Aerobic Exercise Accelerates Skeletal Muscle Atrophy Of Autophagy Atg7 Gene Specific Knockout Mice

Objective:As the most abundant tissue in human body,skeletal muscle plays an important role in maintaining body health in addition to autonomous movement.However,trauma,aging,illness or prolonged bed rest and metabolic disorders often lead to rapid muscle atrophy.At the same time,decreased muscle mass induces disease,reduced physical function,and has substantial negative effects on quality of life.Recent studies have found that skeletal muscle atrophy is usually accompanied by dysfunction of autophagy and increased apoptosis.Autophagy and apoptosis are strictly regulated and play an important role in maintaining muscle mass.Among them,aerobic exercise as a convenient,economical and acceptable effective intervention can improve the occurrence and development of skeletal muscle atrophy,the main mechanism of its regulation of muscle mass has not been fully explained.Therefore,we selected mice with specific knockout of key gene of autophagy in skeletal muscle cells（Atg7）for 12 weeks of aerobic exercise intervention,so as to demonstrate the effect and mechanism of aerobic exercise on the maintenance of skeletal muscle mass under the condition of autophagy gene defects,in order to provide targeted intervention strategies for the prevention or slowing down of skeletal muscle atrophy by exercise.Methods:This study constructed the Atg7 knockout mouse model using the Cre-loxP gene knockout technique.Sixteen four-month-old Atg7 homozygous control mice(Atg7^f/f)and Atg7 gene skeletal muscle-specific knockout mice(Atg7^-/-)were selected and randomly divided into two groups:That is,the control group(Atg7^f/f-NE,Atg7^-/--NE)and the aerobic group(Atg7^f/f-AE,Atg7^-/--AE).Mice in the aerobic exercise group were given treadmill exercise:speed:14m/min,50 minutes of training each time,treadmill slope:10°;5 times a week.After 12 weeks of aerobic exercise,the intervention period was 24 hours of rest,weight weighing,muscle function,and exercise ability tests performed before death.For the left and right hind limbs,we calculated gastrocnemius muscle weight to body weight（GMW/BW）.HE,TUNEL and SDH staining were used to observe the histopathological changes in skeletal muscle.The PCR was used to identify genes,and Western blot was used to detect the expression changes of proteins related to the ubiquitin-proteasome system（UPS）,autophagy,apoptosis and mitochondrial biogenesis.Results:1.Aerobic exercise aggravates gastrocnemius atrophy of Atg7 specific knockout mice:Compared with the Atg7^f/f-NE group,the GMW/BW ratio of the Atg7^f/f-AE group increased significantly（p<0.05）;on the contrary,compared with the Atg7f/f-NE group,the Atg7^-/--NE group had both GMW/BW ratio significantly decreased;with the compare of Atg7^-/--NE group,the Atg7^-/--AE group mice GMW/BW ratio showed a significant decrease（p<0.05）;compared with Atg7^f/f-NE,Atg7^-/--NE GMW/BW ratio was significantly reduced（p<0.05）;compared with Atg7^f/f-AE,the GMW/BW ratio of Atg7^-/--AE also showed a significant reduction（p<0.001）.2.Behavioral test:1)Aerobic exercise decreased the weight test and flipping ability of skeletal muscle Atg7specific knockout mice:There was no significant difference between the Atg7^f/f-AE group and the Atg7^f/f-NE group,while the test results showed an upward trend.There was no significant difference between the Atg7^-/--NE group and the Atg7^f/f-NE group,while the weight test results decreased significantly（p<0.01）.Compared with the Atg7^f/f-NE group,the results of two experimental tests were significantly decreased（p<0.01）;The test results of the Atg7^-/--AE group were significantly decreased compared with the Atg7^-/--NE group（p<0.01;p<0.05）.2)Aerobic exercise decreased the exercise ability of skeletal muscle Atg7 specific knockout mice:Compared with the Atg7^f/f-NE group,the aerobic exhaustion time of the Atg7^f/f-AE group increased significantly（p<0.05）;on the contrary,compared with the Atg7^f/f-NE group,the Atg7^-/--NE group had no significant difference but showed a decreasing trend;with the compare of Atg7^-/--NE group,the aerobic exhaustion time of the Atg7^-/--AE group mice had no significant difference but showed a decreasing trend;compared with Atg7^f/f-AE,the aerobic exhaustion time of Atg7^-/--AE also showed a significant reduction（p<0.001）.3.Histopathological changes:1)HE staining:Compared with the Atg7^f/f-NE group,the cross-sectional area（CSA）of muscle fibers in the Atg7^f/f-AE group was significantly increased（p<0.05）,and their shape was neat and compact.Compared with the Atg7^f/f-NE group,the CSA of muscle fiber in the Atg7^-/--NE group was significantly decreased（p<0.01）.Compared with the Atg7^-/--AE group and the Atg7^-/--NE group,nucleus inmigration and CSA of muscle fibers of gastrocnemius muscle were significantly decreased（p<0.001）,and the phenomenon of nuclear inmigration in skeletal muscle was significantly increased.The CSA of skeletal muscle was more severe in the Atg7^-/--NE group than in the Atg7^-/--AE group（p<0.001）.2)SDH staining analysis:Compared with the Atg7^f/f-NE group,the SDH activity of gastrocnemius muscle in the Atg7^f/f-AE group was significantly increased（p<0.05）;The SDH activity of the Atg7^-/--NE group was significantly decreased compared with the Atg7^f/f-NE group（p<0.01）;The SDH activity of the Atg7^-/--AE group was significantly decreased compared with the Atg7^f/f-AE group（p<0.01）,while the SDH activity of the Atg7^-/--AE group was decreased compared with the Atg7^-/--NE group.4.TUNEL staining analysis:Compared with the Atg7^f/f-NE group,the apoptosis of gastrocnemius in the Atg7^f/f-AE group was decreased.The apoptosis level of gastrocnemius in the Atg7^-/--AE and the Atg7^-/--NE groups was significantly increased（p<0.05）;Apoptosis was significantly increased in the Atg7^-/--NE group compared with the Atg7^f/f-NE group（p<0.001）.The apoptosis of the Atg7-/--AE group was significantly increased compared with Atg7^f/f-AE group（p<0.001）.The apoptosis level of skeletal muscle in the Atg7^-/--AE group was significantly increased compared with the Atg7^-/--NE group（p<0.05）.5.Western blot analysis:1)Aerobic exercise inhibited autophagy related protein expression in skeletal muscle Atg7 specific knockout mice:Compared with the Atg7^f/f-NE group,a significant increase in the expressions of Atg7 and LC3-II/LC3-I were observed in the Atg7^f/f-AE group（p<0.01;p<0.001）,whereas a decrease in p62 expression was observed（p<0.05）;In the Atg7^-/--NE group,the expressions of Atg7 and LC3-II/LC3-I were significantly lower than those in the Atg7^f/f-NE group（p<0.001;p<0.001）,while p62 expression was increased（p<0.01）;there was a significant decrease in the expressions of Atg7 and LC3-II/LC3-I in the Atg7^-/--AE group（p<0.05;p<0.05）compared to the Atg7^f/f-AE group.2)Aerobic exercise increases apoptosis related protein expression in skeletal muscle Atg7specific knockout mice:Compared with the Atg7^f/f-NE group,a significant decrease in Bax expression was observed in the Atg7^f/f-AE group（p<0.01）,whereas an increase in Bcl-2expression was observed.In the Atg7^-/--NE group,Bax expression was significantly increased（p<0.001）,while Bcl-2 expression was decreased compared with the Atg7^f/f-NE group.There was a significant increase in Bax expression of Atg7^-/--AE group（p<0.001）compared to the Atg7^f/f-AE group,but a significant decrease in Bcl-2 expression（p<0.01）.Atg7^-/--AE group expression of Bax was significantly higher（p<0.001）than the Atg7^-/--NE group（p<0.001）,and Bcl-2 expression was significantly lower（p<0.01）.3)Aerobic exercise aggravates the expression of UPS-related proteins in skeletal muscle Atg7 specific knockout mice:Compared with the Atg7^f/f-NE group,the expressions of Atrogin-1 in the Atg7^f/f-AE group was significantly decreased（p<0.01）,while the expressions of Mu RF1 expression showed a downward trend.Compared with the Atg7^f/f-NE group,the expressions of Mu RF1 and Atrogin-1 expressions in Atg7^-/--NE group were significantly increased（p<0.001;p<0.05）;Compared with the Atg7f/f-AE group,the expressions of Mu RF1 and Atrogin-1 in the Atg7^-/--AE group were significantly increased（p<0.01;p<0.001）;The expressions of Mu RF1 and Atrogin-1 in the Atg7^-/--AE group were significantly increased compared with the Atg7^-/--NE group（p<0.01;p<0.001）.4)Aerobic exercise inhibited the expression of mitochondrial biogenesis related proteins in skeletal muscle Atg7 specific knockout mice:The expression of PGC-1αin the Atg7^f/f-AE group was significantly increased compared with the Atg7^f/f-NE group（p<0.01）,and the expression of COXⅣwas increased.The expression of COXⅣin the Atg7^-/--NE group was significantly decreased compared with Atg7^f/f-NE group（p<0.001）,and the expression of PGC-1αwas decreased.The expression of PGC-1αin the Atg7^-/--AE group was significantly decreased compared with the Atg7^-/--NE group（p<0.05）,and the expression of COXⅣwas decreased.The expression of PGC-1αand COXⅣin the Atg7^-/--AE group was significantly decreased compared with the Atg7^f/f-AE group（p<0.001;p<0.001）.Conclusion:Aerobic exercise in mice with normal autophagy function can improve the CSA and GMW/BW ratio of the skeletal muscle,and thus promote the improvement of the mass and exercise ability.Under the condition of skeletal muscle-specific autophagy gene Atg7knockout,aerobic exercise can aggravate the proliferation of apoptosis,reduce the mitochondrial biogenesis,accelerate the degradation of skeletal muscle,thereby reducing the CSA of skeletal muscle and aerobic capacity,and then affect the exercise ability and lead to skeletal muscle atrophy.Aerobic exercise in mice with normal autophagy function can activate the autophagy,inhibit apoptosis,Under the condition of skeletal muscle-specific autophagy gene Atg7 knockout,aerobic exercise can lead to the over-activation of UPS,apoptosis surges and mitochondrial biogenesis decreased.In conclusion,aerobic exercise cannot respond to the benefits of exercise intervention without the autophagy-gene Atg7 in skeletal muscle,and to a certain extent,skeletal muscle mass and exercise ability are affected.