Rehabilitation Exercise Improves Mitochondrial Endoplasmic Reticulum Coupling In Skeletal Muscle Of Chronic Obstructive Pulmonary Disease

Objective: Chronic obstructive pulmonary disease(COPD)is a chronic respiratory disease characterized by persistent airflow limitation,and its morbidity and mortality are steadily increasing.Skeletal muscle dysfunction(SMD)is a severe systemic complication of COPD,characterized by impaired muscle strength or endurance,and up to 30%-40% of COPD patients are associated with SMD.Rehabilitation exercise is currently the most effective intervention for improving SMD,but the molecular mechanisms involved have not been fully elucidated.Mitochondrial endoplasmic reticulum coupling(MERC)is a newly discovered subcellular structure in recent years.As an important communication platform between organelles,its structural damage may lead to abnormal mitophagy.Mitophagy plays an important role in the occurrence and development of COPD-related SMD.Exercise may improve skeletal muscle function by regulating mitophagy.However,the relationship between exercise,mitophagy and MERC in COPD patients is still unclear.Therefore,this topic aims to investigate whether rehabilitation exercise can improve skeletal muscle dysfunction in COPD patients by regulating mitophagy through MERC,and provide a new therapeutic target for improving COPD-related SMD.Methods:(1)Download microarray datasets from the Gene Expression Omnibus database(GEO),and identify differentially expressed genes(DEGs)before and after exercise in COPD.The DEGs were intersected with the human autophagy dataset and the human mitochondrial dataset respectively,to obtain exercise-regulated autophagy genes and mitochondrial genes in COPD skeletal muscle.Mitophagy genes were identified by spearman correlation analysis of these autophagy genes and mitochondrial genes.(2)By analyzing the functions of mitophagy genes and their expressions before and after exercise,the effect of exercise on mitophagy in skeletal muscle of COPD patients was initially explored.(3)A rehabilitation exercise cohort of COPD patients was constructed,and a rehabilitation exercise program was developed and followed up for 12 weeks.Clinical data before and after rehabilitation exercise were collected and evaluated,including 6-minute walk distance(6MWD),diaphragmatic activity during rest and deep breathing,dyspnea scores(m MRC),and quality of life assessment scales(CAT and SGRQ),and anxiety and depression scales(HAMA and HAMD),etc.to determine the effect of rehabilitation exercise.(4)After the cohort was successfully constructed,in order to explore the relationship among exercise,mitophagy and mitochondria-endoplasmic reticulum coupling in COPD patients,we detected the expression of mitophagy genes(Parkin and PINK1),MERC-related genes(MFN2,IP3 R,GRP75 and VDAC1)and reactive oxygen species(ROS)in peripheral blood of COPD patients before and after exercise,and compared them with healthy controls.(5)A COPD rat model was constructed using tobacco smoke combined with lipopolysaccharide,and then given/without exercise intervention for 8 weeks,respectively.We evaluated mitochondrial function by detecting the levels of ROS,mitochondrial membrane potential and ATP in skeletal muscle tissue.We evaluated mitochondrial function by detecting the levels of ROS,mitochondrial membrane potential and ATP in skeletal muscle tissue.We assessed the level of mitochondrial-endoplasmic reticulum coupling(MERC)by observing the structure of MERC in skeletal muscle tissue by electron microscopy and detecting the expression of MFN2,IP3 R,GRP75 and VDAC1 in MERC.Results:(1)Bioinformatics analysis results showed that exercise may upregulate mitophagy in skeletal muscle of COPD patients through genes such as CASP8,BID,RAB1 A,MLST8,TBK1,RGS19,NAMPT,PEA15,NCKAP1,ERBB2,and HDAC6.(2)The mitophagy genes CASP8,BID,RAB1 A,PEA15,ERBB2,and HDAC6 were changed after exercise in COPD and healthy people,but NCKAP1,TBK1,RGS19,NAMPT,MLST8,and HDAC6 only changed after exercise in COPD,suggesting that exercise has an unique effect on COPD patients that is different from healthy people.(3)The effect of exercise compensatory up-regulation of mitophagy in skeletal muscle of COPD patients is limited,and does not persist with the prolongation of exercise time,but gradually declines after reaching a peak at 8 weeks.(4)After the COPD rehabilitation exercise cohort was constructed,the patients were grouped according to the number of weeks,times and time of rehabilitation exercise.When the three data were inconsistent,the two lowest values were used to group them into 5 groups.Control group(control): refused to exercise,the total number of exercise weeks during the follow-up period was less than 1 week,or the total number of exercise times was less than 5,or the total exercise duration was less than 150 minutes;n=46.Rehabilitation exercise group 1(RE-1): 1 week ≤ total number of exercise weeks < 4 weeks,5 times ≤ total number of exercise times < 20 times,150 minutes ≤ total exercise duration < 1200 minutes;n=31.Rehabilitation exercise group 2(RE-2): 4 weeks ≤ total number of exercise weeks < 8 weeks,20 times ≤ total number of exercise times < 40 times,600 minutes ≤ total exercise duration< 2400 minutes;n=23.Rehabilitation exercise group 3(RE-3): 8 weeks ≤ total number of exercise weeks < 12 weeks,40 times ≤ total number of exercise times < 60 times,1200 minutes ≤ total exercise duration < 3600 minutes;n=40.Rehabilitation exercise group 4(RE-4): exercise weeks=12 weeks,total exercise times=60 times,total exercise duration=3600 minutes;n=34.(5)Compared with before rehabilitation,the 6-minute walking distance(6MWD)and the activity of the diaphragm during deep breathing increased significantly in the RE-3 and RE-4 groups(P<0.05).The comparison between groups also showed that compared with the control group,the RE-3 and RE-4 groups had significantly higher 6MWD and diaphragmatic activity during deep breathing.In addition,there was no significant difference between RE-3 group and RE-4 group(P>0.05).(6)After8 weeks of rehabilitation exercise,the levels of ROS in COPD patients were significantly decreased compared with those before exercise,and the expression levels of mitophagy gene Parkin and MERC-related genes MFN2 and IP3 R were significantly increased compared with those before exercise,and the differences were statistically significant(all P<0.05).(7)Compared with normal rats,rats in COPD group developed lung tissue damage,skeletal muscle atrophy and decreased exercise endurance,increased ROS levels in skeletal muscle tissue,and decreased mitochondrial membrane potential(MMP)and ATP levels(all P<0.05).Compared with the rats in the COPD group,the rats in the COPD+ exercise group had less skeletal muscle tissue atrophy,increased exercise tolerance,and increased levels of MMP and ATP(all P<0.05).There was no significant difference in MMP levels in skeletal muscle between the COPD+ exercise group and the normal rat group(P>0.05).(8)Compared with normal rats,the expression of mitophagy genes such as Parkin,PINK1 and LC3 B in the skeletal muscle tissue of COPD group rats decreased(all P<0.05).Compared with the COPD group,the expression levels of Parkin,PINK1 and LC3B(m RNA only)in the COPD+exercise group were all increased(all P<0.05).(9)The structural integrity of MERC in the skeletal muscle of COPD rats was destroyed,which showed that the proportion of MERC structure in the mitochondrial circumference decreased,and the expression of related genes MFN2,IP3 R and GRP75 decreased.Compared with the rats in the COPD group,the structure of MERC in the skeletal muscle of the rats in the COPD+exercise group was restored,the proportion of MERC in the mitochondrial circumference and the expressions of related genes MFN2,IP3 R and GRP75 were all increased(all P<0.05).Conclusion:(1)Exercise may upregulate mitophagy in skeletal muscle of COPD patients,but this effect is limited and does not persist with prolonged exercise,peaking at 8weeks.(2)Rehabilitation exercise over 8 weeks can significantly increase 6MWD and diaphragmatic activity during deep breathing in patients with COPD,reduce the level of ROS in COPD patients,and increase the expressions of Parkin,MFN2,and IP3 R,suggesting that rehabilitation exercise can relieve the exercise tolerance and partially restore the impaired mitophagy and mitochondria-endoplasmic reticulum coupling in COPD patients.(3)COPD rats showed skeletal muscle dysfunction,including muscle fiber atrophy and decreased exercise tolerance,accompanied by mitochondrial abnormalities,including increased ROS level,decreased MMP and ATP.Exercise can improve muscle atrophy and exercise tolerance,and partially reverse mitochondrial damage.(4)Mitophagy and MERC are impaired in skeletal muscle tissue of COPD rats.Exercise may ultimately improve COPD related skeletal muscle dysfunction by restoring MERC and then activating mitophagy.Targeting MERC may be a new strategy to prevent skeletal muscle dysfunction in COPD.