| BackgroundSkeletal muscle will undergo loss and atrophy with age and pathological conditions,but with this process,skeletal muscle satellite cells will proliferate and differentiate to realize the self-renewal of skeletal muscle under physiological conditions and the repair process under pathological conditions.In fact,the regeneration of human skeletal muscle fibers begins with the activation of muscle satellite cells named by myoblasts,and it forms new muscle fibers through the process of proliferation,migration and fusion,however,potential mechanism is unknown.Ezrin protein is widely exist in actin protein on the surface of the plasma membrane and actin cytoskeleton plays a role of connection between the actin microfilament cells connected to the cell membrane,adjusting the shape,movement and adhesion of cells,which takes part in multiple cell process such as tumor cell migration,signal transduction,mitosis,and other important function.Previous studies have shown that Ezrin protein is associated with muscle weakness,muscular dystrophy,muscular dystrophy,and polymyositis.But whether Ezrin is involved in function regultaiton of muscle satellite cells maintain unknown.ObjectiveTo explore the effects of Ezrin on the recruitment,migration,differentiation,myotube fusion and muscle fiber type of skeletal muscle satellite cells;to observe relation between Ezrin-mediated muscle satellite cells differentiation and PKA;to confirm association mechanism between ezrin-mediated changes in NFAT activity and differentiation characteristics of muscle satellite cells.MethodsThe C2C12 cells were used as experimental materials,and the high glucose medium containing 2%horse serum was established to induce their differentiation.After 2,4 and 6 days of culture,MyHC immunofluorescence staining was used to observe the degree of myotubule fusion and the changes of the morphology and number of myotubule.The expression of Ezrin and Ezrin in differentiated and proliferated C2C12 myoblast cells were detected by Western blot and immunofluorescence at day 2,4 and 6.Ad-Ezrin Ad-sh Ezrin were added into culture medium 12 hours before cells differentiation.After 2,4and 6 days of culture,Myosin Skeletal Heavy chain(MyHC)immunofluorescence staining was used to observe the changes of C2C12myoblast cells differentaiotn.On the 6th day of differentiation,MyoG and MEF2C immunofluorescence staining were used to explore the role of Ezrin in the differentiation of C2C12 myoblast cells.MY32(a special antibody for fast MyHC)and NOQ7.5.4D(a special antibody for slow MyHC)immunofluorescence staining were performed on C2C12 myoblast cells transfected with overexpression and knockdown of Ezrin for 6 days.Real-time quantitative PCR was used to detect the m RNA changes of MyHC1,MyHC2A,MyHC2B and MyHC2X,and to analyze the change characteristics of slow(elongated)and fast(short and thick)muscle fibers.The m RNA changes of myogenetic regulatory factors MyOD and MYF-5 were detected.The levels of PKAγ,PKAαCAT,PKAα/β/γCAT,PKAREG Iα,PKAREG Iβ,PKAREG Iα,NFATc1,NFATc2,NFATc3 and NFATc4 were detected by Western blot.PKA inhibitor H-89 and PKA agonist N~6-BZ-c AMP were used to investigate the relation between Ezrin-mediated muscle satellite cells differentiation and PKA.And Ad-NFATc1,Ad-NFATc2,Ad-sh NFATc3 and Ad-sh NFATc4 were used to confirm association mechanism between Ezrin-mediated changes in NFAT activity and differentiation characteristics of muscle satellite cells.Results(1)Ezrin expressions were obsviouly increased in both proliferation and differentiation of C2C12 myoblast cells.The expression of Ezrin increased gradually and decreased at the end of myotube differentiation near maturity.(2)Ad-Ezrin promoted myoblast differentiation and myotuble fusion,resultsing in the increased length of myotubules,and the increased number of myotubules,which was consistent with the increase in MyHC-1 expression,in myotubule numbers with more than 3 nuclei with MyoG and MEF2C positive.Ad-sh-Ezrin significantly inhibited the differentiation of C2C12 myoblasts.(3)Ad-sh Ezrin significantly promoted the differentiation and formation of myoblast II myofiber,but Ad-Ezrin inhibited the differentiation and formation of myoblast II myofiber.(4)PKA inhibitor H-89 can inhibit the differentiation of C2C12 myoblast cells treated with Ad-Ezrin.PKA agonist 8-bro-c AMP can effectively reverse the effect of Ad-sh Ezrin on the differentiation and fusion of C2C12 myoblast cells.(5)Ad-Ezrin treatment increased the NFATc1 and NFATc2 expression levels in differentiating C2C12 myoblasts,and Ad-sh Ezrin increased the NFATC3 and NFATC4 expression levels during C2C12 myoblast differentiation.(6)The inhibitory effect of Ad-sh Ezrin on differentiation of myoblasts distinguished by MyHC,MEF2C and MyoG could be reversed by overexpression of NFATc2 and knockdown of NFATc4 by sh RNA.Conclusions(1)Ezrin-mediated differentiation of myoblast cells and fusion of myoducts are related to PKA-MyoG/MEF2C signaling pathway.(2)NFATc2/c4-MyoG/MEF2C were associated with Ezrin-mediated differentiation of myoblasts and myotube fusion. |
