Study On The Relationship Between Calpain System And Skeletal Muscle Mass Maintenance Mechanism Of Hibernating Daurian Ground Squirrels(Spermophilus Dauricus)

Objectives: Our previous study found that no significant atrophy was observed in soleus muscle(typical slow-twitch muscle,SOL)during hibernation,which is associated with calpain system.Here,we investigated calpain-dependent regulation of anti-atrophy mechanisms in different fast skeletal muscles(lateral gastrocnemius,adductor magnus,plantaris longus and extensor digitorum longus)of Daurian ground squirrels(spermophilus dauricus).Methods:The protein expressions of MuRF1,calpastatin,calpain 1,calpain 2,desmin,troponin T and C were determined by western blots.Cross sectional area(CSA)and fiber type distribution were detected by immunofluorescence.The mRNA expression of atrogin-1 and calpain 2 was investigated by quantitative real-time PCR.Lateral gastrocnemius(LG),adductor magnus(AM),plantaris longus(PL)and extensor digitorum longus(EDL)muscles from summer active(SA),pre-hibernation(PRE),hibernation(HIB),interbout arousals(IBA)and post-hibernation(POST)were used in the study.Results:The indexes of muscle mass,the cross sectional area(CSA)and the markers of muscle atrophy(MuRF1 and atrogin-1)indicated that lateral gastrocnemius,adductor magnus and plantaris longus muscles showed various degrees of anti-disuse muscle atrophy with the hierarchy of effect being lateral gastrocnemius > adductor magnus > plantaris longus,no significant muscle atrophy was observed in extensor digitorum longus muscle.Furthermore,the lateral gastrocnemius,adductor magnus,plantaris longus,and extensor digitorum longus muscles underwent conversion of fast-to-slow fiber type during mid-hibernation.The mechanistic measurements showed that in lateral gastrocnemius muscle,the protein level of calpastatin(endogenous inhibitor of calpain)was significantly decreased whereas calpain 1 and 2 was significantly increased,the downstream substrates of calpains,desmin and titin,was significantly decreased during hibernation compared with summer activity.In adductor magnus muscle,the protein level of calpastatin was lower than that of calpain 1 and 2 during hibernation,the degradation of calpain downstream substrates including desmin,troponin T(TnT)and titin was significantly increased during hibernation compared with summer activity.In plantaris longus muscle,the expression of calpastatin was significantly decreased whereas calpain 1 and 2 was increased during hibernation and interbout arousals,the downstream substrates of calpains,desmin and TnT,was significantly decreased during hibernation compared with summer activity.Our findings suggest that the high expression of calpain during hibernation may be one of the important reasons for significant degradation of its downstream substrates.Differently,in extensor digitorum longus muscle,the expression of calpastatin showed a significant increase during hibernation and interbout arousals,desmin and titin maintained and the expression of TnT was significantly increased during hibernation and interbout arousals compared with that during summer activity.The maintenance of calpain during hibernation may be one of the important mechanisms for the downstream substrates of calpain do not undergo significant degradation.Conclusions:These findings suggest that the decrease of calpastatin and the high expression of calpain 1 and 2 may be one of the important mechanisms of muscle mass loss in lateral gastrocnemius,adductor magnus and plantaris longus muscles during hibernation.The high expression of calpain inhibitor calpastatin during hibernation inhibits the activity of calpain,which may be one of the important mechanisms for extensor digitorum longus muscle to maintain muscle mass.Thus,the differential activation of the calpain system and the selective degradation of downstream substrates may be involved in muscle atrophy of different fast-twitch muscles during hibernation.