Optogenetic induction of contractile ability in immature C2C12 myotubes

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...

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Main Authors:	Asano, Toshifumi, Ishizuka, Toru, Morishima, Keisuke, Yawo, Hiromu
Format:	Online
Language:	English
Published:	Nature Publishing Group 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650824/

id	pubmed-4650824
recordtype	oai_dc
spelling	pubmed-46508242015-11-24 Optogenetic induction of contractile ability in immature C2C12 myotubes Asano, Toshifumi Ishizuka, Toru Morishima, Keisuke Yawo, Hiromu Article 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. Nature Publishing Group 2015-02-09 /pmc/articles/PMC4650824/ /pubmed/25661648 http://dx.doi.org/10.1038/srep08317 Text en Copyright © 2015, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Asano, Toshifumi Ishizuka, Toru Morishima, Keisuke Yawo, Hiromu
spellingShingle	Asano, Toshifumi Ishizuka, Toru Morishima, Keisuke Yawo, Hiromu Optogenetic induction of contractile ability in immature C2C12 myotubes
author_facet	Asano, Toshifumi Ishizuka, Toru Morishima, Keisuke Yawo, Hiromu
author_sort	Asano, Toshifumi
title	Optogenetic induction of contractile ability in immature C2C12 myotubes
title_short	Optogenetic induction of contractile ability in immature C2C12 myotubes
title_full	Optogenetic induction of contractile ability in immature C2C12 myotubes
title_fullStr	Optogenetic induction of contractile ability in immature C2C12 myotubes
title_full_unstemmed	Optogenetic induction of contractile ability in immature C2C12 myotubes
title_sort	optogenetic induction of contractile ability in immature c2c12 myotubes
description	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.
publisher	Nature Publishing Group
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650824/
_version_	1613503250238013440

Optogenetic induction of contractile ability in immature C2C12 myotubes

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