The Role Of Skeletal Muscle Deglycase DJ-1 In Improving Disuse Muscular Atrophy Through Exercise

Research Purpose Muscle atrophy increases the risk of falls,prolongs the recovery period of the disease,and lead to further decline in muscle function,forming a vicious circle,which has become an urgent problem in the field of health medicine.As a safe intervention strategy with low side effects,exercise is of great significance for the prevention and treatment of muscle atrophy.However,there are still many unknowns in its mechanism of action,which needs to be further studied.Our previous study found for the first time that deglycase DJ-1 plays an important role in exercise and muscle atrophy,but the specific mechanism has not been fully elucidated.Therefore,this study aims to:(1)use public databases and animal models to explore the effects of muscle atrophy and resistance training on the expression level of DJ-1 in skeletal muscle;(2)construct skeletal muscle-specific knockout DJ-1 transgenic mice,to clarify the role of DJ-1 in the regulation of exercise capacity and muscle atrophy,and to clarify its potential molecular mechanism;(3)to verify whether resistance training can improve muscle atrophy through skeletal muscle DJ-1;(4)to apply DJ-1 inhibitor-Compound23,mimics the effect it exerts on skeletal muscle atrophy in vivo.This study will help to deepen the understanding of muscle atrophy treatment and provide a theoretical basis for the development of new intervention strategies.Research Methods This article is divided into five parts.Study 1 explored the effects of muscle atrophy and resistance training on the expression level of deglycase DJ-1 through multi-omics analysis and animal models.In terms of multi-omics analysis,use "muscles" as the keyword to search the databases of muscle atrophy-related transcriptome,proteome,and single-cell sequencing in databases such as Pub Med and GEO Data Sets,and use R software to analyze the differences in DJ-1 between the healthy group and the muscle atrophy group.In terms of animal experiments,a mouse model of disuse muscular atrophy and a mouse model of resistance training were established to detect the expression level of skeletal muscle DJ-1.In the mouse model of disuse muscular atrophy,8-week-old male C57BL/6J mice were randomly divided into control group(WT group,n=5)and immobilization group(WT+Immo group,n=5).The immobilization scheme is as follows: use medical tape to wrap the tail of the mouse and suspend it on the top of the cage for 14 days.Immediately after suspension,tissue samples such as gastrocnemius muscle were collected,and the expression level of DJ-1 in gastrocnemius muscle was detected by q RT-PCR and Western blot.In the resistance training mouse model,8-week-old male C57BL/6J mice were randomly divided into a sedentary group(CON group,n=5)and a resistance training group(RE group,n=5).Resistance training mice performed 10 repetitions of resistance training every day,with a 2-min rest between each repetition.For the first 1-2 repetitions,the intensity of resistance training is 50% of the maximum weight-bearing capacity,for the 3rd-4th repetition,the intensity of resistance training is 75% of the maximum weight-bearing capacity,for the 5th-10 th repetition,resistance training strength is 100% of the maximum load capacity.The mice were trained 6 days a week for a total of 12 weeks,and the maximum weight-bearing capacity of the mice was tested every 2 weeks,and the intensity of resistance training was adjusted.Immediately after the training,tissue samples such as gastrocnemius muscle were collected,and the expression level of DJ-1 in gastrocnemius muscle was detected by q RT-PCR and Western blot.In the second study,skeletal muscle-specific knockout DJ-1 transgenic mice were constructed to clarify the regulatory role of skeletal muscle DJ-1 in exercise capacity and muscle atrophy.DJ-1-flox mice were mated with Myl1-cre tool mice,and skeletal muscle-specific knockout DJ-1(muscle specific knockout DJ-1,MDKO)mice were obtained through the Cre-Lox P system.After the mice grow to 10-12 weeks old,the grip strength,maximum load-bearing capacity,and exercise endurance are tested;the body composition of the mice is tested,and gastrocnemius muscle and other tissue samples are collected for morphological testing(H&E staining,muscle fiber typing,fluorescent staining,and electron microscopy).Gastrocnemius muscle energy metabolism analysis(OROBOROS O2K),fast-twitch muscle fibers,slow-twitch fibers,mitochondrial quantity,mitochondrial complex related gene expression level detection(q RT-PCR),DJ-1,mitochondrial complex and other related protein expression level analysis(Western blot).Study 3,combined with in vivo animal experiments and in vitro cell experiments,clarified the molecular mechanism of skeletal muscle DJ-1 regulating exercise capacity and muscle atrophy.In the animal experiment,an animal model of atrophy of the suspension muscle of the hindlimb was established,and the model construction scheme was the same as that in the first part.After successful modeling,the gastrocnemius muscle tissue was collected for transcriptome sequencing,morphological detection(H&E staining,muscle fiber typing fluorescent staining,ROS reactive oxygen species staining),gastrocnemius muscle energy metabolism(OROBOROS O2K),fast muscle fiber,slow muscle fiber,atrophy,mitochondrial complex Detection of animal-related gene expression level(q RT-PCR),autophagy pathway,muscle development pathway,muscle atrophy,ubiquitination modification and other related protein expression level analysis(Western blot),extraction of gastrocnemius muscle cytoplasmic protein and nuclear protein to detect the expression level of Fox O1.In the cell experiment,C2C12 myoblasts were cultured,and after they were differentiated into myotube cells,the sh-DJ-1 lentivirus infection experiment was performed,and the expression levels of muscle development and atrophy-related proteins(Western blot)were detected after 48 hours;HEK 293 T cells were cultured Plasmids such as human Trim63 promoter and human Fbxo32 promoter were transfected respectively,and dual luciferase experiments were performed.Study 4 is to verify whether resistance training can improve muscle atrophy through skeletal muscle DJ-1.The atrophy models of hindlimb suspensor muscles were established in 10-week-old male control mice and MDKO mice,respectively.After successful modeling,a 4-week resistance training intervention was carried out,and the specific plan was the same as the first part.Immediately after the last training session,gastrocnemius muscle tissues were collected,and the expression levels of genes and proteins related to muscle atrophy were detected by q RT-PCR and Western blot.Study five,the application of DJ-1 inhibitor-Compound23 to simulate the role of DJ-1 in skeletal muscle in vivo.10-week-old male control mice and MDKO mice were subjected to hindlimb suspension.After 7 days of suspension,Compound23 was injected in situ into the gastrocnemius muscle.After 14 days of suspension,the gastrocnemius tissue and other tissues were collected,and H&E staining,q RT-PCR and Western blot were performed.Evaluation of the effect of Compound23 in skeletal muscle in vivo.ResultsStudy 1,transcriptome sequencing results showed that hindlimb suspension significantly down-regulated the expression level of mouse muscle Dj-1 gene(p<0.05),it was further estimated in mouse skeletal muscle samples(p<0.01).Through transcriptome sequencing analysis of clinical muscle samples,it was found that the expression of Dj-1 gene in muscle tissue of patients with ankle injuries was positively correlated with the time to resume activities(p<0.01).In addition,12 weeks of resistance training can promote the expression of DJ-1 in gastrocnemius muscle tissue of mice(p<0.01).In Study 2,knocking out DJ-1 in skeletal muscle did not affect the body weight of mice,but resulted in a decrease in lean body mass(p<0.05),a decrease in muscle weight(p<0.05)and an increase in fat mass(p<0.05).The exercise ability test showed that knocking out DJ-1 in skeletal muscle weakened the mouse’s grip strength(p<0.001),maximum weight-bearing ability(p<0.01),suspension ability(p<0.01),and fatigue resistance ability(p<0.01)and exercise tolerance(p<0.01).Further morphological observations revealed that the knockout of DJ-1 in skeletal muscle decreased the cross-sectional area of gastrocnemius muscle cells(p<0.01),resulting in gastrocnemius muscle mitochondrial cristae rupture,swelling,and decreased respiratory metabolic function(p<0.05).The results of q RT-PCR and Western blot showed that the knockout of DJ-1 in skeletal muscle down-regulated the expression levels of some genes related to type I and type IIA muscle fibers,but had no significant effect on the types of skeletal muscle fibers,the number of mitochondria,and the expression of genes and proteins related to mitochondrial complexes.Study 3,under the condition of hindlimb suspension,knocking out DJ-1 in skeletal muscle exacerbated the loss of lean body mass and muscle weight(p<0.01);promoted the inward movement of gastrocnemius muscle cells(p<0.01),and reduced the crosssectional area of muscle cells(p<0.05);in addition,significantly down-regulate the gene expression levels of type I muscle fiber and type IIA muscle fiber markers;damage the mitochondrial energy metabolism function of gastrocnemius muscle,reduce the number of mitochondria(p<0.01),but does not change the muscle fiber type,does not affect the level of oxidative stress in skeletal muscle.Further transcriptome sequencing,Western blot,and q RT-PCR detection found that skeletal muscle knockout DJ-1 up-regulated muscle atrophy genes Trim63(p<0.001),Fbxo32(p<0.05),Mstn(p<0.01)regulated by the Fox O pathway.It increases the ubiquitination level of muscle proteins,but has no effect on the expression of muscle satellite cell marker genes and muscle development-related proteins,and does not affect the expression of autophagy pathway-related molecules.Dual-luciferase reporter experiments verified that skeletal muscle DJ-1 transcriptionally regulates the occurrence and development of muscle atrophy through the Fox O1-195/-189 binding site on Trim63 and the Fox O1-38/-31 binding site on Fbxo32 promoter.In the fourth part,Western blot and q RT-PCR were used to detect the expression levels of atrophy genes and proteins in control and MDKO mice under suspension conditions before and after exercise intervention.The results showed that the loss of skeletal muscle DJ-1 weakened the effect of resistance training on muscle improvement of atrophy.In the fifth part,under the condition of suspension,gastrocnemius injection of Compound23 reduces the cross-sectional area of myocytes(p<0.05).Knockout of DJ-1 in skeletal muscle partially counteracts the effect of Compound23 on muscle atrophy under suspension stimulation(p<0.05).Conclusion1.The expression level of DJ-1 in skeletal muscle is negatively correlated with muscle atrophy,and exercise can promote the expression of DJ-1.Mice lacking DJ-1 in skeletal muscle have impaired exercise capacity,accompanied by muscle atrophy.2.The role of DJ-1 in exercise capacity and muscle atrophy is related to the AKTFox O1-Mur F1 signaling pathway.Deletion of DJ-1 in skeletal muscle inhibits the phosphorylation level of AKT-Fox O1,promotes the entry of Fox O1 into the nucleus,and finally activates the expression of atrophic genes such as Trim63 and Fbxo32 atthe transcriptional level.3.The effect of resistance training on improving muscle atrophy is partly related to the promotion of DJ-1 expression in skeletal muscle.