| Objective sarcopenia is an age-related skeletal muscular disease,characterized by generalized,progressive decline of muscle mass and function.Sarcopenia increased risks of the patients underwent surgery and adversely impact the quality of life and life span of old individuals.The mechanism of sarcopenia remains unclear and there’s no proofed pharmacological treatment for sarcopenia by now.Exercise interventions are regarded to be the primary treatment for sarcopenia,but the individual treatment of exercise interventions is hard to performed because its complicate mechanism.In current study,we focused on the improvement of sarcopenia by exercise interventions,to investigate the role of Nrf2 on regulating the morphology and function of mitochondria in skeletal muscle tissue during exercise interventions.Besides,we also investigate the effects of the activator of Nrf2,sulforaphane(SFN),as a pharmacological treatment for sarcopenia.Our study aims to illustrate the function of activating Nrf2 by exercise in improving sarcopenia,and in order to provide new ideas for the treatment of sarcopenia by targeting Nrf2.Methods 1.The exercise intervention model was established by treadmill,aged(20-22 months old)Nrf2 knockout(Nrf2-KO)mice and age-matched wild-type(WT)C57BL6/J mice were randomly divided into 4 groups: WT-Sed,WT-Ex,KO-Sed and KO-Ex.For exercise group,8-week exercise interventions were performed,while the mice in sedentary group were raised in the cage without daily exercise intervention by treadmill.The skeletal muscle function of each group was examined by treadmill test,hanging test,rotarod test and etc.And the frailty score was also assessed.Ultrasonography,H&E staining and Sirius red staining were used to evaluate effects of exercise interventions on skeletal muscle tissue.Western blot,q PCR and activity assay of specific enzymes were performed to examine the changes of age-related markers,genes regulating energy metabolism of mitochondria and enzymes of aerobic respiration.Besides,transmission electron microscope(TEM)was carried out to observe the morphology of mitochondria in skeletal muscle tissue of each group,and the number of abnormal mitochondria was also calculated to investigate the effects of Nrf2 on the morphology and function of mitochondria during the improvement of sarcopenia by exercise interventions.2.Knockdown of Nrf2 or Keap1(overexpressing Nrf2)in senescent mouse C2C12 myoblasts were performed in vitro and C2C12 myoblasts were then induced to differentiate into mature C2C12 myotubes.Western blot,q PCR and other methods were used to explore the effects of Nrf2 on the senescent phenotype of myotubes and the expression of mitochondrial internal reference proteins.The differences in energy metabolism and mitochondrial function of senescent C2C12 myotubes with different Nrf2 expression levels were evaluated by ATP content and Seahorse XF cell mito stress test.Mitochondrial membrane proteins were stained by immunofluorescence to observe the effects of knockdown or overexpression of Nrf2 on mitochondrial morphology in C2C12 cells.The expressions of the proteins that related to mitochondrial dynamics in the skeletal muscle tissue in each group were examined,and Drp1 was identified as the potential target that may be regulated by Nrf2 both in vitro and in vivo.The possible mechanism of Nrf2 regulating Drp1 was further verified by transcription site prediction of transcription factor binding sites(TFBS),protein phosphorylation level determination of Drp1 protein stability.3.The treatment of sulforaphane(SFN),a specific activator of Nrf2,was performed both in vitro and in vivo.The protein expression of Nrf2,Drp1 and OXPHOS complex in senescent C2C12 myotubes after SFN treatment was examined.The improvement of metabolic level of senescent C2C12 myotubes by SFN was determined by ATP content assay and Seahorse XF cell mito stress test.As for the in vivo study,C57BL6/J mice were divided into 4 groups according to the genotype and whether SFN treatment was performed,WT-Control,WT-SFN,KO-Control and KO-SFN group.The skeletal muscle function and frailty score were assessed,and effects of SFN on the expression of Drp1 protein,age-related markers and mitochondrial internal reference proteins were examined.Results 1.The results showed that exercise intervention can significantly improve the skeletal muscle function of aged wild-type mice.The ultrasonography,section staining of skeletal muscle tissue indicated that exercise intervention significantly enhanced SWE of skeletal muscle,and increased diameter of muscle fiber,and reduces interstitial fibrosis of skeletal muscle tissue.Besides,exercise intervention reduced the expression of aging-related genes and increase the Sirt1 and Sirt3 proteins,which were reported to exert anti-aging functions in skeletal muscle tissue,and increased activity of succinate dehydrogenase(SDH)and citrate synthase(CS).TEM showed that the average mitochondrial size was significantly reduced and the proportion of abnormal mitochondria increased after exercise intervention in wild-type mice.However,Nrf2 knockout impaired the improvements of exercise intervention on senescent skeletal muscle tissue,suggesting the important role of Nrf2 in improving mitochondrial morphology and function in skeletal muscle tissue,as well as sarcopenia-like phenotypes.2.Knockdown of Nrf2 in senescent C2C12 myoblasts aggravated the senescent phenotypes of myotubes and decreased the expression of mitochondrial internal reference proteins.ATP content and oxygen consumption rate(OCR)were also declined after knockdown of Nrf2,indicating the impaired metabolic process of myotubes.In contrast,knockdown of Keap1 restored the senescence phenotypes of C2C12 myotubes and the expression of mitochondrial internal reference proteins.Besides,the ATP content and OCR were increased,suggesting that Nrf2 overexpression significantly improved the energy metabolism of senescent myotubes.In addition,immunofluorescence of TOM20 demonstrated the elongated mitochondria in C2C12 Nrf2-si cells and granular-shaped mitochondria in C2C12 Keap1-si cells,indicating that Nrf2 was related to the alternation of mitochondrial morphology and maintain the mitochondrial function in senescent C2C12 cells.The further study in vivo and in vitro screened the proteins that associated with mitochondrial dynamics to verify the possible regulatory mechanism of Nrf2.We found that Nrf2 is likely to stabilize Drp1 protein and increase its intracellular protein level by mediating the ubiquitination of Drp1 to promote Drp1-induced mitochondrial fission process in aging myotubes,which contributes to clearance of abnormal mitochondria,mitochondrial quality control and ultimately improving mitochondrial function in skeletal muscle cells.3.The vitro experiments of senescent C2C12 myotubes indicated that SFN treatment could significantly increase the levels of Nrf2 and Drp1 proteins in myotubes in a dose-dependent manner.Besides,SFN treatment restored the senescent phenotype of myotubes and significantly increased cellular ATP content and the OCR of cells.In vivo experiments further confirmed that SFN can significantly improve the skeletal muscle function and the pathological phenotypes of sarcopenia,and increase the protein expression level of Drp1 in skeletal muscle tissue in aged wildtype mice.However,the therapeutic effects of SFN were not significantly observed in Nrf2-KO mice.These results suggests that the effects of SFN in improving the sarcopenia-like phenotypes may carry out in a Nrf2-dependant manner.Conclusions Activating Nrf2 signaling is necessary for the improvement of sarcopenia and mitochondrial function in skeletal muscle tissue during exercise.Conversely,Nrf2 deficiency could significantly blunt the effects of exercise interventions in improving sarcopenia.The underlying mechanism indicates that increase the stability of Drp1 through deubiquitinating and promote Drp1-dependent mitochondrial fission to attenuate mitochondrial disorder.The present study illustrated the mechanism of activating Nrf2 by exercise interventions.Further studies investigate the effects of Nrf2 activator on improving mitochondrial function of senescent skeletal muscle tissue and sarcopenia-like phenotypes,which provided a theoretical basis for exploring pharmacological treatments of sarcopenia targeting Nrf2. |
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