Effects Of Aerobic Exercise On Muscle Stem Cell Depletion,phenotypes And Cell Cycle Regulation In Mouse Skeletal Muscle

Muscle stem cells are confirmed as a post-natal somatic stem cells in muscle tissue,which is an important factor for repairing muscle fiber injury and retarding skeletal muscle aging.Physical exercise is widely considered as the main way to maintain skeletal muscle function.However,there are some unclearly questions,such as how exercise maintains the normal state of muscle stem cells,how the increasing of muscle stem cells helps skeletal muscle function,etc.With the extension of human expectative life,motor dysfunction has become an important factor affecting the quality of life of the elderly.Therefore,the aim of this research is twofold,including?i?how to rationally apply physical exercise to maintain healthy skeletal muscle for a long time until agedness;?ii?how to understand the proliferous feature?cell cycle?of muscle stem cells to provide application value for the treatment of muscle atrophy.Objective:The aim of this research are as follows.Firstly,we study the effects of moderate intensity aerobic exercise on the number and proliferation capacities of muscle stem cells in mice at different life stages.Secondly,analyze the relationship between the numbers of muscle stem cells,myonuclei numbers and phenotypic indexes such as muscle mass,muscle strength and cross-sectional area of myofibers.Finally explore the cell cycle regulation mechanisms of exercise in slowing down skeletal muscle aging by affecting muscle stem cells.This research provides improved techniques and solutions for delaying aging of people's body through exercises.At the same time,single muscle fiber culture,analysis technology and small animal skeletal muscle function test methods were established to provide corresponding research technology for basic research of animal models in sports science.Methods:Male mice of BALB/c strain were used in this study.The mice were divided into five groups,sedentary control group?group C?,aged from 6 to 12 weeks exercise group?group Q?,aged from 20 to 26 weeks exercise group?group M?,aged from 50 to56 weeks exercise group?group L?,aged from 6 to 56 weeks exercise group?group Z?.After exercises of treadmill with moderate intensity,the right gastrocnemius muscle of each mouse was isolated.Then the single myofibers were separated and cultured.The muscle stem cells were stained by Pax7 through immunofluorescence.The numbers of total nuclei and muscle stem cells per myofibers were counted for further analysis.After paraffin embedding,the left gastrocnemius muscle of each mouse was transected and stained with HE.Subsequently,the cross-sectional areas of gastrocnemius and myofibers were measured.Quadriceps femoris was used for Western blotting of cell cycle regulation related proteins.Before sampling,the phenotypic indexes such as peak grip strength,grip endurance and body weight of mice need to be tested.Results:1.With growing age,the quantity of the muscle stem cells will gradually decline,and through long-term aerobic exercise such decline trend can be effectively slow down.Furthermore,Six-week aerobic exercise intervention can reverse the number of reduced muscle stem cells.2.The number of myofibrillar nuclei in gastrocnemius muscle of mice decreased with aging,while the degree of decline is small.Aerobic exercise intervention can increase the number of myonuclei,while the degree of decline is small.3.Before aging,weight and gastrocnemius muscle quality increased with the increase of age.In the old age,the weight and gastrocnemius muscle quality of the control group decreased significantly,while the weight and gastrocnemius muscle quality of the group with aerobic exercise intervention decreased less than the control group.Meanwhile,the gastrocnemius muscle mass fraction showed a downward trend after 12 weeks of age.4.The peak value of mouse grip strength appeared at the 12^th week old,and then gradually decreased.Aerobic exercise could maintain the grip strength of mice in different groups.5.Before aging,the cross-sectional area of mouse gastrocnemius muscle increased with the increase of age.In the old age,the cross-sectional area of gastrocnemius muscle decreased rapidly.The cross-sectional area of myofibers was the largest at the 12^th week old,and gradually decreased with the increase of age.In the samples of young age,aerobic exercise has a significant effect on increasing cross-sectional area of myofibers,while in old age,this effect is weakened.6.The number of muscle stem cells was positively correlated with the number of myonuclei.At the same time,the number of myonuclei was positively correlated with the cross-sectional area of myofibers,muscle strength and gastrocnemius muscle mass fraction,but not with body weight and muscle mass.7.Aerobic exercise significantly reduced the protein levels of Visfatin,Sirt1,CDK6,Cyclin A2,Cyclin B1,Cyclin D3,Cyclin E2,p16 and p21 in skeletal muscle of mice.Conclusions:1.Aerobic exercise can increase the numbers of muscle stem cells and myonuclei in adult mice.2.The number of muscle stem cells is highly correlated with the number of myonuclei,and the increase of myonuclei can improve the cross-sectional area of myofibers and positive affect the strength level of skeletal muscle.3.Aerobic exercise intervention of mice at any stage of life is helpful to maintain their weight and muscle quality in old age,but has little influence on changing the trend of decreasing skeletal muscle proportion in body composition.4.Promoting cell cycle signals is not the main regulatory pathway for aerobic exercise to maintain a certain number of muscle stem cells.On the contrary,aerobic exercise can promote the steady state of myofibers,keep muscle stem cells at quiescent state,and down-regulate the expression of cyclins.5.The expression of p16 gene in skeletal muscle increases with aging.The accumulation of p16 protein is related to the decline of skeletal muscle and muscle stem cells.Aerobic exercise can reduce the expression of p16 and slow down the decline of skeletal muscle.6.The down-regulation of Sirt1 expression in skeletal muscle is beneficial to protein synthesis of muscle fibers and can potentially inhibit the proliferation of muscle stem cells.