The Role And Mechamism Of Isoproterenol On Myoblast Differetiation And Fusion

Background:Skeletal muscle is an important tissue of human body,accounting for 40%of human body weight.In addition to the classic function of skeletal muscle as an organ of movement,it is also an organ that is helpful for health.The metabolic ability and endocrine function of skeletal muscle have an important influence on insulin sensitivity and blood lipid level,some of which are the risk factors of life-style diabetes,hypertension,hyperlipidemia and obesity.Muscular dystrophy（MD）is a group of diseases that cause progressive weakness and loss of muscle mass,at least including Duchenne and Becker MD.Apart from intrinsic causes including abnormal genes that disturbs the production of proteins needed to shape healthy muscle,the autonomic dysfunction acting as a possibly extrinsic reason can give rise to pathological changes in MD.However,previous studies have primarily focused on the influence of excessive sympathetic nerve system（SNS）on heart structure and function in muscular atrophy with rare interest in its probable role in the progression of skeletal myopathy.Muscle satellite cells conduce to the need for physiological self-renewal and the repair of pathological injury.As one of pathological conditions,excessive SNS aggravated muscle weakness and loss of muscle mass,ascribing to activated beta1 adrenergic receptor（β₁-Ad R）,desensitizedβ₂-Ad R and diminished anabolism in skeletal muscle.However,the mechanism of these changes in satellite cells is still unclear.Objective:To determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion,and to clarify possible molecular mechanismsMethods:Using 2%horse serum（HS）plus high glucose（HG）-DMEM to induce C2C12 myoblast cell differentiation system were established.The distinct role of ISO on myoblast differentiation and fusion treated with different methods including single,interval single or continuous single,the changes in morphology of myotube were determined by My HC immunostaining.Myotube was defined as 3+nucleuses within a cellular structure in order to rule out myoblast cells undergoing mitosis.Two types muscle fibers including slow（slim-long）and fast（thick-short）myofibers were detected by real-time PCR using primers for My HC1,My HC2a,My HC2b and My HC2X.The PKA,AKT,p38MAPK,ERK1/2,NFATs and FOXO1 in C2C12myoblast cells were analyzed by Western blot.Nuclear translocation of FOXO1 and MEF2C were observed by using cellular immunofluorescence staining.Application with PKA inhibitor H-89 and activator N6 Bz-c AMP in vitro was helpful for understanding of ISO’effects and PKA alteration.Usingβ1-Ad R blocker CPG-20712A,we better identify the inhibitory effects of ISO on myoblast differentiation and fusion throughβ1-Ad R.ERK1/2 blocker PD98059 was used to evaluate the role of activating-ERK1/2 by ISO in changes of AKT,p38MAPK and FOXO1.Ad-NFATc1,Ad-NFATc2,Ad-sh NFATc3 and Ad-sh NFATc4 were used to confirm the relationship between NFATs and ISO-mediated C2C12 myoblast differentiation and fusion.Results:（1）β₁andβ₂-adrenergic receptors（Ad R）were expressed in C2C12myoblast cells.The differentiated satellite cells exhibited an increased expression ofβ₂-Ad R,as compared with the proliferating cells.Continuous single-dose ISO increasedβ₁-Ad R expression in C2C12 myoblast cells,accompanied by the increased phospho-β₂-Ad R levels while decreasingβ₂-Ad R levels.（2）Continuous exposure of isoprenaline（ISO）,aβ-Ad R agonist,delayed C2C12myoblast cell differentiation and myoblast fusion in time-and dose-dependent manner.ISO also increased short myotube numbers while decreasing long myotube numbers,consistent with the greater reduction in My HC1,My HC2a and My HC2x expression.（3）Continuous exposure of ISO gradually decreased the ratio of PKA RI/RII,and PKA RI activator efficiently reversed the ISO effect on C2C12 myoblast cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects.（4）Continuous single-dose ISO increased phospho-ERK1/2 levels,resulting in increasing phospho-β₂-Ad Rlevels while decreasingβ2-Ad R levels,and the specific effects could be abolished by ERK1/2 inhibitor.（5）Continuous exposure of ISO time-and dosage-dependently altered NFATc1-c4signaling during halting or diminishing myotube size,especially in reducing the levels of NFATc1/c2.Overexpression of NFATc1 or NFATc2,NFATc1 in particular,markedly abolished the inhibition of differentiation and myoblast fusion of myoblast cells by ISO,showing the recovery of myotube number and size characterized by more than 3 nuclei in either My HC or MEF2C positive myotubes,resulting in more strongly restoration of the inhibited type I muscle fiber by ISO,in a MEF2C dependent manner.By contrast,knockdown of NFATc4 by sh RNA in myoblast cells prominently increased number of My HC⁺or MEF2C⁺cells with 1～2 nuclei,causing fewer myotubes and smaller myotube size,triggering the further reduction in typeⅡmuscle fiber compared to ISO group in a MEF2C independent way.（6）Continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing p AKT,p-p38MAPK and p FOXO1 levels.Conversely,blockade of ERK1/2 signaling partially abrogated ISO’effects on AKT,p38MAPK and FOXO1signaling,which partially restored C2C12myoblast differentiation and myoblast fusion,leading to an increase in the numbers of medium myotube along with the increased expression of My HC1 and My HC2a.Conclusion:（1）Pattern of altered NFATs controlled the traits of myoblast differentiation and fusion,myotube size and muscle fiber specialization mediated by ISO,in MEF2C-dependent and independent way.（2）Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion,at least in part,through PKA-ERK1/2-FOXO1 signaling pathways,which were associated with the reducedβ₂-Ad R and increasedβ₁-Ad R levels.