Relationship And Regulation Of Skeletal Muscle Differentiation By Circadian Clock Gene

Objective:All tissues of the body posses a functional circadian clock.The core clock mechanism eligible for generation of the rhythmicity within the SCN and peripheral rhythmic cells is formed by interactive transcriptional–translational feedback loops between the clock genes,including Bmal1,Clock,Per,Cry,Rev-erb? and ROR?.Skeletal muscle,similar to almost every cell of the body,has circadian rhythms.Recent studies suggest that circadian clocks could play a more important role in developmental and regenerative processes of skeletal muscle.However,the mechanisms involved in circadian clocks of during skeletal muscle development are not fully understood.Disruption of the circadian rhythms are associated with many diseases,such as cardiovascular disease,cancer.Circadian misalignment can be detrimental to skeletal muscle health.Increased levels of circulatory lipopolysaccharide(LPS)are observed in above patients.Critical unanswered questions are how skeletal muscle are affected.In this study,RT-PCR and Western blot were used to observe the spatio-temporal expression characteristics of circadian clock genes in mouse gestational age whole embryos and differentiating C2C12 myoblasts.At the same time,to examine what role Bmal1,Clock and MyoD play in the development of skeletal muscle.In addition,we used C2C12 myoblasts to investigate the effects of LPS on myogenesis.The results will present the possible interrelationship between circadian clock genes and skeletal muscle decay.Methods:1.Total RNA was extracted using TRIZol from E10~E17 whole embryos and differentiating C2C12 myoblasts.Bmal1,Clock,Per1,Cry1,Rev-erb?,MyoD,myogenin,Tcap and MAZ mRNA were examined by RT-PCR.2.Western blot was used to investigate the expression of BMAL1,CLOCK and MYOD in differentiating C2C12 myoblasts.3.The relationship of Bmal1,Clock and MyoD are analyzed by luciferase reporter assay.4.C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS(0.1 or 1?g/mL).Expression of Bmal1,Clock,Per1,Cry1,Rev-erb?,MyoD,myogenin,myostain,Tcap and MAZ mRNA were measured using RT-PCR.Western blot was used to examine the expression of BMAL1,CLOCK and MYOD at 48 h,96h,and 144 h.Results:1.The expression of Bmal1,Clock and Per1 mRNA in mouse whole embryo in the early developmental stages were lower.With development,the expression of these three circadian clock genes were increased,especially Per1.At later developmental stages,the expression of Bmal1 and Clock were slightly increased.Remarkably,the expression of Per1 at E17 was more than three times of that of E15.Cry1 was expressed at very slightly higher level than Rev-erb? that was weakly expressed at baseline level.The weakly expression of Rev-erb? might have the potential to promote skeletal muscle development.Per1 functions could be a mechanism to prevent circadian molecular rhythms in embryos and promote organs development including skeletal muscle.With the development of the embryo,the expression intensity of MyoD and myogenin were gradually increased.The expression of myogenin increased quickly from E12 and got a peak at E17.The level of MyoD reached small peaks at E15 and decreased gradually.In addition,the level of Tcap expression at E15 synchronously increased.These data suggest that sarcomeres in muscle develop rapidly.The expression of MAZ maintained a higher level and sustained increased in embryos.MAZ may play a role in positive transcriptional regulation,concern with the development and differentiation of skeletal muscle.2.With differentiation inducement of C2C12 myoblasts,more and more myotubes appeared,and the expression of Bmal1 and Clock were synchronously increased.At the same time,the expression of Cry1 was up-regulated and reached small peaks at 120 h,then decreased gradually.Per1 and Rev-erb? were very weakly expressed.The expression intensity of MyoD and myogenin were gradually increased,specially myogenin.Its mean that C2C12 myoblasts are optimal model for research on myogenic differentiation.Using Western blot analysis,the protein level of BMAL1 and CLOCK were highly expressed in C2C12 myoblasts and differentiating C2C12 myoblasts.The process of skeletal muscle development is complicated,in which too many factors are involved with.BMAL1 and CLOCK may be necessary for active of myogenic regulatory factor.It's interesting that theband of MYOD did not achieve statistical significance.MyoD is a key regulator of myogenesis.It may help differentiation of myogenic precursors to become mature multinucleated myotubes.3.By luciferase reporter assay,we found that BMAL1/CLOCK and MYOD work in synergistic fashion to regulate the expression of Tcap(Titin-cap).4.C2C12 myoblasts were cultured in DM for 144 h in the absence or presence of0.1?g/mL or 1?g/mL LPS.LPS treatment time-dependently down-regulated the expression of circadian clock genes,especially Bmal1 and Clock.Per1 and Cry1 decreased slightly.Rev-erb? expression was initially reduced with LPS treatment for 24 h,and then decreased in time-dependent manner.But the mRNA expression of the circadian clock genes did not show dose-dependent between 0.1 and 1?g/mL LPS.Western blot analysis,the protein level of BMAL1 and CLOCK were highly expressed at 48 h,96h,and 144 h in the presence of 0.1or 1?g/mL LPS.Higher LPS concentrations(1?g/mL)did not further increase the darkness of band.It did not show dose-dependent between 0.1 and 1?g/mL LPS.5.In LPS-treated C2C12 myoblasts,the expression of MyoD and myogenin were lower.And LPS treatment for 120 h,the expression of MyoD and myogenin decreased significantly.Tcap,one of the titin interacting Z-disc proteins,involved regulation and development of normal sarcomeric structure.In LPS-treated C2C12 myoblasts for 72 h,the expression of Tcap remained relatively lower and stable.Its mean that LPS inhibits sarcomere differentiation.The expression of MAZ was down-regulated after LPS treatment for 24 h.After LPS(1?g/mL)treatment for 120 h,the expression of myostain was significantly up-regulated.myostain may be involved in inhibiting skeletal muscle cell differentiation.Conclusion:1.The intrinsic circadian clock regulates the timing of genes involved in the development of skeletal muscle.Bmal1,Clock and MyoD are highly expressed in the skeletal muscle and have a critical role in regulation of myogenic differentiation.2.Bmal1 potently activates myogenic regulatory factors(MRF)transcription.3.The low level of Rev-erb? and higher activity of Per1 may have the potential to promote the development of skeletal muscle and other organ.There are abundant potential signals from maternal rhythms but the circadian clock gene in whole embryo are insensitive to them.The higher activity of Per1 may suppress the biological time in thewhole embryos.4.LPS time-dependently suppressed the formation of multinucleated myotubes.But the expression of BMAL1 and CLOCK are unchanged.Maybe higher BMAL1 and CLOCK concentrations can protect and rescue the impairment of myogenesis.