| BackgroundMaintaining good movement function is essential to the human body,and muscular atrophy can lead to limitation or deprivation of motor function,the most common type of disuse muscular atrophy.Hibernating animals choose hibernation to survive the cold and food-deprived environment of winter.The typical small hibernating mammal,Spermophilus dauricus,is in a state of prolonged inactivity during hibernation.However,unlike nonhibernating animals that undergo disuse atrophy when activity is reduced,the skeletal muscles of ground squirrels do not undergo significant muscular atrophy during monthslong hibernation.Therefore,they are considered to be a natural animal model for anti-disuse atrophy.The skeletal,muscular,and nervous systems work together to achieve mammalian movement,with the motor nerve and skeletal muscle connected by a neuromuscular junction.In the rat model of disuse atrophy,the morphology of the neuromuscular junction undergoes degenerative changes that affect its normal function.The morphology of the neuromuscular junction in hibernating mammals has not been investigated yet.In this study,the hindlimb soleus and extensor digitorum longus muscles of Daurian ground squirrels were selected as the subjects to investigate the morphology and related regulatory mechanisms of the neuromuscular junction in skeletal muscles hibernating ground squirrels.Objects and methodsThe typical slow-twitch muscle soleus and the typical fast-twitch muscle extensor digitorum longus were studied in the pre-hibernation,hibernation,interbout awakening,and post hibernation groups of Daurian ground squirrels.In this study,the immunohistochemical staining technique was used to measure the neuromuscular junction-related indicators.Possible causes affecting the neuromuscular junction morphology are the expression of target proteins such as Agrin,Lrp4,Mu SK,Rapsyn,and HSP90 beta Agrin-Lrp4-Mu SK,a critical pathway for the formation and maintenance of the neuromuscular junction,was chosen to be detected in skeletal muscle.In addition,the expression of REDD1,FOXO4,FOXO1,PGC-1?,and Atrogin-1 was examined to confirm the relationship between oxidative stress and the morphological changes of neuromuscular junctions in different twitch types of skeletal muscles of ground squirrels during hibernation.The drastic metabolic changes in ground squirrels during hibernation make the housekeeping proteins unsuitable for use as the internal reference,and the improvement of the experimental technique is reported in Chapter 5.Results1.Morphometric measurement of neuromuscular junctions in different types of skeletal muscles in hibernating ground squirrelsThe morphological indices of the neuromuscular junction in the soleus muscle during hibernation,such as the total branch length and branch complexity of the presynaptic junction,the perimeter and area of the postsynaptic endplate area,and the perimeter and area of the acetylcholine receptor staining area,were significantly reduced compared with those before hibernation and recovered to a certain extent in the out-of-hibernation group.In contrast,in the extensor digitorum longus muscle,the morphology of the neuromuscular junction did not change significantly during hibernation.2.Detection of protein expression of neuromuscular junction-related regulatory pathways in different types of skeletal muscle in hibernating ground squirrelsThe expression of Agrin increased to 1.67-fold in the extensor digitorum longus in the interbout arousal group compared to the pre-hibernation group;the expression of Lrp4 decreased by 46.2% in the soleus muscle compared to the hibernation group.The expression of other proteins in both skeletal muscles did not change significantly during hibernation.3.Detection of indexes related to antioxidative stress strategies in different twitch types of muscle fibers of hibernating ground squirrelsThe expression of REDD1 in soleus muscle increased to 1.58-fold in the interbout arousal group compared to the pre-hibernation group,but its expression did not change significantly in extensor digitorum longus muscle during different periods.The phosphorylation level of FOXO4 in soleus muscle decreased by 63.2% in the interbout arousal group;the phosphorylation level in extensor digitorum longus muscle did not change significantly.In the hibernation group,PGC-1? increased to 1.57-fold in soleus muscle compared with the pre-hibernation group,while the expression level of PGC-1? did not change significantly in extensor digitorum longus muscle.The total protein expression and phosphorylation levels of FOXO1 in soleus muscle increased to 1.72-fold and 1.91-fold,respectively,in the hibernation group compared with the pre-hibernation group,and the phosphorylation level of FOXO1 in extensor digitorum longus muscle decreased by 77.4%compared with the hibernation group.The expression of Atrogin-1 was reduced by 42.3% in the hibernation group compared to the pre-hibernation group in extensor digitorum longus muscle,and no significant changes were observed in soleus muscle.ConclusionsIn this study,we found that the morphology of neuromuscular junctions in the soleus muscle of Daurian ground squirrels during hibernation underwent degenerative changes similar to those in the disused state of non-hibernating animals recovered to a certain extent after emergence from hibernation.In contrast,the neuromuscular junctions in the long toe extensor muscle of hibernating ground squirrels showed no morphological changes during hibernation.In addition,we found that Agrin,Lrp4,PGC-1?,and related signaling pathways may affect the morphology of neuromuscular junctions in hibernating ground squirrels;we found that skeletal muscles of different twitch types in Daurian ground squirrels developed adaptive and differential anti-oxidative stress strategies according to their respective energy metabolism patterns during hibernation;the degenerative changes in the morphology of neuromuscular junctions in Daurian ground squirrel soleus muscles during hibernation were not caused by oxidative stress. |
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