The Determinant Role Of Beclin1 Gene In The Regulation Of Skeletal Muscle Atrophy By Resistance Exercise

Objective:As the most massive peripheral tissue in human body,skeletal muscle plays a major role in maintaining the body’s physiological functions,such as exercise,energy metabolism,and immune regulation.The mass loss of skeletal muscle increases with the extension of age and lifestyle changes.In recent years,previous studies have found that the occurrence of skeletal muscle atrophy is often accompanied by a low level of autophagy and the increase of apoptosis.Exercise,as a convenient,economical and environmentally effective intervention,can improve the occurrence and development of skeletal muscle atrophy.Among them,resistance exercise is recommended as the preferred intervention,and its mechanism is closely related to the activation of autophagy and the inhibition of apoptosis.The complex interaction between autophagy and apoptosis plays an important role in the maintenance of skeletal muscle mass.Therefore,we chose skeletal muscle autophagy gene（Beclin1）knockout mice to offer resistance exercise for 8 weeks and to explore the determinant role of resistance exercise on autophagy and skeletal muscle quality maintenance under abnormal functional status of autophagy.It is expected to provide vital targets and intervention strategies for skeletal muscle atrophy and then execute relevant exercise risk references for people with abnormal functional status of autophagy caused by exogenous drugs or gene defects in physical fitness.Methods:Cre-loxP gene knockout technology was used to construct skeletal muscle autophagy gene Beclin1 knockout mice.The experiment used 3-month-old knockout mice for 8 weeks of resistance exercise.Among them,totally 12 mice of loxP homozygous mice(BECN1^f/f)and Beclin1 gene skeletal muscle-specific knockout homozygous mice(BECN1^cko)were allocated in each group.The mice from each group were divided into two sub-groups randomly.Each sub-group had 6 mice,namely the non-exercise groups(BECN1^f/f-NE,BECN1^cko-NE)and the resistance exercise group(BECN1^f/f-RE,BECN1^cko-RE).In the resistance exercise group,the mice were subjected to loading on their tails and climbing training on a 1 m-high 85°ladder.The climbing training was composed of 3 repeated cycles as a group,with 3 times of climbing in each cycle.The 1-minute resting interval between climbing and 2-minute resting interval between cycles were set.The training frequency was once every other day（3 times of a week）.After the completion of entire resistance exercise intervention,the mice were provided the rest for 24 hours and the free accessibility to food and water.Finally,the body weights of the mice were recorded and the mice were sacrificed by cervical dislocation method.Similarly,the rectus femoris weights of the left and right hind limbs of the mice were collected and measured,respectively,and the rectus femoris weight/body weight（RFW/BW）ratio was calculated to determine the rate of skeletal muscle atrophy.In addition,the partial skeletal muscle samples were subjected to HE and TUNEL staining,and transmission electron microscopic observed for evaluating the changes in skeletal muscle structure,functional status of autophagy and apoptosis,mitochondrial quality,and muscle fiber ultrastructure.WB was used to evaluate the expression of proteins in skeletal muscle associated with mitophagy and apoptosis.Results:1.RFW/BW ratio:under normal condition of the skeletal muscle Beclin1 gene,resistance exercise significantly promotes an increased ratio of RFW/BW in BECN1^f/f group of mice（p<0.05）;on the contrary,after knockout Beclin1 gene in skeletal muscle,resistance exercise significantly inhibited RFW/BW of mice in BECN1^cko group（p<0.05）;suggesting that the Beclin1 gene in skeletal muscle plays a key role in maintaining rectus femoris mass.2.The changes in the structure of skeletal muscle:1).The results of HE staining:under the normal condition of the skeletal muscle Beclin1 gene,resistance exercise significantly increased in the cross-sectional area（CSA）of the rectus femoris muscle fiber in the BECN1^f/f group（p<0.05）,narrowed the muscle fiber gap,and improved the shape of the muscle fiber;conversely,after the knockout of the Beclin1 gene in the skeletal,resistance exercise significantly exacerbated decrease the CSA of the rectus femoris muscle fibers in the BECN1^cko group（p<0.05）,the irregular shape of the cross-sectional muscle area,and the increase of inward nuclear movement;thus indicating that the Beclin1 gene in skeletal muscle plays an important role in maintaining the CSA of rectus femoris muscle fibers and inhibits muscle fiber aging and degeneration.2).Transmission electron microscopy:under the normal condition of the Beclin1 gene in skeletal muscle,resistance exercise was beneficial to the more compact arrangement of rectus femoris fibers in BECN1^f/f group,with neat sarcomas and clear bright and dark bands accompanied by increased mitochondrial fusion and the formation of autophagosomes.However,when knockout of Beclin1 gene in skeletal muscle,resistance exercise accelerated the destruction of rectus femoral muscle fiber structure,increased vacuolation and swelling mitochondrial,and even increased abnormal accumulation of autophagosomes in the BECN1^ckogroup.These results indicate that the Beclin1 gene plays an important role in maintaining rectus femoris fibers’ultrastructure.3.The result of TUNEL staining analysis:under the normal condition of the Beclin1 gene in skeletal muscle,the number of positive cells in the rectus femoris muscle of the mice in the resistance exercise BECN1^f/f group was significantly reduced（p<0.01）;conversely,after the knockout of the Beclin1 gene in the skeletal,resistance exercise promoted the number of positive cells in the rectus femoris muscle fibers of the BECN1^cko group（p<0.05）;indicating that the deletion of the skeletal muscle Beclin1 gene may lead to a significant increase in the apoptosis of rectus femoris fibers.4.The result of WB analysis:1).The expression of mitophagy related protein expression:under the normal condition of the skeletal muscle Beclin1 gene,resistance exercise significantly increased the protein expression levels of PINK1,Parkin,LC3-II/LC3-I in the rectus femoris muscle of mice in the BECN1^f/fgroup（p<0.001;p<0.01;p<0.001）,reduce the expression of p62 protein（p<0.05）;compared with BECN1^f/f group,the mouse rectus femoris protein PINK1 and Parkin were significantly reduced in the BECN1^cko group,while the LC3-II/LC3-I ratio and p62 expression were significantly increased;under the condition of skeletal muscle Beclin1 gene knockout,compared with the BECN1^cko-NE group,resistance exercise significantly increased the protein PINK1,Parkin,LC3-II/LC3-I,and p62 expression levels in the rectus femoris muscle of mice in the BECN1^cko group（p<0.001;p<0.001;p<0.001;p<0.001）;thus indicating that resistance exercise can effectively activate mitophagy,but cannot reverse the inhibited of autophagy caused by Beclin1 knockout,indicating that Beclin1 is in ensuring the unobstructed autophagy has an vital role.2).The expression level of apoptosis-related proteins:under normal Beclin1 gene condition,resistance exercise significantly inhibited Bax,Cyt-c,Caspase-9,and Cleaved-caspase 3proteins expression in rectus femoris of BECN1^f/f group（p<0.001;p<0.001;p<0.05;p<0.05）,and significantly increased Bcl-2 expression（p<0.001）;when knockout of Beclin1gene in skeletal muscle,resistance exercise significantly increased Bax,Cyt-C,Caspase-9,and Cleaved-caspase 3 proteins expression level in rectus femoris of BECN1^cko mice compared to the BECN1^cko-NE group（p<0.001;p<0.001;p<0.001;p<0.001）and significantly decreased the expression of Bcl-2（p<0.001）;these results indicate that Beclin1 gene plays a role in inhibiting the apoptosis of skeletal muscle cells.Conclusion:Resistance exercise can inhibit apoptosis by activating skeletal muscle mitophagy in normal mice,improving the CSA of the rectus femoris muscle,the arrangement of muscle fibers,and increasing the wet weight ratio of the rectus femoris to promote the increase of the RFW/BW;However,resistance exercise showed that under the condition of knocking out the skeletal muscle autophagy gene Beclin1,it blocked the occurrence of mitophagy and aggravated the apoptosis,induced abnormal changes in the CSA,morphology and nucleus position of the rectus femoris muscle fibers and the abnormality of muscle fiber structure together leads to a significant decrease in the RFW/BW,indicating that resistance exercise can exacerbate abnormal mitophagy and abnormal morphology when the skeletal muscle autophagy gene Beclin1 is knocked out,thereby inducing intrinsic apoptosis and promoting skeletal muscle atrophy.