Comparative Study Of Protein Synthesis,Autophagy And Regeneration In The Maintenance Of Fast/slow Muscle In Hindlimb Unloading Rats And Hibernating Ground Squirrels

Objective:Our previous studies found that calpain system had muscle-specific regulation in soleus muscle(SOL)and extensor digitorum longus muscle(EDL)of non-hibernating animals and hibernating animals Therefore,this study would focus on comparing the differential regulation mechanism of protein synthesis in slow muscle SOL and fast muscle EDL between hindlimb unloading rats and hibernating daurian ground squirrels(Spermophilus dauricus),and further clarify the difference of muscle protein turnover and muscle cell regeneration with protein synthesis in disuse atrophy of non-hibernating animals and anti-disuse atrophy of hibernating animals.Methods:The rats were divided into a control group(CON,n=8)and a hindlimb unloading group(HLU,n=8).The daurian ground squirrels(as known as ground squirrels)were divided into a summer active group(SA,n=8)and a hibernation group(HIB,n=8).The muscle fiber cross-sectional area(CSA)and satellite cell count were detected by frozen sections and immunofluorescence assay.The m RNA expression of key ribosomal biogenetic molecules(c-myc and IST1)was detected by RT-PCR.Detecting expression of anabolism-related proteins(positive regulatory proteins p-Akt,m TORC1,p-m TORC1,p-S6K1,p-4E-BP1 and negative regulatory proteins myostatin),autophagy(Beclin1,LC3 and P62)and regeneration marker proteins(Myo D and myogenin)by western Blot.The activity of cathepsin L was measured by immunoenzyme link assay.Results:(1)Compared with CON rats,muscle mass,muscle weight/body weight and CSA were significantly decreased in SOL and EDL of the HLU rats.Compared with SA ground squirrels,only muscle weight/body weight of ground squirrel in HIB group increased significantly,and there is no significant change in other indicators.(2)Compared with CON rats,the expressions of protein synthesis positive regulatory proteins P-Akt,m TORC1,P-m TORC1 and P-S6K1 in SOL of rats with hindlimb unloading were obviously decreased,but there was no obvious change in EDL.The expression of synthetic positive regulatory protein in SOL of ground squirrels in the HIB group was significantly lower than that in the SA group,While in EDL,the protein expression of P-Akt and P-m TORC1 was not visibly changed,while the protein expression of downstream P-S6K1 substrate increased significantly.(3)Compared with CON rats,the expression of synthetic negative regulatory protein myostatin in SOL of HLU rats had no significant change,but it was obviously lower in EDL muscle than that in CON rats.Compared with the SA group,the expression of myostatin in the HIB group had no obvious change in EDL,but decreased visibly in SOL muscle.(4)The increase of Beclin1 expression can promote autophagy(turnover),and the expression of Beclin 1 protein in EDL of hindlimb unloaded rats and SOL of hibernating ground squirrels increased obviously compared with their control group.Cathepsin L is another index to measure autophagy.Compared with CON,the expressions of cathepsin L in the SOL and EDL of rats have no significant changes,while the expression of cathepsin L in the SOL of hibernating group is significantly higher than that in SA group,but no change in EDL muscle compared with SA group.(5)Compared with CON,the muscle-derived factor Myo D and myogenin in SOL and EDL of the hindlimb unloading rats was down-regulated and the skeletal muscle fibrosis factor type ? collagen(collagen?)was up-regulated.Compared with the summer group,the fibrosis factor collagen? and myogenic factor myogenin in SOL of hibernating squirrel were significantly increased,but in EDL of hibernating squirrel,the fibrosis factor collagen?was significantly increased,while myogenic factor myogenin had no significantly difference.Conclusion:In the process of muscle atrophy in non-hibernating animals and anti-muscle atrophy in hibernating animals,protein synthesis,autophagy and regeneration signaling occur in slow/fast muscles in a muscle-specific way.The atrophy of SOL and EDL in rats after 14 days of hind limb unloading is related to the decrease of Akt-m TORC1 signaling,and the damaged SOL and EDL activation of myogenin in repairing damaged muscle fibers while was inhibited myostatin and skeletal muscle fibrosis occurred.The limited shrinkage of SOL and EDL during hibernation in Daurian ground squirrels is related to the balance of protein turnover.At the same time,satellite cells are not involved in maintaining the mass of fast and slow muscles of hibernating squirrels,and there is no obvious fibrosis.