Time-dependent Changes Of MyoD And Clock Gene BMAL1 In Skeletal Muscle Of EIMD Rats

Object: The immunofluorescence co-localization of MyoD,a myogenic regulator,and BMAL1,a clock gene,was observed before and after exercise-induced skeletal muscle damage in rats,and the expression of BMAL1 and MyoD mRNA genes at different times was analyzed to explore the relationship between the two,as well as the changes in rhythmic oscillations between BMAL1 and MyoD after skeletal muscle injury,thus providing a new basis for the mechanism by which clock genes are involved in regulating exercise-induced skeletal muscle damage and repair processes.Methods: A total of 208 8-week-old male Sprague-Dawley rats were randomly divided into group C(control group)and group M(model group).Samples were collected every6 hours for 13 time periods(specific groups were as follows,group C: C0h-C72h;group M: M0h-M72h)at 0h-72 h after modeling by one-time high-load downhill centrifugal exercise.The gross condition of the experimental rats was observed,the ultrastructure of skeletal muscle was observed by transmission electron microscopy,the serum CK-MM level of the experimental rats was detected by ELISA,the expression of BMAL1 and MyoD genes in skeletal muscle was detected by PCR,and the co-expression of BMAL1 and MyoD was observed by immunofluorescence.The parameters of the fitted cosine curve were obtained using the cosine analysis software circacompare(R package),and the obtained data were statistically analyzed using graphpadprism8.0 statistical software.Results: 1.Gross observation: After skeletal muscle injury modeling,the rats in group M had arrested movement,decreased exercise capacity,shortness of breath and increased body temperature.2.Ultrastructural observation: The ultrastructure of skeletal muscle at 0h-72 h after model establishment was damaged to different extents:manifested as myofibril and mitochondrial disarrangement,and mitochondrial swelling.Afterwards,it showed that the injury tended to recover with time to 72 hours,which tended to be closer to group C.3.CK-MM content: The serum CK-MM concentration in group M increased immediately after exercise,and then gradually approached that in group C.At 0 h,12 h and 24 h after exercise,the serum CK-MM concentration in group M was significantly increased compared with that in group C,and there was no significant difference between the two groups thereafter.4.Immunofluorescence co-localization observation: The co-localization of BMAL1 and MyoD can be obviously observed in the control model at 12 hours,a small amount of co-localization markers can still be observed at 24 hours,and there is no significant change in other phases.5.Real-time PCR observation: In the control group,BMAL1 gene expression showed rhythmic expression,showing a total of three complete rhythmic cycles within72 hours,and the trend of each cycle first decreased and then increased;MyoD gene expression also showed rhythmic expression,showing a total of three complete rhythmic cycles within 72 hours,and the trend of each cycle first decreased,then increased and then decreased.In the model group,the expression of BMAL1 gene lost its rhythmicity in 72 hours.During the analysis on each day,it was found that the rhythm showed a disordered state on the first day,the rhythm had recovered in the second day,and the expression trend had been similar to that in the control group on the third day;MyoD gene was still rhythmically expressed in the model group at 72 hours,and it was found during the analysis on each day that it also showed a disordered state on the first day,the rhythm gradually recovered in the second day,and there was no significant difference in the rhythm between the model group and the control group on the third day.Conclusion: 1.the clock gene BMAL1 colocalizes with the myogenic regulator MyoD in exercise-induced skeletal muscle damage and repair.2.The gene expression of BMAL1 and MyoD showed time-acting rhythmic oscillations in the normal state,and their expression appeared disordered after heavy load exercise,and the trend was similar.3.The rhythmic changes of MyoD gene are disturbed during exercise-induced skeletal muscle damage and can return to normal state over time,and the mechanism is related to the regulation of MyoD by BMAL1.