Studies On The Developmental Regulation Of Mouse Skeletal Myoblast Differentiation By TMEM16A

TMEM16A(Transmembrane Protein 16A)is the molecular basis of Ca2--dependent chloride channels(CaCCs).It is widely expressed in the body and has important physiological functions,such as regulating smooth muscle contraction and epithelial cell secretion.So far,the function of TMEM16A has not been reported in skeletal muscle.Our previous study found that TMEM16A has important regulatory functions for the differentiation and development of mouse skeletal muscle,but the specific mechanism is not clear.We first studied the localization of TMEM16A in skeletal muscle myoblasts.After overexpression of TMEM16A in myoblasts.it was found found that they were mainly distributed in the cytoplasm,and after induction of differentiation into myotubes,TMEM16A was mostly located on the cell membrane.Subcellular localization studies revealed that TMEM16A is colocalized with endoplasmic reticulum,lysosomes,and endosomes in myoblasts.In the freshly isolated mouse skeletal muscle fibers,TMEM16A and Actinin were distributed alternately.The above results suggest that TMEM16A may play a role in the proliferation and differentiation of skeletal muscle cells.In this paper,the cell proliferation was analyzed by MTT assay.The results showed that the deletion of TMEM16A promoted the proliferation of mouse primary myoblast.Further studies found that the expression of cyclin D1 and p-ERK1/2 in TMEM16A-inhibited or TMEM16A-deficient mouse myoblasts significantly increased,while p21 decreased.Our previous studies have found that muscle differentiation is inhibited after loss of TMEM 16A.In the present study,we further found that p-ERK1/2 and activated caspase 3 were significantly higher after induction of differentiation in vitro in TMEM16A-deficient primary mouse myoblasts,which is suggested that both them may be involved in the regulation of skeletal muscle differentiation by TMEM16A.In this article,we treated with p-ERK1/2 and caspase inhibitors respectively,and found that the muscle differentiation of primary myoblasts in TMEM16A-deficient mice was significantly improved.In summary,our article found that in myoblasts,TMEM16A is located in the endoplasmic reticulum,lysosomes and intracellular bodies;In mouse skeletal muscle fibers,TMEM16A may be located on the T-tube.TMEM16A regulates myoblast proliferation through ERK1/2,cyclin D1 and p21,and regulates skeletal muscle differentiation by decreasing the activation of p-ERK1/2 and caspase 3.The results of this study preliminarily revealed the mechanism by which TMEM16A regulates the differentiation and development of skeletal muscle.