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SIRT5 safeguards against primate skeletal muscle ageing via desuccinylation of TBK1

Abstract

Ageing-induced skeletal muscle deterioration contributes to sarcopenia and frailty, adversely impacting the quality of life in the elderly. However, the molecular mechanisms behind primate skeletal muscle ageing remain largely unexplored. Here, we show that SIRT5 expression is reduced in aged primate skeletal muscles from both genders. SIRT5 deficiency in human myotubes hastens cellular senescence and intensifies inflammation. Mechanistically, we demonstrate that TBK1 is a natural substrate for SIRT5. SIRT5 desuccinylates TBK1 at lysine 137, which leads to TBK1 dephosphorylation and the suppression of the downstream inflammatory pathway. Using SIRT5 lentiviral vectors for skeletal muscle gene therapy in male mice enhances physical performance and alleviates age-related muscle dysfunction. This study sheds light on the molecular underpinnings of skeletal muscle ageing and presents the SIRT5–TBK1 pathway as a promising target for combating age-related skeletal muscle degeneration.

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Fig. 1: Phenotypic alterations in skeletal muscles of old cynomolgus monkeys.
Fig. 2: Inflammation is a pronounced feature of primate skeletal muscle ageing.
Fig. 3: SIRT5 deficiency induces senescence in hMyotubes.
Fig. 4: SIRT5 interacts with and desuccinylates TBK1.
Fig. 5: SIRT5 desuccinylates TBK1 at Lys137 and facilitates its dephosphorylation.
Fig. 6: Inactivation of TBK1 cascade alleviates senescence and inflammation in hMyotubes.
Fig. 7: SIRT5-based gene therapy ameliorates skeletal muscle dysfunction in aged mice.

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Data availability

RNA-seq data have been deposited in the Genome Sequence Archive in the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation) of the Chinese Academy of Sciences, under accession numbers HRA007459 and CRA017135 (refs. 96,97). The data based on LC–MS/MS have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) through the iProX partner repository under accession numbers PXD052992 and PXD052984 (refs. 98,99). Source data are provided with this paper.

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Acknowledgements

We thank Z. Diao for helping with the generation and characterization of SIRT5+/+ and SIRT5−/− hES cells. We thank L. Bai, R. Bai, J. Lu, Y. Yang and X. Li for their administrative assistance, and J. Jia and C. Xie for their help with animal experiments. We also thank J. Jia (Institute of Biophysics, Chinese Academy of Sciences) for help with FACS experiments, and J. Wang (Institute of Biophysics, Chinese Academy of Sciences) for his help in LC–MS/MS. This work was supported by the National Natural Science Foundation of China (82488301 to G.-H.L. and J.Q.; 82122024 to S.W.; 82125011 to J.Q.; 82071588 to S.W.; 92468303 to S.W. and X.F.; 81921006 to G.-H.L. and J.Q.; 92149301 to G.-H.L. and S.W.; 92168201 to G.-H.L.; 82361148131 to W.Z.; 82172447 to X.Z.; 82472459 to X.Z.), the STI2030-Major Projects (2021ZD0202400 to S.W. and Q.Z.), the National Key Research and Development Program of China (2020YFA0804000 to G.-H.L. and S.W.; 2022YFA1103700 to W.Z., S.M. and J.Q.; 2023YFC3605400 to Q.Z.), Non-Communicable Chronic Diseases-National Science and Technology Major Project (2024ZD0530400 to J.Q.), the National Natural Science Foundation of China (82330044 to G.-H.L.; 32341001 to G.-H.L.; 32121001 to W.Z.; 82192863 to W.Z.; 82361148130 to G.-H.L. and J.Q.; 8231101626 to J.Q.; 32300980 to H.H.; 82322025 to S.M.; 82271600 to S.M.; 82471586 to S.W.; 32400968 to Y.J.; 82422031 to H.S.), CAS Project for Young Scientists in Basic Research (YSBR-076to G.-H.L. and J.Q.; YSBR-012 to W.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDC0200000 to S.M.; XDA0460403 to W.Z.), the Program of the Beijing Natural Science Foundation (Z240018 to G.-H.L. and S.W.; JQ24044 to W.Z.; Z230011to J.Q.), the Informatization Plan of Chinese Academy of Sciences (CAS-WX2022SDC-XK14 to G.-H.L.), New Cornerstone Science Foundation through the XPLORER PRIZE (2021-1045 to G.-H.L.), Beijing Municipal Public Welfare Development and Reform Pilot Project for Medical Research Institutes (JYY2023-13 to W.Z.), CAS Youth Interdisciplinary Team to W. Z., Key Laboratory of Alzheimer’s Disease of Zhejiang Province (ZJAD-2024001 to J.Q.), Shenzhen Medical Research Fund (C2406001 to G.-H.L.), Beijing Hospitals Authority Youth Programme (QML20230806 to Q.Z.), Excellent Young Talents Program of Capital Medical University (12300927 to S.W.), The Project for Technology Development of Beijing-affiliated Medical Research Institutes (11000023T000002036310 to S.W.), Excellent Young Talents Training Program for the Construction of Beijing Municipal University Teacher Team (BPHR202203105 to S.W.), Young Elite Scientists Sponsorship Program by CAST (2021QNRC001 to S.M.), Youth Innovation Promotion Association of CAS (2022083 to S.M.), Beijing Science and Technology Nova Cross Program (20220484180 to X.Z.) and Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences (2023IOZ0202 to J.Q.; 2023IOZ0102 to S.M.; 2024IOZ0103 to J.Q.).

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Authors and Affiliations

Authors

Contributions

G.-H.L., S.W. and J.Q. conceived the work and supervised the overall experiments. Q.Z. and Y.J. performed the experiments related to the phenotypic and mechanistic analyses. X.J. performed bioinformatic analyses. G.-H.L., S.W., Q.J., Q.Z., Y.J., X.J., X.Z., F.L., H.H., Z.Z., H.W., S.S., S.M., W.Z., Y.Y., X.F. and G.Z. performed manuscript writing, reviewing and editing. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Jing Qu, Si Wang or Guang-Hui Liu.

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The authors declare no competing interests.

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Nature Metabolism thanks Danica Chen, Vera Gorbunova and Young Jang for their contribution to the peer review of this work. Primary Handling Editors: Jean Nakhle and Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Information summary of cynomolgus monkeys and phenotypical analysis in young and old monkey skeletal muscles.

a. Information summary of the cynomolgus monkeys analyzed in this study. b.WGA staining in young and old monkey skeletal muscles. Left, representative images. Scale bars, 100 μm. Right, the percentages of central nuclei were quantified. Arrows indicate the central nuclei in skeletal muscles. c.The percentage of type IIX (MYH1-positive) fires in young and old monkey skeletal muscles was quantified. d, e. Immunofluorescence staining of LAP2 (d) and HP1γ (e) in young and old monkey skeletal muscles. Left, representative images. Scale bars, 100 μm and 25 μm (zoomed in). Right, the proportion of positively stained nuclei was quantified. Arrows indicate the positively stained nuclei in b, d and e. Two-tailed Student’s t-test was used for statistical analysis and data are shown as the mean ± s.e.m. n = 8 monkeys from both genders per group in be. P values are annotated in the figures.

Source data

Extended Data Fig. 2 Gender-based analysis of transcriptome and quantitative proteome of young and old monkey skeletal muscle.

a. Ring plots showing upregulated and downregulated DEGs in male old versus male young monkey skeletal muscles, and female old versus female young monkey skeletal muscles. b. Representative GO terms and pathways for upregulated and downregulated DEGs in male old versus male young monkey skeletal muscles, and female old versus female young monkey skeletal muscles. c. Ring plots showing upregulated and downregulated DEPs in male old versus male young monkey skeletal muscles, and female old versus female young monkey skeletal muscles. d. Representative GO terms and pathways for upregulated and downregulated DEPs in male old versus male young monkey skeletal muscles, and female old versus female young monkey skeletal muscles. e. Lollipop plots showing the shared upregulated and downregulated GO terms and pathways for upregulated and downregulated DEGs and DEPs in male old versus male young monkey skeletal muscles, and female old versus female young monkey skeletal muscles. Hypergeometric tests were performed in b, d and e. mkMuscle, monkey muscle.

Extended Data Fig. 3 Generation and characterization of WT and SIRT5-deficient hES cells.

a. A quantitative PCR with reverse transcription (RT-qPCR) analysis of the relative transcript levels of SIRT1-7 genes in young and old monkey skeletal muscles. Two-tailed Student’s t-test was used for statistical analysis. n = 8 monkeys from both genders per group. b. Schematic representation of CRISPR-Cas9-mediated SIRT5 targeting strategy in hES cells. c. No off-target (OT) cleavage in top 10 predictive off-target sites against SIRT5 sgRNA in SIRT5−/− hES cells by genomic DNA PCR and DNA sequencing. d. Western blotting analysis of SIRT5 protein level in SIRT5+/+ and SIRT5−/− hES cells. β-tubulin was used as loading control. e. Immunostaining of SOX2, OCT4 and NANOG in SIRT5+/+ and SIRT5−/− hES cells. Scale bars, 50 μm. f. Karyotyping analysis of SIRT5−/− hES cells. g. Immunostaining of TUJ1, SMA and FOXA2 to evaluate the differentiation potentials of SIRT5+/+ and SIRT5−/− hES cells. Scale bars, 250 μm. h. Immunostaining of Ki67 in SIRT5+/+ and SIRT5−/− hES cells. Scale bars, 100 μm. n = 3 biological replicates. Two-tailed Student’s t-test was used for statistical analysis. Data are presented as the mean ± s.e.m. in a and h. The representative results from one of three independent experiments in d and e. P values are annotated in the figures.

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Extended Data Fig. 4 Analysis of mitochondrial activity in SIRT5-deficient human myotubes.

a. Snapshot showing the transcript levels of SIRT1-7 gene in SIRT5+/+ and SIRT5−/− hMyotubes. b. Left, representative images of MyHC-positive myotubes in SIRT5+/+ and SIRT5−/− hMyotubes. Scale bars, 150 μm. Right, the differentiation efficiency of hMyotube was quantified. c. Western blotting analysis of the expression levels of oxidative respiratory chain complex proteins in SIRT5+/+ and SIRT5−/− hMyotubes. GAPDH was used as loading control. The representative results from one of three independent experiments. d. Left, analysis of mitochondria superoxide levels in SIRT5+/+ and SIRT5−/− hMyotubes by using MitoSOXTM Red probe. Scale bars, 100 μm. Right, positively stained area was quantified. e. Western blotting analysis of 4-HNE modified protein and SOD1 protein levels in SIRT5+/+ and SIRT5−/− hMyotubes. GAPDH was used as loading control. Two-tailed student’s t-test was used for statistical analysis and data are presented as the mean ± s.e.m. n = 3 biological replicates in be. P values are annotated in the figures.

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Extended Data Fig. 5 SIRT5 interacts with TBK1 and protects skeletal muscle from aging.

a. TBK1 and SDHA were identified as SIRT5-interacting proteins by Co-IP and mass spectrometry. b. Immunofluorescence staining of TOM20, FLAG-TBK1 and HA-SIRT5 in hMyotubes. Scale bars, 160 μm and 20 μm (zoomed in). c. Western blotting analysis of TBK1 and p-TBK1 (S172) protein expression levels in hMyotubes transduced with lentivirus vectors expressing FLAG-Luc, FLAG-TBK1-WT or FLAG-TBK1-K137R. β-tubulin was used as loading control. d. RT-qPCR analysis of the transcript levels of IL6, IL8 and MCP1 in hMyotubes transduced with lentivirus vectors expressing FLAG-Luc, FLAG-TBK1-WT or FLAG-TBK1-K137R. e. Immunofluorescence staining of IL-6 in mouse skeletal muscle of Young-Luc, Old-Luc and Old-SIRT5 groups. Left, representative images. Scale bars, 100 μm and 25 μm (zoomed in). Right, IL6-positive cells were quantified. n = 6 male mice per group. f. Principal component (PC) analysis of RNA-seq data of mouse skeletal muscles in Young-Luc, Old-Luc and Old-SIRT5 groups. g. Volcano plot showing upregulated and downregulated DEGs of the skeletal muscles of Old-Luc versus Young-Luc and Old-SIRT5 versus Old-Luc-transduced mice. Benjamini-Hochberg P values were performed. Two-tailed student’s t-test was used for statistical analysis and data are presented as the mean ± s.e.m. in ce. n = 3 biological replicates in c and d. P values are annotated in the figures. mMuscle, mouse muscle.

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Supplementary information

Reporting Summary

Supplementary Tables 1–6

Supplementary Table 1: DEPs between old and young cynomolgus monkey skeletal muscles. Supplementary Table 2: DEGs identified by RNA-seq analysis between SIRT5+/+ and SIRT5−/− human myotubes. Supplementary Table 3: The candidate SIRT5-interacting proteins identified by LC–MS/MS. Supplementary Table 4: DEGs identified by RNA-seq analysis in mouse skeletal muscles. Supplementary Table 5: Antibodies used in this study. Supplementary Table 6: The sequences of siRNAs and primers used in this study.

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Zhao, Q., Jing, Y., Jiang, X. et al. SIRT5 safeguards against primate skeletal muscle ageing via desuccinylation of TBK1. Nat Metab 7, 556–573 (2025). https://doi.org/10.1038/s42255-025-01235-8

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