| Background and purpose:When the rate of protein synthesis in the muscle cells exceeds the rate of protein degradation,the muscles will grow;when the rate of protein degradation exceeds the rate of protein synthesis,the muscles will atrophy.Under normal conditions,the main function of autophagy is to degrade intracellular aging and damaged organelles,and to achieve intracellular organelle renewal.In an unfavorable state,autophagy degrades macromolecular substances such as non-essential proteins in cells,providing energy and synthetic substrates for cell survival,and is a self-rescue mechanism.Therefore,autophagy is a basic mechanism for biological survival,so this mechanism exists in all eukaryotes during evolution.There are two main pathways regulating autopahgy in mammals,TOR pathway and Beclin1 pathway.In the normal situation,actived m TOR will inhibite of initiation of autophagy.Beclin1-Vps34 complex in the Beclin1 pathway is the key step to control autophay mechanism.Under the stress conditions such as starvation and hypoxia,Beclin1-Vps34 complex induces the initiation of autophagy.LC3 protein is the key step of the autophagy,which is responsible for the maturation of autophagic vesicles.The relationship between autophagy and disuse muscle atrophy is one of the hot issues in current research.Many studies have reasearched this from different angles.The results showed that muscle state were alleviated by inhibition of autophagy in muscle atrophy,indicating that over-activated autophagy caused muscle atrophy;inhibition of autophagy in skeletal muscle of non-hibernators,resulting in skeletal muscle morphology and function could not be maintained normally,indicating that the maintenance of skeletal muscle structure and function requires moderate autophagy.Hibernation is a strategy for many animals to resist the bad environment.After a long period of inactivity,the skeletal muscle mass loss is less,and the structure and function are more intact.Therefore,in order to explore whether autophagy is involved in the adaptation and maintenance of skeletal muscle during hibernation in hibernators,we measured the markers of autophagy and the key indicators in the signal pathway in different types of skeletal muscle in different periods of hibernation of hibernating Daurian ground squirrels(Spermophilus dauricus).Experimental method:40 Daurian squirrels were divided into five groups(n=8)as follows,summer active group(SA),pre-hibernation group(PRE),hibernation group(HIB),interbout arousal group(IBA)and post-hibernation(POST).(1)Recorded the body weight and muscle weight,and used the frozen section cutting and immunofluorescence to measure the fiber cross-sectional area in soleus and extensor digitorum longus as the basic data of the skeletal muscle.(2)The morphology of the autophagic vesicles was observed using a fluoroscopy electron microscope to determine whether autophagy occurred.(3)Real-time quantitative PCR was used to measure the expression of LC3,P62,Mu RF-1,Atrogin-1,m TOR,Raptor,AKT and Beclin1(4)Protein expression of Mu RF-1,PI3 K,P-PI3 K,AKT,P-AKT,m TOR,Raptor,P-m TOR,Beclin1,Cathepsin L,Atg7,LC3,and P62 were detected by Western blot.(5)The immunofluorescence histochemistry was used to detect the change of protein aggregation point of LC3 and P62.Results and discussions:(1)The changes of body weight,muscle weight and cross-sectional area of muscle fibers of Spermophilus dauricus during different periods of hibernation Compared with the summer group,the body weight of Daurian ground squirrels in the pre-hibernation group was significantly increased(P<0.05),which may be that the ground squirrels experienced a period of fattening before hibernation.Compared with pre-hibernation group,body weight in the hibernation group,interbout arousal group and the post-hibernation group was significantly reduced(P<0.05).Compared with pre-hibernation group,the muscle weight of soleus and extensor digitorum longus in the hibernation state did not change significantly(P>0.05).Compared with the pre-hibernation group,there was no significant change in the fiber cross-sectional area of the soleus and the extensor digitorum longus during hibernation.These results showed that although the Daurian ground squirrels experienced the long period of inactivity during hibernation,the soleus and the extensor digitorum longus didn`t represent significant muscle atrophy.(2)Detection of autophagic vesicles Electron microscopy is the gold marker for observing whether autophagy occurs.In the two types muscles we discovered the autophagic vesicles,myofilament scattered,mitochondrial swelling and other phenomena in hibernation group,but in the interbout arousal group we didn`t detect the autophagic vesicles.These results showed that autophay occurs during hibernation,the interbout arousal may have the role in reducing autophagy levels.(3)The change of Beclin1 pathway LC3 is the key protein that promotes the maturation of autophagic vesicles.Increased expression of LC3 protein represents the increased in autophagy levels.Western blot results showed that compared with the pre-hibernation group,the LC3 protein expression of the soleus in the hibernation group increased by 60%(P<0.05),but there were no significant change in the interbout arousal group(P>0.05).The number of LC3 aggregation points can represent the expression level of LC3,which can also reflect changes in autophagy levels.Immunofluorescence was used to measure the LC3 points.Compared with the pre-hibernation group,the number of LC3 points in the hibernation group was significantly increased by 68%(P<0.05),but there were no significant change in the interbout arousal group of the soleus.The results of Western blot of the extensor digitorum longus showed that the LC3 protein levels in the hibernation group and the interbout arousal group were reduced by 61% and 84%(P<0.05),respectively.The results of LC3 points showed that compared with the hibernation group,the number of LC3 points in the hibernation group and the interbout arousal group were reduced by 33% and 54%(P<0.05),respectively.This results indicated that the autophagy level in the soleus increased in the hibernation group and decreased in the interbout arousal group.The levels of autophagy of the extensor digitorum longus in the hibernating group and the interbout arousal group all maintained the low expression state.Beclin1 is the key protein in the Beclin1 pathway,and it is also one of the basic substances that constitute autophagy.Under adverse conditions,Beclin1 induces autophagy initiation,and its expression level is directly proportional to the level of autophagy.The western blot results showed that compared with pre-hibernation group Beclin1 protein espression in the hibernation group increased by 18%(P<0.05)and decreased by 45%(P<0.05)in the interbout arousal group in the soleus.Compared with pre-hibernation group,Beclin1 protein espression of the extensor digitorum longus decreased by 32.5% and 19.5%(P<0.05)in the hibernation group and the interbout arousal group,respectively.This indicated that the expression of Beclin1 protein also increased when the level of autophagy increased,and the expression level of Beclin1 protein decreased when the level of autophagy decreased.The trend of Beclin1 in the two groups of muscles was consistent with the trend of autophagy.Therefore,this result suggested that the Beclin1 pathway is the main regulatory pathway regulating autophagy in skeletal muscle during hibernation.(4)The change of TOR signal pathway The key protein of the TOR pathway is m TOR,which is activated by phosphorylation to inhibit the occurrence of autophagy,and its expression level is inversely proportional to the level of autophagy.The results showed that the expression levels of phospho-m TOR of soleus and extensor digitorum longus in the hibernation group and the interbout arousal group were significantly lower than that in the pre-hibernation group.Since the level of autophagy in the soleus was increased in the hibernation group and decreased in the interbout arousal group;The level of autophagy in the extensor digitorum longus was decreased in both the hibernation group and the interbout arousal group.From the above results,we conclude that the change of TOR pathway during hibernation is not consistent with the change of autophagy level,suggesting that TOR is not involved in the regulation of autophagy during hibernation,and there may be other pathways regulating autophagy.Conclusions:The results of LC3 immunofluorescence and immunoblotting of the soleus in the hibernation group showed the increased in protein expression in the hibernation group and decreased in the interbout arousal group.This result indicated that the level of autophagy of the soleus in the hibernation group increased,and decreased in the interbout arousal group.In the extensor digitorum longus,The results of LC3 immunofluorescence and immunoblotting showed decreased in the hibernation group and the interbout arousal group,,and the results showed that the levels of autophagy in the hibernation group and the interbout arousal group were decreased.During hibernation,the key protein Beclin1,which promotes autophagy,is consistent with the changes in autophagy levels in the two groups of muscles,suggesting that the Beclin1 pathway is the main pathway regulating autophagy during hibernation.The key protein phospho-m TOR,its protein expression,is down-regulated in the two groups of muscles during hibernation,and its change trend is inconsistent with the level of autophagy,suggesting that the TOR pathway may not participate in the regulation of autophagy during hibernation.In the regulation of autophagy in skeletal muscle during hibernation,there may be other pathways regulating autophagy. |
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