| Objective:Skeletal muscle accounts for approximately 40% of an adult’s body weight and plays a dynamic role during exercise while maintaining multiple homeostatic mechanisms in the body,including glucose metabolism and thermogenesis.Skeletal muscle is plastic,which can respond to changes in the environment,and is an important energy buffer pool for the body.Alcohol abuse can impair the function of multiple organs,including the liver,skeletal muscle,heart,and pancreas.Alcoholic myopathy is a muscle disease caused by alcohol intoxication.Long-term alcoholism can lead to skeletal muscle atrophy.Whether acute intoxication or chronic alcoholism,alcohol abuse leads to a huge disease burden and is a public health concern.Nuclear factor erythroid 2-related factor 1(NFE2L1,also known as Nrf1),a member of the CNC-b ZIP family,is a key factor in cellular adaptive responses.It has been found in multiple tissues(including heart,fat,liver,kidney,and brain,etc.)that NFE2L1 plays an important role in maintaining cellular homeostasis and organ integrity,but the specific role of NFE2L1 in skeletal muscle tissue has not been studied.This paper is divided into three parts: In the first part,we analyze whether human NFE2L1 gene polymorphism is associated with lean body mass and grip strength in the UK Biobank cohort;the content and function of skeletal muscle in striated muscle cell-specific Nfe2l1 knockout mice.In the second part,we explore the mechanism of Nfe2l1 deletion leading to muscle atrophy.In the third part,we study the expression levels of NFE2L1 in skeletal muscle tissue and C2C12 cells after acute alcohol exposure.We explore the effect of Nfe2l1 deletion on the occurrence of acute alcoholic myopathy.Through three parts of the study,the aim is to take NFE2L1 as the starting point,to explore its role in the maintenance of skeletal muscle homeostasis and the occurrence of alcoholic myopathy.We try to explore the potential of NFE2L1 as a targeted therapy for myopathy,and to provide a scientific basis for precise prevention.Methods:1.To assess the association of NFE2L1 variants with lean mass and hand grip strength,a candidate gene association study was performed in the UKB cohort.Variants within NFE2L1(GRCH37 chr17: 48048359-48061545)and its flanking 10 kb length(GRCH37 chr17: 48038359-48071545)were extracted as test genotypes.Appendicular lean mass(ALM)and hand grip strength(HGS)were used as phenotypes to assess muscle strength.2.The changes of body weight and body composition were monitored in striated muscle cell-specific Nfe2l1 knockout mice,and the motor function of the mice was evaluated by means of a wheel-type fatigue apparatus,a rotarod-type fatigue apparatus,and a grasping force meter.The skeletal muscle tissues and other important organs of mice in different ages were harvested,weighed and stored.The total RNA of skeletal muscle tissue was extracted,and the transcription levels of genes such as inflammatory indicators and protein degradation were detected.The total protein of the tissue was extracted to detect mitochondrial function and other related proteins.The skeletal muscle tissues were collected for multi-omics detection and analysis;Skeletal muscle tissue was fixed with paraformaldehyde,H&E stainings,immunohistochemistry and immunofluorescence experiments were performed.The fresh skeletal muscle tissues were frozen sectioned,and oil red staining,immunofluorescence and other experiments were performed to determine the changes of muscle fibers.3.Using the C2C12 myoblast cell line in vitro experiments,acute alcohol treatment was given,and samples at different time points were collected.Total RNA was extracted to detect the transcription level of Nfe2l1 gene;total protein was extracted to detect NFE2L1 protein,which indicated whether alcohol exposure affected the expression of NFE2L1.4.The mice were treated with acute alcohol,collect mouse skeletal muscle tissue at the specified time point.The total RNA of skeletal muscle tissue was extracted,and the transcription levels of genes such as inflammatory indicators and protein degradation were detected.The total protein of the tissue was extracted to detect related proteins such as NFE2L1 and mitochondrial function.The skeletal muscle tissue was fixed with paraformaldehyde and then embedded.H&E staining and immunohistochemical experiments were performed to determine the damage of skeletal muscle.Results:1.NFE2L1 variants were associated with human muscle composition.There were 13 and 6 NFE2L1 variants were associated with ALM and HGS through a candidate gene association analysis,respectively.2.The deletion of Nfe2l1 results in muscle atrophy characterized by reduced type IIb myofibers.The results showed that the deletion of Nfe2l1 in striated muscle cells can lead to weight loss in mice.The results of body composition and organ coefficient showed that the mass of skeletal muscle tissue in different parts was significantly reduced compared with the control group(P < 0.05),accompanied by weakened muscle strength(P < 0.05).Organ coefficients were all calibrated using tibia length(no statistical difference between groups).Pathological examination showed that age-related muscle atrophy appeared after H&E staining in skeletal muscle tissue,which was characterized by a decrease in the area of muscle cells(P < 0.05)and an increase in nuclei.At the same time,the combination of oil red stainings and Sirius red stainings proved the occurrence of fat accumulation and fibrosis in muscle fibers.The occurrence of inflammatory reaction was confirmed by mRNA levels and immunohistochemical experiments.Immunofluorescence results showed that the type IIb myofibers in adult Nfe2l1(SM)-KO mice were significantly reduced.Notably,compared with female mice,male mice developed phenotype earlier and had relatively more severe muscle tissue lesions.Although severe skeletal muscle atrophy and weakening of muscle strength occurred in adult mice,skeletal muscle did not change significantly in juvenile mice.3.Deletion of Nfe2l1 leads to weakened proteasome function in muscle cells.Single-nucleus RNA sequence,proteomics and non-targeted metabolomics experiments were performed in juvenile mice.The combined analysis of multiple omics found that the function of proteasome was significantly weakened.This change may be the root cause of skeletal muscle atrophy..4.Acute alcohol exposure increases the expression of NFE2L1 in skeletal muscle tissue and cells.After acute alcohol exposure,there was no significant change in skeletal muscle tissue,and the expression of serum creatine kinase showed a trend of increasing,suggesting signs of damage to skeletal muscle tissue.Acute alcohol exposure increased the expression of NFE2L1 in skeletal muscle tissue and cells,and showed a time-toxic effect relationship in C2C12 cells.That is,as a key responsive molecule to environmental exposure,the transcription factor NFE2L1 can be activated during alcohol exposure.5.Binge alcohol exposure leads to muscle damage in Nfe2l1(SM)-KO mice,and mitochondrial function is weakened.It was found by mRNA and immunohistochemical experiments that compared with the control group,the muscle tissue had an inflammatory response after binge alcohol exposure in Nfe2l1(SM)-KO mice(P < 0.05).The H&E staining experiment found that the skeletal muscle cells had obvious morphological changes,increased nuclei,and disordered muscle fiber structures after binge alcohol exposure.Detection of mitochondrial function-related indicators found that alcohol binge drinking led to a weakening trend of mitochondrial function in mice.Conclusion:1.NFE2L1 gene variants is associated with human muscle composition.2.Deletion of Nfe2l1 in striated muscle cells leads to age-related muscle atrophy characterized by reduced type IIb myofibers,and the function of proteasome in muscle cells is weakened.3.Acute alcohol exposure leads to increased expression of NFE2L1 in skeletal muscle tissue and cells.The muscle in Nfe2l1(SM)-KO mice were significantly damaged after binge alcohol exposure,suggesting that NFE2L1 exerts a protective effect on muscle tissue. |
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