Single-nucleus profiling unveils a geroprotective role of the FOXO3 in primate skeletal muscle aging

Abstract

Age-dependent loss of skeletal muscle mass and function is a feature of sarcopenia, and increases the risk of many aging-related metabolic diseases. Here, we report phenotypic and single-nucleus transcriptomic analyses of non-human primate skeletal muscle aging. A higher transcriptional fluctuation was observed in myonuclei relative to other interstitial cell types, indicating a higher susceptibility of skeletal muscle fiber to aging. We found a downregulation of FOXO3 in aged primate skeletal muscle, and identified FOXO3 as a hub transcription factor maintaining skeletal muscle homeostasis. Through the establishment of a complementary experimental pipeline based on a human pluripotent stem cell-derived myotube model, we revealed that silence of FOXO3 accelerates human myotube senescence, whereas genetic activation of endogenous FOXO3 alleviates human myotube aging. Altogether, based on a combination of monkey skeletal muscle and human myotube aging research models, we unraveled the pivotal role of the FOXO3 in safeguarding primate skeletal muscle from aging, providing a comprehensive resource for the development of clinical diagnosis and targeted therapeutic interventions against human skeletal muscle aging and the onset of sarcopenia along with aging-related disorders.

Keywords

single-nucleus RNA sequencing / primate / aging / skeletal muscle / FOXO3

References

[1] Aibar S, González-Blas CB, Moerman T et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods 2017;14:1083–1086.

[2] Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–169.

[3] Anderson RM, Colman RJ. Prospects and perspectives in primate aging research. Antioxid Redox Signal 2011;14:203–205.

[4] Angelidis I, Simon LM, Fernandez IE et al. An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat Commun 2019;10:963.

[5] Askanas V, Engel WK. Inclusion-body myositis, a multifactorial muscle disease associated with aging: current concepts of pathogenesis. Curr Opin Rheumatol 2007;19:550–559.

[6] Askanas V, Engel WK, Nogalska A. Pathogenic considerations in sporadic inclusion-body myositis, a degenerative muscle disease associated with aging and abnormalities of myoproteostasis. J Neuropathol Exp Neurol 2012;71:680–693.

[7] Baskin KK, Winders BR, Olson EN. Muscle as a “mediator” of systemic metabolism. Cell Metab 2015;21:237–248.

[8] Butler A, Hoffman P, Smibert P et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 2018;36:411–420.

[9] Cadot B, Gache V, Gomes ER. Moving and positioning the nucleus in skeletal muscle – one step at a time. Nucleus 2015;6:373–381.

[10] Cai Y, Song W, Li J et al. The landscape of aging. Sci China Life Sci 2022; 65(12), 2354–2454.

[11] Calissi G, Lam EW, Link W. Therapeutic strategies targeting FOXO transcription factors. Nat Rev Drug Discov 2021;20:21–38.

[12] Dalle S, Koppo K. Cannabinoid receptor 1 expression is higher in muscle of old vs. young males, and increases upon resistance exercise in older adults. Sci Rep 2021;11:18349.

[13] De Micheli AJ, Spector JA, Elemento O et al. A reference single-cell transcriptomic atlas of human skeletal muscle tissue reveals bifurcated muscle stem cell populations. Skelet Muscle 2020;10:19.

[14] Dell’Orso S, Juan AH, Ko KD et al. Single cell analysis of adult mouse skeletal muscle stem cells in homeostatic and regenerative conditions. Development 2019;146:dev174177.

[15] Demontis F, Piccirillo R, Goldberg AL et al. Mechanisms of skeletal muscle aging: insights from Drosophila and mammalian models. Dis Model Mech 2013;6:1339–1352.

[16] Dirks AJ, Hofer T, Marzetti E et al. Mitochondrial DNA mutations, energy metabolism and apoptosis in aging muscle. Ageing Res Rev 2006;5:179–195.

[17] Domingues-Faria C, Vasson MP, Goncalves-Mendes N et al. Skeletal muscle regeneration and impact of aging and nutrition. Ageing Res Rev 2016;26:22–36.

[18] Dos Santos M, Backer S, Saintpierre B et al. Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers. Nat Commun 2020;11:5102.

[19] Fleming SJ, Marioni JC, Babadi M. CellBender remove-background: a deep generative model for unsupervised removal of background noise from scRNA-seq datasets. bioRxiv 2019.

[20] Frontera WR, Ochala J. Skeletal muscle: a brief review of structure and function. Calcif Tissue Int 2015;96:183–195.

[21] Fuchs E, Blau HM. Tissue stem cells: architects of their niches. Cell Stem Cell 2020;27:532–556.

[22] Giordani L, He GJ, Negroni E et al. High-dimensional single-cell cartography reveals novel skeletal muscle-resident cell populations. Mol Cell 2019;74:609–621.e6.

[23] Gomarasca M, Banfi G, Lombardi G. Myokines: the endocrine coupling of skeletal muscle and bone. Adv Clin Chem 2020;94:155–218.

[24] González JP, Queralt-Rosinach N, Bravo A et al. DisGeNET: a discovery platform for the dynamical exploration of human diseases and their genes. Database 2015;2015:bav028.

[25] Guang-Hui Liu YB, Qu J, Zhang W et al. Aging Atlas: a multi-omics database for aging biology. Nucleic Acids Res 2021;49:D825–d830.

[26] He X, Memczak S, Qu J et al. Single-cell omics in ageing: a young and growing field. Nat Metab 2020;2:293–302.

[27] Hershberg EA, Camplisson CK, Close JL et al. PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments. Nat Methods 2021;18:937–944.

[28] Hu H, Ji Q, Song M et al. ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin. Nucleic Acids Res 2020;48:6001–6018.

[29] Iizuka K, Machida T, Hirafuji M. Skeletal muscle is an endocrine organ. J Pharmacol Sci 2014;125:125–131.

[30] Irrthum A, Wehenkel L, Geurts P. Inferring regulatory networks from expression data using tree-based methods. PLoS One 2010;5:e12776.

[31] Jiang C, Wen Y, Kuroda K et al. Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy. Dis Model Mech 2014;7:997–1004.

[32] Jones RA, Harrison C, Eaton SL et al. Cellular and molecular anatomy of the human neuromuscular junction. Cell Rep 2017;21:2348–2356.

[33] Kalinkovich A, Livshits G. Sarcopenic obesity or obese sarcopenia: a cross talk between age-associated adipose tissue and skeletal muscle inflammation as a main mechanism of the pathogenesis. Ageing Res Rev 2017;35:200–221.

[34] Kanehisa M, Furumichi M, Sato Y et al. KEGG: integrating viruses and cellular organisms. Nucleic Acids Res 2021;49:D545–D551.

[35] Kang W, Jin T, Zhang T et al. Regeneration Roadmap: data-base resources for regenerative biology. Nucleic Acids Res 2022;50:D1085–D1090.

[36] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 2015;12:357–360.

[37] Kim M, Franke V, Brandt B et al. Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells. Nat Commun 2020;11:6375.

[38] Kishi JY, Lapan SW, Beliveau BJ et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 2019;16:533–544.

[39] Krishnaswami SR, Grindberg RV, Novotny M et al. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc 2016;11:499–524.

[40] Larsson L, Degens H, Li M et al. Sarcopenia: aging-related loss of muscle mass and function. Physiol Rev 2019;99:427–511.

[41] Leger B, Derave W, De Bock K et al. Human sarcopenia reveals an increase in SOCS-3 and myostatin and a reduced efficiency of Akt phosphorylation. Rejuvenation Res 2008;11:163–175B.

[42] Lei J, Wang S, Kang W et al. FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction. Protein Cell 2021;12:145–151.

[43] Leng SX, Pawelec G. Single-cell immune atlas for human aging and frailty. Life Med 2022:lnac013.

[44] Li J, Zheng Y, Yan P et al. A single-cell transcriptomic atlas of primate pancreatic islet aging. Natl Sci Rev 2021;8:nwaa127.

[45] Liberzon A, Birger C, Thorvaldsdóttir H et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 2015;1:417–425.

[46] Limbad C, Doi R, McGirr J et al. Senolysis induced by 25-hydroxycholesterol targets CRYAB in multiple cell types. iScience 2022;25:103848.

[47] Lipina C, Hundal HS. Lipid modulation of skeletal muscle mass and function. J Cachexia Sarcopenia Muscle 2017;8:190–201.

[48] Liu L, Liu X, Bai Y et al. Neuregulin-1β modulates myogenesis in septic mouse serum-treated C2C12 myotubes in vitro through PPARγ/NF-κB signaling. Mol Biol Rep 2018;45:1611–1619.

[49] Liu Z, Li W, Geng L et al. Cross-species metabolomic analysis identifies uridine as a potent regeneration promoting factor. Cell Discov 2022;8:6.

[50] Livshits G, Kalinkovich A. Inflammaging as a common ground for the development and maintenance of sarcopenia, obesity, cardiomyopathy and dysbiosis. Ageing Res Rev 2019;56:100980.

[51] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

[52] Ma S, Sun S, Geng L et al. Caloric restriction reprograms the single-cell transcriptional landscape of Rattus Norvegicus aging. Cell 2020;180:1001.e22.

[53] Ma S, Sun S, Li J et al. Single-cell transcriptomic atlas of primate cardiopulmonary aging. Cell Res 2021;31:415–432.

[54] Ma S, Wang S, Ye Y et al. Heterochronic parabiosis induces stem cell revitalization and systemic rejuvenation across aged tissues. Cell Stem Cell 2022;29:1005.e10.

[55] Maeda S, Miyagawa S, Kawamura T et al. Notch signaling-modified mesenchymal stem cells improve tissue perfusion by induction of arteriogenesis in a rat hindlimb ischemia model. Sci Rep 2021;11:2543.

[56] Maffioletti SM, Gerli MF, Ragazzi M et al. Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells. Nat Protoc 2015;10:941–958.

[57] Mammucari C, Milan G, Romanello V et al. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007;6:458–471.

[58] McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst 2019;8:329–337.e4.

[59] McKellar DW, Walter LD, Song LT et al. Large-scale integration of single-cell transcriptomic data captures transitional progenitor states in mouse skeletal muscle regeneration. Commun Biol 2021;4:1280.

[60] Mercken EM, Capri M, Carboneau BA et al. Conserved and species-specific molecular denominators in mammalian skeletal muscle aging. NPJ Aging Mech Dis 2017;3:8.

[61] Morris BJ, Willcox DC, Donlon TA et al. FOXO3: A major gene for human longevity — a mini-review. Gerontology 2015;61:515–525.

[62] Murgia M, Toniolo L, Nagaraj N et al. Single muscle fiber proteomics reveals fiber-type-specific features of human muscle aging. Cell Rep 2017;19:2396–2409.

[63] Navarro G. A guided tour to approximate string matching. ACM Comput Surv 2001;33:31–88.

[64] Ohsawa N, Koebis M, Suo S et al. Alternative splicing of PDLIM3/ALP, for α-actinin-associated LIM protein 3, is aberrant in persons with myotonic dystrophy. Biochem Biophys Res Commun 2011;409:64–69.

[65] Orchard P, Manickam N, Ventresca C et al. Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits. Genome Res 2021;31:2258–2275.

[66] Podnar J, Deiderick H, Hunicke-Smith S. Next-generation sequencing fragment library construction. Curr Protoc Mol Biol 2014;107:7.17.11–7.17.16.

[67] Rubenstein AB, Smith GR, Raue U et al. Single-cell transcriptional profiles in human skeletal muscle. Sci Rep 2020;10:229.

[68] Salih DA, Brunet A. FoxO transcription factors in the maintenance of cellular homeostasis during aging. Curr Opin Cell Biol 2008;20:126–136.

[69] Sandri M, Sandri C, Gilbert A et al. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 2004;117:399–412.

[70] Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003;13:2498–2504.

[71] Simon M, Van Meter M, Ablaeva J et al. LINE1 derepression in aged wild-type and SIRT6-deficient mice drives inflammation. Cell Metab 2019;29:871–885.e5 e875.

[72] Stokes T, Hector AJ, Morton RW et al. Recent perspectives regarding the role of dietary protein for the promotion of muscle hypertrophy with resistance exercise training. Nutrients 2018;10:180.

[73] Tieland M, Trouwborst I, Clark BC. Skeletal muscle performance and ageing. J Cachexia Sarcopenia Muscle 2018;9:3–19.

[74] Verma M, Asakura Y, Murakonda BSR et al. Muscle satellite cell cross-talk with a vascular niche maintains quiescence via VEGF and notch signaling. Cell Stem Cell 2018;23:530–543.e9.

[75] Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell 2005;122:659–667.

[76] Wang S, Cheng F, Ji Q et al. Hyperthermia differentially affects specific human stem cells and their differentiated derivatives. Protein Cell 2022;13:615–622.

[77] Wang S, Hu B, Ding Z et al. ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells. Cell Discov 2018;4:2.

[78] Wang S, Yao X, Ma S et al. A single-cell transcriptomic landscape of the lungs of patients with COVID-19. Nat Cell Biol 2021a;23:1314–1328.

[79] Wang S, Zheng Y, Li J et al. Single-cell transcriptomic atlas of primate ovarian aging. Cell 2020;180:585–600.e19.

[80] Wang S, Zheng Y, Li Q et al. Deciphering primate retinal aging at single-cell resolution. Protein Cell 2021b;12:889–898.

[81] Wang Y, Ng S-C. Sphingolipids mediate lipotoxicity in muscular dystrophies. Life Med 2022:lnac015.

[82] Wickham, H. 2016. ggplot2: Elegant Graphics for Data Analysis. Springer

[83] Wilkinson DJ, Piasecki M, Atherton PJ. The age-related loss of skeletal muscle mass and function: measurement and physiology of muscle fibre atrophy and muscle fibre loss in humans. Ageing Res Rev 2018;47:123–132.

[84] Yan P, Li Q, Wang L et al. FOXO3-engineered human ESC-derived vascular cells promote vascular protection and regeneration. Cell Stem Cell 2019;24:447–461.e8.

[85] Yates AD, Achuthan P, Akanni W et al. Ensembl 2020. Nucleic Acids Res 2020;48:D682–D688.

[86] Yu W, Clyne M, Khoury MJ et al. Phenopedia and genopedia: disease- centered and gene-centered views of the evolving knowledge of human genetic associations. Bioinformatics 2010;26:145–146.

[87] Zhang W, Li J, Suzuki K, et al. Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Sci 2015;348:1160–1163.

[88] Zhang H, Li J, Ren J et al. Single-nucleus transcriptomic landscape of primate hippocampal aging. Protein Cell 2021;12:695–716.

[89] Zhang W, Wan H, Feng G et al. SIRT6 deficiency results in developmental retardation in cynomolgus monkeys. Nature 2018;560:661–665.

[90] Zhang W, Zhang S, Yan P et al. A single-cell transcriptomic landscape of primate arterial aging. Nat Commun 2020a;11:2202.

[91] Zhang W, Qu J, Liu G-H, Belmonte JCI. The ageing epigenome and its rejuvenation. Nat Rev Mol Cell Biol 2020b;21:137–150.

[92] Zhang Y, Zheng Y, Wang S et al. Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging. Protein & Cell 2022:pwac038.

[93] Zhao D, Chen S. Failures at every level: breakdown of the epigenetic machinery of aging. Life Med 2022:lnac016.

[94] Zhao J, Brault JJ, Schild A et al. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 2007;6:472–483.

[95] Zhou Y, Zhou B, Pache L et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 2019;10:1523.

[96] Zou, X., Dai, X., Mentis, A.F.A., Esteban, M.A., Liu, L., and Han, L. From monkey single-cell atlases into a broader biomedical perspective. Life Med. 2022:lnac028.

[97] Zou Z, Long X, Zhao Q et al. A single-cell transcriptomic atlas of human skin aging. Dev Cell 2021;56:383–397.e8.

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The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.