Effects And Mechanism Study Of Muscle-derived E₂ In The Exercise-induced Improvement Of Mitochondrial Function In Skeletal Muscle

ObjectiveEstrogen is a steroid hormone that plays a crucial role in the development of female secondary sex characteristics,regulation of fertility,and protection against oxidative stress and mitochondrial damage.Postmenopausal women,who experience a decline in estrogen levels,are more susceptible to systemic skeletal muscle dysfunction accompanied by mitochondrial dysfunction.Estrogen replacement therapy（ERT）has been found to mitigate these effects,suggesting a direct or indirect relationship between estrogen levels and skeletal muscle mitochondrial status in postmenopausal women.Exercise has been identified as one of the most effective ways to prevent skeletal muscle functional decline and maintain muscle health.Our previous research has demonstrated that exercise training promotes aromatase expression,the rate-limiting enzyme for estradiol（E₂）synthesis in skeletal muscle tissue,and elevates skeletal muscle E₂ levels.However,it remains unclear whether myogenic E₂ mediates the protective effects of exercise on skeletal muscle by enhancing antioxidant capacity and mitochondrial function.This study will establish a mouse model with low estrogen levels and combine it with muscle-specific aromatase（ARO）gene knockout,supplementing them with E₂ or intervening with exercise.We aim to clarify the impact of exercise training on skeletal muscle aromatase expression,endogenous E₂ production,skeletal muscle antioxidant capacity,and mitochondrial function in mice under low estrogen conditions.Additionally,we will explore the role and mechanism of myogenic E₂ in improving skeletal muscle function in mice through exercise training and provide a theoretical and experimental basis for preventing skeletal muscle function decline in postmenopausal women through exercise.MethodsPart I Experiment:Eight-week-old female C57BL/6J mice were randomly assigned to a sham surgery group（Sham）,an ovariectomy surgery group（OVX）,and an ovariectomy surgery plus exercise training group（OVX+ET）（n=12）.The OVX mouse model was constructed by bilateral ovariectomy surgery,and the exercise group mice underwent moderate-intensity treadmill training for eight weeks.During the intervention period,the mice’s body composition,grip strength,and anti-fatigue capacity were measured.After the intervention,changes in the skeletal muscle morphology,antioxidant capacity,mitochondrial function,related molecular expression,and changes in skeletal muscle aromatase activity/expression,E₂ level,and estrogen receptor expression were examined.Specific indicators were measured as shown in the in vivo experimental technology roadmap I.This part of the experiment aimed to verify the exercise adaptation effect of the skeletal muscle in OVX mice and to clarify whether its mechanism is related to changes in skeletal muscle antioxidant capacity,mitochondrial function,and skeletal muscle E₂ levels.Part II Experiment:The prepared aromatase gene（ARO）Flox heterozygous mice and muscle-specific Cre-positive mice were used for breeding to obtain progeny Flox mice and muscle tissue ARO gene pure knockout（MS-ARO-CKO）mice.Female Flox mice and MS-ARO-CKO mice were randomly divided into the Sham group,OVX group,KO+Sham group,and KO+OVX group（n=6）.In addition,male Flox mice and MS-ARO-CKO mice were assigned to the WT and KO groups.Female mice were subjected to the same ovariectomy protocol as in the first part.Body weight,body composition,grip strength,fatigue resistance,and skeletal muscle E₂ levels of mice in the different groups were examined,as shown in the experimental technical roadmap II.The main objective of this part of the experiment was to elucidate the effect of skeletal muscle ARO gene deletion on skeletal muscle E₂ levels and function in mice under normal or low estrogen conditions.Part III Experiment:Eight-week-old MS-ARO-CKO mice were used as experimental subjects and randomly divided into the Sham,OVX,OVX+E₂（E₂ supplementation group）,and OVX+ET group（n=6）.The ovariectomy surgery and exercise training program was the same as in Part I.E₂ was supplemented using subcutaneously implanted sustained-release pellets,and the supplementation time was synchronized with the exercise intervention time.After the intervention,changes in skeletal muscle mitochondrial function,antioxidant capacity,related molecular expression,and skeletal muscle E₂ receptor expression were measured in the mice,with specific detection indicators as shown in the experimental technical roadmap III.This part of the experiment aimed to demonstrate whether myogenic E₂ is involved in the protective effect of exercise training on skeletal muscle mitochondria in OVX mice.Results1.Results of Part I of the experiment:（1）The effects of exercise on skeletal muscle mass and function in OVX miceCompared with the Sham group,the OVX group mice had mainly white blood cells in vaginal secretions,uterine atrophy,and decreased serum E₂ levels（P<0.01）;There were no significant changes in skeletal muscle wet weight,fiber cross-sectional area,and collagen fiber level,but grip strength and anti-fatigue ability decreased（P<0.01）;The phosphorylation level of the myosin regulatory light chain associated with muscle strength in the gastrocnemius muscle decreased（P<0.05）.Compared with the OVX group,the OVX+ET group mice had a reduced body fat rate and increased lean body weight rate（P<0.01）;The anti-fatigue ability of skeletal muscle rose（P<0.05）,and the phosphorylation level of the myosin regulatory light chain associated with muscle strength in the gastrocnemius muscle increased（P<0.01）.（2）The effects of exercise on skeletal muscle mitochondria in OVX miceCompared with the Sham group,the number of gastrocnemius muscle mitochondria decreased,and some mitochondrial abnormalities in the interstitial fibers of the muscle increased in the OVX group mice;ATP production decreased（P<0.01）,and respiratory rates of mitochondrial respiratory chain complex I state 4,complex I and II state 3,complex II state 3,and complex IV state 3 decreased（P<0.05）;The expression levels of mitochondrial respiratory chain complex I,complex II,complex IV,biogenesis-related PGC-1αand TFAM proteins,and fusion protein mfn2 decreased（P<0.05）,while the level of division protein drp1 increased（P<0.01）.Compared with the OVX group,exercise training in the OVX+ET group improved the above effects of gastrocnemius muscle mitochondrial quantity,morphology,and respiratory function induced by OVX;At the same time,the expression of PGC-1α,TFAM,and mfn2 proteins in the gastrocnemius muscle increased（P<0.05）,and the expression of drp1 protein decreased（P<0.01）.（3）The effects of exercise on the antioxidant capacity of skeletal muscle in OVX mice.Compared to the Sham group,the SOD,CAT,and GPx activities related to antioxidant defense in the gastrocnemius muscle of OVX mice decreased（P<0.05）,while MDA content increased（P<0.01）,and the expression levels of Sirt1,p Nrf2,Nrf2,and HO-1related to antioxidant signaling pathways were downregulated（P<0.05）.Compared to the OVX group,the expression of Sirt1,p Nrf2,Nrf2,and HO-1 proteins in the gastrocnemius muscle of OVX+ET mice increased（P<0.05）,and SOD,CAT,and GPx activities increased（P<0.05）.（4）The effects of exercise on the expression and activity of aromatase,E₂ production,and receptor expression in the skeletal muscle of OVX mice.Compared to the Sham group,there were no significant changes in the expression and activity of aromatase in the gastrocnemius muscle of OVX mice,and the E₂ content in the muscle decreased（P<0.01）,with downregulation of estrogen receptor ERαexpression（P<0.01）and upregulation of ERβexpression（P<0.01）.Compared to the OVX group,the expression and activity of aromatase in the gastrocnemius muscle of OVX+ET mice increased（P<0.05）,and the E₂ level in the muscle increased（P<0.01）,with upregulation of ERαexpression（P<0.05）and no significant changes in ERβexpression.2.Results of Part II of the experiment:（1）Validation of Muscle Tissue-specific ARO Gene Knockout（KO）in MiceCompared to the WT group,ARO gene KO led to a lack of ARO expression in the gastrocnemius,tibialis anterior,soleus,and extensor digitorum longus muscles in mice,and a decrease in ARO expression in the heart muscle,while the ARO protein expression levels in other tissues such as the kidney,cerebral cortex,and liver remained unchanged.（2）The Effects of Muscle Tissue-specific ARO KO on Body Composition and Skeletal Muscle Function in Mice:Compared to the same-sex WT group,ARO KO led to a decrease in skeletal muscle fatigue resistance in male and female mice（P<0.05;inverted grid test,male KO group vs.WT group,P=0.08）;body weight,fat content,and lean body weight were not significantly affected.（3）The Effects of Muscle Tissue-specific ARO KO on Skeletal Muscle E₂ Levels in Male and Female Mice:Compared to the same-sex WT group,ARO KO led to a decrease in E₂ levels in the gastrocnemius muscles of male and female mice（P<0.05）.In addition,compared to the KO+Sham group,KO+OVX mice had a lower E₂ level in the gastrocnemius muscles（P<0.05）.3.Results of Part III of the experiment:（1）Effects of exercise or E₂ supplementation on skeletal muscle mitochondria in KO+OVX mice.Compared with the KO group,the KO+OVX group of mice showed an abnormal increase in partially enlarged mitochondria between muscle fibers in the gastrocnemius muscle tissue,disordered or missing cristae,vacuolated mitochondria,decreased relative mitochondrial DNA content and ATP production（P<0.01）,and a decrease in respiratory rates at complex I state 4,complex I state 3,complex I and II state 3,complex II state 3,and complex IV state 3（P<0.05）;The protein expression of complex I-V,PGC-1α,TFAM,and mfn2 decreased（P<0.01）,and the protein expression of drp1increased（P<0.01）.Compared with the KO+OVX group,the KO+OVX+E₂ group showed that E₂ supplementation improved the changes in mitochondrial number,morphology,and respiratory function induced by OVX（P<0.05）,upregulated ATP content and mitochondrial DNA level（P<0.05）,promoted the protein expression of complex I-V,PGC-1α,TFAM,and mfn2（P<0.05）,and inhibited the protein expression of drp1（P<0.05）.Compared with the KO+OVX group,the KO+OVX+ET group showed similar effects as E2 supplementation with exercise training,improved abnormal mitochondrial morphology in the gastrocnemius muscle induced by OVX,increased respiratory rates at complex I state 3,complex I and II state 3,complex II state 3,and complex IV state 3（P<0.01）,increased ATP production and relative mitochondrial DNA content（P<0.05）,upregulated the protein expression levels of complex I,complex II,complex III,complex V,PGC-1α,TFAM,and mfn2（P<0.05）,and inhibited the protein expression of drp1（P<0.01）.（2）Effects of exercise or E₂ supplementation on the antioxidant capacity of skeletal muscle in KO+OVX miceCompared with the KO group,the SOD activity related to antioxidant capacity in the gastrocnemius muscle of KO+OVX mice decreased（P<0.05）,and the protein expressions of Sirt1,p Nrf2,Nrf2,and HO-1 also decreased（P<0.05）.Compared with the KO+OVX group,the SOD,CAT,and GPx activities in the gastrocnemius muscle of KO+OVX+E₂ mice increased（P<0.01）,and the MDA content decreased（P<0.01）,along with an increase in the protein expressions of Sirt1,p Nrf2,Nrf2,and HO-1（P<0.01）.However,there were no significant changes in the antioxidant enzyme activity,MDA content,and antioxidant signal-related protein expressions in the skeletal muscle of KO+OVX+ET mice compared with the KO+OVX group.（3）Effects of exercise on ERs expression in the skeletal muscle of OVX miceCompared with the KO group,the expression of ERαin the gastrocnemius muscle of KO+OVX mice was downregulated（P<0.01）.However,the expression levels of ERαin the gastrocnemius muscle of KO+OVX+E₂ and KO+OVX+ET mice increased（P<0.01）compared with the KO+OVX group.（4）Effects of exercise on the phosphorylation level of myosin regulatory light chain in the skeletal muscle of OVX miceCompared with the KO group,the phosphorylation level of the myosin regulatory light chain in the gastrocnemius muscle of KO+OVX mice decreased（P<0.05）.However,the phosphorylation level of the myosin regulatory light chain increased（P<0.05）in the gastrocnemius muscle of KO+OVX+E₂ and KO+OVX+ET mice compared with the KO+OVX group.ConclusionThis study constructed an animal model with low estrogen levels,combined with in vivo muscle-specific aromatase（ARO）gene knockout,and used interventions such as exercise and E₂ supplementation to analyze the experimental results.The main conclusions are as follows:（1）Ovariectomy combined with muscle-specific aromatase knockout technology can cause low circulation and skeletal muscle estrogen levels.（2）Under conditions of low estrogen levels,exercise training can upregulate aromatase expression and activity,promote the generation of muscle-derived E₂,upregulate the Sirt1/Nrf2/HO-1 signaling pathway to enhance antioxidant capacity,and maintain the redox homeostasis of muscle cells.（3）Under conditions of low estrogen levels,exercise training may upregulate the expression of ERα,promote mitochondrial biogenesis and dynamics,improve mitochondrial quality and function,and thereby improve skeletal muscle function.