training significantly decreased the fraction of contractile myotubes, whereas tetrodotoxin was less effective. These results suggest that oscillations of membrane potential and intracellular Ca2+ accompanied by OS promoted sarcomere assembly and the development of contractility during the myogenic process. These results also suggest that optogenetic techniques could be used to manipulate the activity-dependent process during myogenic development.