Differential Regulation Mechanism In Hypertrophic Diaphragm And Atrophic Gastrocnemius Muscles In Hibernating Daurian Ground Squirrels

Background and objective: Skeletal muscle has a high degree of plasticity,which can adapt to changes in the environment.Skeletal muscle maintenance exhibits distinct muscle type specificity in hibernating mammals.The balance of protein metabolism regulates in the size of muscle fibers reflect the balance of protein metabolism,causing muscle hypertrophy when protein synthesis is greater than protein degradation,and muscle atrophy when protein degradation is greater than protein synthesis.In addition,when muscles are damaged,the regenerative potential is activated.Therefore,in this study,we selected two fast-type muscles(diaphragm and gastrocnemius muscle)of Daurian ground squirrels(Spermophilus dauricus)in the summer active and hibernating group,to detect whether differences in regulation of protein metabolism and muscle regeneration are involved in related to the opposite phenotypic adaptation mechanism of muscle hypertrophy and atrophy in Daurian ground squirrels.Methods: The diaphragm and gastrocnemius muscle of the summer active group(SA)and hibernation group(HIB)of Daurian ground squirrels were used as the research objects.The cross-sectional area of muscle fiber(CSA)and fiber type distribution were measured by freezing section technique and immunofluorescent analysis.The expression levels of protein related to calpain degradation pathway(calpain-1,calpain-2,calpastatin,desmin and troponin T),ubiquitin-proteasome pathway(Mu RF1 and atrogin-1),autophagy-lysosomal pathway(Beclin1 and LC3-II),Akt-m TOR signaling pathway(P-Akt,P-m TORC1,P-S6K1 and P-4E-BP1)and muscle regeneration(Myo D,myogenin and myostatin)were determined by western blot.Enzyme activity of chymotrypsin and trypsin was measured by enzyme activity assay.Results: The result of morphological showed that the muscle weight and muscle fiber cross-sectional area of the diaphragm muscle were increased significantly,while both of them were decreased significantly in the gastrocnemius muscle in the hibernation group compared with those in the summer active group.Besides,in the diaphragm,the proportion of MHC I,MHC IIb and MHC IIx muscle fibers increased significantly,while the proportion of MHC IIa muscle fibers was decreased significantly in hibernation group as compared with the summer active group.In the gastrocnemius muscle,the proportion of MHC I,MHC IIa,and MHC IIx muscle fibers was increased significantly,while the proportion of MHC IIb muscle fibers was decreased significantly in hibernation group as compared with the summer active group.In the protein degradation pathway,calpain-1,calpain-2,and desmin protein expression levels,as the marker molecules of the calpain pathway,were significantly reduced in the diaphragm,but significantly increased in the gastrocnemius muscle in hibernation group as compared with the summer active group.On the contrary,the expression level of calpastatin was increased significantly in the diaphragm,but reduced significantly in the gastrocnemius muscle in hibernation group as compared with the summer active group.Additionally,troponin T protein expression level showed no change in the diaphragm and gastrocnemius muscles between the hibernation and summer active group.The protein expression level of Mu RF1 and atrogin-1,as ubiquitin-proteasome pathway molecules markers,as well as the activities of chymotrypsin and trypsin,were all significantly reduced in the diaphragm and significantly increased in the gastrocnemius muscles in hibernation group as compared with the summer active group.Besides,the expression levels of autophagy-related proteins Beclin1 and LC3-II were significantly increased in the diaphragm and significantly decreased in the gastrocnemius muscles in hibernation group as compared with the summer active groups.In the protein synthesis pathway,the protein expression levels of P-Akt,P-m TORC1,P-S6K1,and P-4E-BP1 in the Akt-m TOR signaling pathway were significantly increased in the diaphragm and significantly decreased in the gastrocnemius muscle in hibernation group as compared with the summer active group.The expression levels of Myo D and myogenin the critical protein in muscle regeneration,were significantly increased in the diaphragm,and reduced significantly in the gastrocnemius muscle,while the protein expression level of myostatin was significantly decreased in the diaphragm and significantly increased in the gastrocnemius muscle in hibernation group as compared with the summer active group.Conclusions: The mass of diaphragm increased and the mass of gastrocnemius decreased in hibernating ground squirrels.Moreover,both the diaphragm and gastrocnemius muscles displayed a significant fast-to-slow fiber-type transition in hibernation,indicating that not all skeletal muscle mass remains unchanged in ground squirrels during hibernation,with different types of skeletal muscles showing different adaptive characteristics and mechanisms.Our study further indicated that increased protein synthesis,decreased protein degradation,and increased muscle regeneration potential contributed to diaphragm muscle hypertrophy,whereas decreased protein synthesis,increased protein degradation,and decreased muscle regeneration potential are involved in the mechanism of gastrocnemius muscle atrophy.In conclusion,the differences in muscle regeneration and regulatory pattern of protein metabolism may contribute to the different adaptive changes observed in the diaphragm and gastrocnemius muscles of Daurian ground squirrels.