Effects And Mechanism Of Copper Chelator TETA On Skeletal Muscle Of Aged Mice

Background:Sarcopenia is a syndrome that can lead to adverse outcomes,such as reduced physical function,poor quality of life,and even death.Sarcopenia is characterized by a progressive and widespread decline in skeletal muscle mass and strength.Sarcopenia is highly prevalent among the elderly population and increases the risk of falls and fractures.It also affects post-surgical prognosis,prolongs hospitalization,and can even lead to increased mortality.As a result,sarcopenia is a major disorder that significantly impacts the independence and quality of life of older adults.As life expectancy continues to increase,the prevalence of sarcopenia is expected to rise.As a result,standardized diagnostic criteria,early intervention guidance,and aggressive therapeutic recommendations have been a major focus in both basic and clinical research.It has been demonstrated that the dysregulation of skeletal muscle protein homeostasis,reduction of satellite cell number,and mitochondrial dysfunction are important mechanisms in the onset and progression of sarcopenia.Although research focusing on the key factors of the sarcopenia mechanism is ongoing,no drugs targeting sarcopenia have been approved for clinical use.Therefore,pharmacological studies to effectively alleviate sarcopenia remain a necessary component of sarcopenia management.Caloric restriction（CR）is a mild biological stress response.Reducing caloric intake without causing malnutrition improves overall health and extends lifespan by regulating energy and nutrient sensing pathways,activating autophagy,promoting mitochondrial biogenesis,enhancing antioxidant defense,and improving protein turnover.In addition,CR not only extends the lifespan of living organisms but also enhances skeletal muscle satellite cell regeneration and increases skeletal muscle resistance to atrophy.This,in turn,improves age-related skeletal muscle atrophy and enhances skeletal muscle function.However,achieving the CR effect by altering food intake requires sustained self-regulation accompanied by varying degrees of subjective discomfort.As a result,caloric restriction mimetics（CRMs）that can induce a CR response in the body and avoid fasting intolerance have gained attention.Studies have confirmed that certain CRMs can help delay skeletal muscle atrophy,including acetyltransferase inhibitors like spermine,and deacetylase/SIRT1 activators like resveratrol.They can increase the levels of acetyl coenzyme A in tissue cells and activate the deacetylase SIRT1,which promotes systemic protein deacetylation and activates autophagy.Meanwhile,spermidine has been found to promote satellite cell activation and proliferation.Additionally,resveratrol has been shown to inhibit skeletal muscle protein degradation,improve mitochondrial function,and attenuate skeletal muscle atrophy.In recent years,triethylenetetramine（TETA）has been found to induce CR by increasing the level of tissue cell deacetylation,and is thus considered a novel CRM.Objective:The objective of this study is to investigate whether TETA can inhibit skeletal muscle atrophy in aged mice,explore its mechanism of action on aged skeletal muscle and its relationship with intracellular copper content,and identify new directions for pharmacological treatment of age-related skeletal muscle atrophy.Methods:Six-month-old and 18-month-old C57BL/6 male mice were categorized into a young control group（Y）,an old control group（O）,a group treated with CuSO₄（O-Cu）,a group treated with TETA（O-TETA）,and a group co-treated with CuSO₄and TETA（O-Cu-TETA）.CuSO₄ and TETA treatments were applied for 8 weeks according to the grouping,and isometric pure water was used as a blank control in the Y and O groups.Skeletal muscle strength and anti-fatigue function were assessed through behavioral experiments,including the grip strength test,hanging time test,and treadmill endurance test.Skeletal muscle cross-sectional area was measured using HE staining.Immunohistochemical staining was performed to determine the percentage of Pax7+satellite cells among nucleated cells.Immunofluorescence staining revealed the presence of SDH+mitochondrial oxidative activity and an increase in mitochondrial number.Biochemical assays were conducted to evaluate the intracellular copper content,mitochondrial oxidase（citrate synthase,CS）and antioxidant enzyme activities（superoxide dismutase,SOD,and catalase,CAT）,as well as the oxygen free radical（ROS）content in skeletal muscle cells.Real-time fluorescence quantitative PCR was conducted to evaluate the expression levels of PGC-1α,as well as the mitochondrial fusion and fission factors Mfn2 and Drp1 mRNA.Western blot analysis was conducted to measure the protein expression levels of MyHC,MyoD,MyoG,MuRF-1,MAFbx,SIRT1,FoxO3a,HDAC4,and PGC-1α.Results:Compared with group Y,the copper content in the gastrocnemius muscle of group O was decreased.Compared with the O group,the copper content in the skeletal muscle cells of the O-TETA group did not significantly decrease.In contrast,CuSO₄ treatment significantly increased the copper content in the gastrocnemius muscle cells of aged mice（P<0.05）.The copper content in the gastrocnemius muscle of the O-Cu-TETA group was significantly higher than that of the O group,O-Cu group,and O-TETA group（P<0.0001,P<0.0001,P<0.0001）.Compared with the O group,there was no statistical difference in the behavioral testing indicators between the O-TETA group and the O-Cu-TETA group.However,the grip strength test,hanging time test,and treadmill endurance test of the O-Cu group all showed significant decreases（P<0.0001,P<0.05,P<0.01）.The grip strength of the O-TETA group was significantly greater than that of the O-Cu group（P<0.05）.The cross-sectional area of the gastrocnemius muscle cells in the O-TETA group was the largest at 1199.45±612.89μm²（149.868～7093.28μm²）,while the cross-sectional area of the O-Cu group was the smallest at 787.53±457.82μm²（98.56～3319.16μm²）.Compared to the O group,the myocyte area of the gastrocnemius muscle was significantly reduced in the O-Cu group（P<0.0001）,while it was significantly increased in the O-TETA group and the O-Cu-TETA group（P<0.0001,P<0.01）.The cross-sectional area of myocytes in the O-TETA group and O-Cu-TETA group was significantly larger than that in the O-Cu group,showing a statistically significant difference（P<0.0001 and P<0.0001）.Compared to the O group,the proportion of satellite cells in the O-Cu group was significantly reduced（1.42%±0.48%）（P<0.01）,while the proportion of satellite cells in the O-TETA group was significantly increased（3.19%±0.52%）（P<0.01）.However,the number of satellite cells in the O-Cu-TETA group did not change significantly.Compared with the O-Cu group and the O-Cu-TETA group,the O-TETA group had significantly more satellite cells（P<0.0001,P<0.001）.The proportion of satellite cells in the O-Cu-TETA group was also significantly higher than that in the O-Cu group（P<0.01）.Myocyte biochemical testing revealed that the CS activity of the O-TETA group was significantly increased compared to the O group and the O-Cu group（P<0.001,P<0.01）.However,there was no statistical difference in the increased CS activity between the O group and the O-Cu group,as well as between the O-Cu group and the O-Cu-TETA group.Moreover,the O-Cu-TETA group exhibited an increasing trend compared to the O-Cu group and a decreasing trend compared to the O-TETA group.There was no statistically significant difference in the reactivity of SDH among the treatment groups.Real-time fluorescence quantitative PCR analysis of gastrocnemius muscle mitochondria revealed that,compared to the elderly control group,the relative expression level of Mfn2 mRNA,a promoter of mitochondrial fusion,was decreased in all treatment groups,with the O-Cu group showing the most significant reduction（P<0.0001）.Compared with the O-TETA group and the O-Cu-TETA group,the relative expression level of Mfn2 mRNA in the O-Cu group was significantly lower（P<0.0001,P<0.0001）.Compared to the O group,the relative expression level of Drp1 mRNA,which promotes mitochondrial fission,also decreased in each treatment group.The decrease in the O-Cu group was statistically significant（P<0.0001）.The relative expression levels of Drp1 mRNA in the O-TETA group and O-Cu-TETA group were both higher than those in the O-Cu group（P<0.0001,P<0.0001）.Compared to the O-Cu-TETA group,the O-TETA group showed a statistically significant increase in the relative expression level of Drp1 mRNA（P<0.05）.Compared to the O group,there were no statistically significant differences in the expression levels of PGC-1α mRNA and protein in each treatment group.However,the expression levels of mRNA and protein in the O-TETA group showed an increasing trend.Western blot testing revealed that the MyHC protein levels in the O-TETA group and O-Cu-TETA group were significantly increased compared to the O group（P<0.0001,P<0.001）.Moreover,the levels of MyHC protein in the O-TETA group and O-Cu-TETA group were significantly higher than those in the O-Cu group（P<0.0001,P<0.001）.Additionally,the MyHC protein level in the O-Cu-TETA group was higher than that in the O-Cu group.The protein level was lower in the O-TETA group compared to that in the O-TETA group（P<0.05）.When compared with the O group or the O-Cu group,the MyoD protein levels were significantly increased in the O-TETA group and the O-Cu-TETA group（O group vs O-TETA group,P<0.01;O group vs O-Cu-TETA group,P<0.05;O-Cu group vs O-TETA group,P<0.01;O-Cu group vs O-Cu-TETA group,P<0.05）.However,there was no statistical difference between the O-TETA group and the O-Cu-TETA group.There was no statistically significant difference in MyoG protein levels among the groups,but a noticeable downward trend was observed in the two CuSO₄-treated groups.Compared to the O group,the MAFbx protein level significantly increased in the O-Cu group（P<0.05）.However,there was no statistically significant difference between Mu RF-1 and MAFbx in the other groups.The changes in SIRT1 as upstream regulators of skeletal muscle morphology and function are exciting.Compared to the O group,the SIRT1 protein content in the gastrocnemius muscle of mice increased in each treatment group,and there was a statistically significant difference between the O-TETA group and the O-Cu group（P<0.001,P<0.001）.In addition,the SIRT1protein level in the O-Cu-TETA group was significantly lower than that in the O-Cu group and O-TETA group（P<0.01,P<0.01）.Compared to the O group,there was no statistically significant difference in the FoxO3a protein level among the treatment groups.However,there was an increasing trend in the O-Cu group and O-TETA group,while a downward trend was observed in the O-Cu-TETA group.The protein level of HDAC4 in each treatment group showed an increasing trend compared to the O group,with the trend being more pronounced in the O-Cu group and O-Cu-TETA group than in the O-TETA group.Conclusion:As age increases,the copper content in skeletal muscle cells decreases.TETA can up-regulate SIRT1 independently of copper content and improve the resistance to atrophy of aging skeletal muscle without causing copper deficiency in skeletal muscle cells.TETA increases the expression levels of MyHC and MyoD by up-regulating the expression of SIRT1.This leads to an increase in the cross-sectional area of skeletal muscle cells and promotes the activation and proliferation of satellite cells.Consequently,it increases the number of satellite cells,enhancing the regeneration and repair potential of skeletal muscle.TETA can also reduce the expression of mitochondrial Mfn2 and Drp1,down-regulate the balance of mitochondrial dynamics,and increase the activity of mitochondrial CS without increasing mitochondrial volume density,thereby improving age-related mitochondrial dysfunction.Since TETA is the main active ingredient of trientine,we speculate that trientine may be an effective drug for the treatment of primary sarcopenia.Therefore,this study represents a novel exploration of pharmacological treatment for sarcopenia and offers new directions for future clinical research.