Calcium-dependent Chloride Channel-TMEM16A Expression In Mouse Skeletal Muscle Development

Chloride channels play important roles in diverse processes,such as in maintaining osmotic balance inside and outside cells,regulating the excitability of nerves and muscles.previous studies have found that abnormal expression of chloride channels with directly relation to a number of important human diseases such as Huntington's disease,myotonia,asthma,the occurrence of tumors,cystic fibrosis and so on.Transmembrane protein 16(TMEM16)as a newly discovered family of chloride channels,including TMEM16A,TMEM16B and other 10 members,of which TMEM16A widely expressed in mammals,and has important physiological functions,such as regulating the secretion of epithelial cells,smooth muscle cells contraction.However TMEM16A expression and functional status in skeletal muscle has not yet been reported.This paper systematically studied the expression of TMEM16A during the development of mouse skeletal muscle,so as to provide a basis for the study of its function in skeletal muscle.Vertebrate embryonic muscle formation consists of two phases,the starting of the muscle formation is generally referred to as "primary myogenesis",which approximately in mouse embryonic 10.5 days(E10.5)when skeletal muscle progenitor cells begin to form;Secondary muscle fiber formation is mainly for the differentiation,proliferation,migration and integration to myotubes of muscle cells,etc.,the beginning of this process in mouse embryonic day 14(E14).postnatal,the number of mouse skeletal muscle fibers remained stable,and the development is mainly including the mature of muscle fibers and the increased of volume.Firstly,in vitro,we studied the expression of TMEM16A during the process of differentiation of mouse myoblast cell line C2C12.The result show that,with the differentiation of induced,the relative expression of TMEM16A at protein and mRNA levels are both significantly elevated,indicating that it has correlation with the state of muscle differentiation.During mouse development,the expression of TMEM16A in skeletal muscle also showed dynamic changes,during E11.5,E14.5 and E17.5,high protein expression of TMEM16A is detected in mouse hind limb muscles;postnatal meanwhile,the expression of TMEM16A in tibialis anterior muscle is showing a declining trend.In embryonic development,the expression level of mRNA is consistent to that of protein.after birth remains high RNA expression level and protein level was significantly reduced.Immunofluorescence studies further confirmed that TMEM16A expression in muscle fibers.The maturated mouse muscle contains fast and slow muscle fibers,all muscle combining two different ratio of them accordingly called fast and slow muscle.We find that the protein expression of TMEM16A in slow muscle of mice is significantly higher than of fast-twitch.The high level expression of TMEM16A during the development of skeletal muscle suggests that it may have important functions.We use knockout mice to conduct a preliminary study and find that mice lacking TMEM16A gene,induced a serious shortage of skeletal muscle development,perform as very thin muscles.The separation of muscle cells and then induce to differentiate in vitro process find that the TMEM16A gene deletion myoblast differentiation myotubes significantly smaller than the wild type,differentiation index is also significantly lower than that of wild type.In summary,this paper through a series of studies have found that,TMEM16A in C2C12 cells,in vivo mouse skeletal muscle development process as well as in vivo knockout mice all exhibit association with the development and differentiation of skeletal muscle,which suggesting that TMEM16A may plays an important role in mouse embryonic skeletal muscle cells proliferation and the subsequently differentiation and development,in the process,the specific molecular mechanisms involved remain to be further studied.