The Effect Of Calcium-dependent Chloride Channel-TMEM16A On The Proliferation And Differentiation Of Mouse Skeletal Muscle

Excitable cells such as skeletal muscle cells in adult,change of membrane potential and membrane polarization state is the important foundation of cells excitation/contraction coupling.Membrane potential is also involved in cell proliferation capacity,which is lower in proliferating cells.Chloride ions have been considered the most important anion in membrane potential maintainence.So far,a variety of chloride channels including ClC-1,CFTR,ClC-2,ClC-3,Bestrophines,TMEM16A,and TMEM16B have been discovered.Our previous studies suggest that TMEM16A may play critical roles in regulation of cell membrane depolarization during mouse skeletal muscle development.TMEM16A(Transmembrane protein 16 A,also called Anol)is the first determined calcium activated chloride channel(CaCCs)molecule,which is widely expressed in various tissues from Xenopus to the humans.TMEM16A has been reported to mediate chloride ions outflow and subsequent cell depolarization.To investigate the founctions of TMEM16A in the regulation of skeletal muscle development,we detected the effects of T16Ainh-A01,the only TMEM16A specific inhibitor(IC50=1.8 ?M),on the proliferation of myoblast C2C12 cells by using of MTT assay.The results showed that the proliferation level of inhibitor treated cells was obviously higher than that of control cells(25.1%increase on average).The effect of T16Ainh-A01 on cell proliferation was further determined by EdU(5-ethynyl-2'-deoxyuridine)incoporation analysis.To confirm the role T16Ainh-A01 played in skeletal development,we isolated and cultured primary myoblasts from mouse muscle,and similar proliferation promoting effect was observed in T16Ainh-A01 treated cells.We next explored the effects of T16Ainh-A01 on myogenesis in vitro.At concentration of 20?M,it apparently suppressed myogenic differentiation,indicated by thinner and shorter myotubes,as well as lower differentiation index than the control cells(fusion index 45.8%decrease;myofiber length 42.3%decrease;myofiber diameter 31.6%decrease).This paper also showed that a general chloride channels blocker,niflumic acid(NFA)(IC50=7.2 ?M),has a weaker inhibitory effect on the myogenesis process than that of T16Ainh-A01.The above data suggest that TMEM16A may play critical roles in skeletal muscle development through inhibition of proliferation and promotion of myogenic differentiation of myoblasts.As to the under mechanisms,our preliminary data showed that p-ERK1/2 level was higher in TMEM16A over expression C2C12 cells,which was reported to attributed to myogenesis.Since we have found that TMEM16A abundantly expressed in the adult mouse skeletal muscle,we are interested in its physiological founction.So we next detected the expression and distribution characteristics of TMEM16A in skeletal muscle.By immunofluorescence and Western Blotting analysis,we found that from 5 weeks age old to 9 weeks,the location of TMEM16A protein gradually shift from mainly in the cytoplasm to cell membranes-In addition,the protein level was apparently higher in mixture muscle in the tibialis anterior muscle(TA),which is composed of both fast-twitch fiber and slow-twitch fiber,than in the quadriceps(QUA)that mainly contained fast-twitch fibers.Due to the fast-twitch muscle fibers distinguished from slow-twitch fibers is that the latter contains a much more bigger amount of mitochondria than the former,we speculated that TMEM16A may locate on the mitochondria in the cytoplasm.However,no obvious TMEM16A distribution in mitochondria was detected by subcellular fractionation analysis of skeletal muscle cell.In summary,we show here that T16Ainh-A01,the TMEM16A specific inhibitor,'promots myoblast proliferation and inhibit its muscle differentiation,suggesting that the change of membrane potential and deplorization in myoblast cells results from TMEM16A mediated chloride ions flow inside and outside of cell is a crucial regulatory factor in skeletal development.The characterize of spatial distribution and expression level of TMEM16A in skeletal muscle fibers during different development period,suggests it may have a variety of functions in skeletal muscle.