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• W2912606098 abstract "•Aging alters the myogenic support of FAPs to MuSCs•Aged FAPs produce less matricellular WISP1•FAP-derived WISP1 is required for MuSC expansion and commitment•Restoring WISP1 levels rejuvenates the myogenic potential of aged MuSCs Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis. Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis. The regenerative capacity of skeletal muscle depends on the activity of tissue-resident muscle stem cells (MuSCs), also termed satellite cells. As a consequence of aging, the regenerative function of MuSCs is dramatically impaired, leading to inefficient muscle repair following injury (Almada and Wagers, 2016Almada A.E. Wagers A.J. Molecular circuitry of stem cell fate in skeletal muscle regeneration, ageing and disease.Nat. Rev. Mol. Cell Biol. 2016; 17: 267-279Crossref PubMed Scopus (180) Google Scholar, Blau et al., 2015Blau H.M. Cosgrove B.D. Ho A.T. The central role of muscle stem cells in regenerative failure with aging.Nat. Med. 2015; 21: 854-862Crossref PubMed Scopus (243) Google Scholar, Brack and Muñoz-Cánoves, 2016Brack A.S. Muñoz-Cánoves P. The ins and outs of muscle stem cell aging.Skelet. Muscle. 2016; 6: 1Crossref PubMed Scopus (82) Google Scholar). The number of MuSCs decreases in aged muscle (Shefer et al., 2006Shefer G. Van de Mark D.P. Richardson J.B. Yablonka-Reuveni Z. Satellite-cell pool size does matter: Defining the myogenic potency of aging skeletal muscle.Dev. Biol. 2006; 294: 50-66Crossref PubMed Scopus (346) Google Scholar), and remaining cells display impaired activation, adhesion, migration, proliferation, self-renewal, and differentiation and gradually switch to a senescent phenotype (Lukjanenko et al., 2016Lukjanenko L. Jung M.J. Hegde N. Perruisseau-Carrier C. Migliavacca E. Rozo M. Karaz S. Jacot G. Schmidt M. Li L. et al.Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice.Nat. Med. 2016; 22: 897-905Crossref PubMed Scopus (159) Google Scholar, Price et al., 2014Price F.D. von Maltzahn J. Bentzinger C.F. Dumont N.A. Yin H. Chang N.C. Wilson D.H. Frenette J. Rudnicki M.A. Inhibition of JAK-STAT signaling stimulates adult satellite cell function.Nat. Med. 2014; 20: 1174-1181Crossref PubMed Scopus (257) Google Scholar, Sousa-Victor et al., 2014Sousa-Victor P. Gutarra S. García-Prat L. Rodriguez-Ubreva J. Ortet L. Ruiz-Bonilla V. Jardí M. Ballestar E. González S. Serrano A.L. et al.Geriatric muscle stem cells switch reversible quiescence into senescence.Nature. 2014; 506: 316-321Crossref PubMed Scopus (621) Google Scholar, Tierney et al., 2018Tierney M.T. Stec M.J. Rulands S. Simons B.D. Sacco A. Muscle stem cells exhibit distinct clonal dynamics in response to tissue repair and homeostatic aging.Cell Stem Cell. 2018; 22: 119-127Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Alterations in both cellular signaling pathways and metabolism have been shown to impair MuSC function during aging. Aged MuSCs exhibit increased p38-mitogen-activated protein kinase (MAPK), ERK-MAPK, and JAK-STAT signaling (Bernet et al., 2014Bernet J.D. Doles J.D. Hall J.K. Kelly Tanaka K. Carter T.A. Olwin B.B. p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice.Nat. Med. 2014; 20: 265-271Crossref PubMed Scopus (362) Google Scholar, Chakkalakal et al., 2012Chakkalakal J.V. Jones K.M. Basson M.A. Brack A.S. The aged niche disrupts muscle stem cell quiescence.Nature. 2012; 490: 355-360Crossref PubMed Scopus (548) Google Scholar, Cosgrove et al., 2014Cosgrove B.D. Gilbert P.M. Porpiglia E. Mourkioti F. Lee S.P. Corbel S.Y. Llewellyn M.E. Delp S.L. Blau H.M. Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.Nat. Med. 2014; 20: 255-264Crossref PubMed Scopus (423) Google Scholar, Price et al., 2014Price F.D. von Maltzahn J. Bentzinger C.F. Dumont N.A. Yin H. Chang N.C. Wilson D.H. Frenette J. Rudnicki M.A. Inhibition of JAK-STAT signaling stimulates adult satellite cell function.Nat. Med. 2014; 20: 1174-1181Crossref PubMed Scopus (257) Google Scholar, Sousa-Victor et al., 2014Sousa-Victor P. Gutarra S. García-Prat L. Rodriguez-Ubreva J. Ortet L. Ruiz-Bonilla V. Jardí M. Ballestar E. González S. Serrano A.L. et al.Geriatric muscle stem cells switch reversible quiescence into senescence.Nature. 2014; 506: 316-321Crossref PubMed Scopus (621) Google Scholar), as well as Hoxa9 induction in response to epigenetic stress (Schwörer et al., 2016Schwörer S. Becker F. Feller C. Baig A.H. Köber U. Henze H. Kraus J.M. Xin B. Lechel A. Lipka D.B. et al.Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.Nature. 2016; 540: 428-432Crossref PubMed Scopus (78) Google Scholar). In addition, impaired mitochondrial function as well as decreased autophagy perturb the physiology of MuSCs during aging and accelerate their transition toward senescence (García-Prat et al., 2016García-Prat L. Martínez-Vicente M. Perdiguero E. Ortet L. Rodríguez-Ubreva J. Rebollo E. Ruiz-Bonilla V. Gutarra S. Ballestar E. Serrano A.L. et al.Autophagy maintains stemness by preventing senescence.Nature. 2016; 529: 37-42Crossref PubMed Scopus (807) Google Scholar, Zhang et al., 2016Zhang H. Ryu D. Wu Y. Gariani K. Wang X. Luan P. D’Amico D. Ropelle E.R. Lutolf M.P. Aebersold R. et al.NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice.Science. 2016; 352: 1436-1443Crossref PubMed Scopus (690) Google Scholar). Importantly, recent studies have begun to elucidate the nature of the extracellular signals, which are disrupted in the niche and mediate some of these long-lasting intrinsic adaptations. For example, alterations in the levels of Fibronectin, Notch ligands, fibroblast growth factor (FGF)-2, Wnt ligand C1q, transforming growth factor β (TGF-β), oxytocin, or apelin have been reported to affect cellular aging pathways and thereby disturb MuSC function (Brack et al., 2007Brack A.S. Conboy M.J. Roy S. Lee M. Kuo C.J. Keller C. Rando T.A. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis.Science. 2007; 317: 807-810Crossref PubMed Scopus (1105) Google Scholar, Carlson et al., 2008Carlson M.E. Hsu M. Conboy I.M. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.Nature. 2008; 454: 528-532Crossref PubMed Scopus (373) Google Scholar, Chakkalakal et al., 2012Chakkalakal J.V. Jones K.M. Basson M.A. Brack A.S. The aged niche disrupts muscle stem cell quiescence.Nature. 2012; 490: 355-360Crossref PubMed Scopus (548) Google Scholar, Elabd et al., 2014Elabd C. Cousin W. Upadhyayula P. Chen R.Y. Chooljian M.S. Li J. Kung S. Jiang K.P. Conboy I.M. Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration.Nat. Commun. 2014; 5: 4082Crossref PubMed Scopus (259) Google Scholar, Lukjanenko et al., 2016Lukjanenko L. Jung M.J. Hegde N. Perruisseau-Carrier C. Migliavacca E. Rozo M. Karaz S. Jacot G. Schmidt M. Li L. et al.Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice.Nat. Med. 2016; 22: 897-905Crossref PubMed Scopus (159) Google Scholar, Naito et al., 2012Naito A.T. Sumida T. Nomura S. Liu M.L. Higo T. Nakagawa A. Okada K. Sakai T. Hashimoto A. Hara Y. et al.Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes.Cell. 2012; 149: 1298-1313Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Price et al., 2014Price F.D. von Maltzahn J. Bentzinger C.F. Dumont N.A. Yin H. Chang N.C. Wilson D.H. Frenette J. Rudnicki M.A. Inhibition of JAK-STAT signaling stimulates adult satellite cell function.Nat. Med. 2014; 20: 1174-1181Crossref PubMed Scopus (257) Google Scholar, Vinel et al., 2018Vinel C. Lukjanenko L. Batut A. Deleruyelle S. Pradère J.P. Le Gonidec S. Dortignac A. Geoffre N. Pereira O. Karaz S. et al.The exerkine apelin reverses age-associated sarcopenia.Nat. Med. 2018; 24: 1360-1371Crossref PubMed Scopus (163) Google Scholar, Wang et al., 2015Wang Y. Wehling-Henricks M. Samengo G. Tidball J.G. Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle-derived nitric oxide.Aging Cell. 2015; 14: 678-688Crossref PubMed Scopus (114) Google Scholar). Yet, in spite of these fundamental advancements in our understanding of the muscle stem cell niche, the cellular origin of extrinsic MuSC regulatory signals that are affected by the aging process remain largely enigmatic. During adult myogenesis, MuSC function is under the control of a wide range of paracrine signals originating from different cell types in the niche (Mashinchian et al., 2018Mashinchian O. Pisconti A. Le Moal E. Bentzinger C.F. The Muscle Stem Cell Niche in Health and Disease.Curr. Top. Dev. Biol. 2018; 126: 23-65Crossref PubMed Scopus (56) Google Scholar). The regulatory interplay between MuSCs, immune cells, fibrogenic cells, and adipogenic progenitors has emerged to be of particular complexity (Heredia et al., 2013Heredia J.E. Mukundan L. Chen F.M. Mueller A.A. Deo R.C. Locksley R.M. Rando T.A. Chawla A. Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration.Cell. 2013; 153: 376-388Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, Varga et al., 2016Varga T. Mounier R. Horvath A. Cuvellier S. Dumont F. Poliska S. Ardjoune H. Juban G. Nagy L. Chazaud B. Highly dynamic transcriptional signature of distinct macrophage subsets during sterile inflammation, resolution, and tissue repair.J. Immunol. 2016; 196: 4771-4782Crossref PubMed Scopus (107) Google Scholar, Verma et al., 2018Verma M. Asakura Y. Murakonda B.S.R. Pengo T. Latroche C. Chazaud B. McLoon L.K. Asakura A. Muscle satellite cell cross-talk with a vascular niche maintains quiescence via VEGF and notch signaling.Cell Stem Cell. 2018; 23: 530-543Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Reciprocal control of MuSCs and fibroblasts is indispensable for efficient expansion of the MuSC pool and for keeping interstitial fibrosis in check (Fry et al., 2016Fry C.S. Kirby T.J. Kosmac K. McCarthy J.J. Peterson C.A. Myogenic progenitor cells control extracellular matrix production by fibroblasts during skeletal muscle hypertrophy.Cell Stem Cell. 2016; 20: 56-69Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, Murphy et al., 2011Murphy M.M. Lawson J.A. Mathew S.J. Hutcheson D.A. Kardon G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.Development. 2011; 138: 3625-3637Crossref PubMed Scopus (757) Google Scholar). Similarly, ablation of lineages with adipogenic potential leads to dysfunctional muscle repair (Liu et al., 2012Liu W. Liu Y. Lai X. Kuang S. Intramuscular adipose is derived from a non-Pax3 lineage and required for efficient regeneration of skeletal muscles.Dev. Biol. 2012; 361: 27-38Crossref PubMed Scopus (56) Google Scholar). Both fibroblast-like cells and adipocytes residing in skeletal muscle are derived from a common bipotent mesenchymal fibro-adipogenic progenitor (FAP) marked by the expression of the platelet-derived growth factor receptor (PDGFR) α (Joe et al., 2010Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat. Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (1013) Google Scholar, Liu et al., 2012Liu W. Liu Y. Lai X. Kuang S. Intramuscular adipose is derived from a non-Pax3 lineage and required for efficient regeneration of skeletal muscles.Dev. Biol. 2012; 361: 27-38Crossref PubMed Scopus (56) Google Scholar, Uezumi et al., 2010Uezumi A. Fukada S. Yamamoto N. Takeda S. Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.Nat. Cell Biol. 2010; 12: 143-152Crossref PubMed Scopus (809) Google Scholar). Upon muscle injury, FAPs activate, enter a proliferative phase at the same time as MuSCs, and support myogenic commitment (Joe et al., 2010Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat. Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (1013) Google Scholar). Altered FAP lineage decisions during regeneration perturb extracellular matrix (ECM) remodeling and impair myogenesis (Fiore et al., 2016Fiore D. Judson R.N. Low M. Lee S. Zhang E. Hopkins C. Xu P. Lenzi A. Rossi F.M. Lemos D.R. Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration.Stem Cell Res. (Amst.). 2016; 17: 161-169Crossref PubMed Scopus (90) Google Scholar, Mozzetta et al., 2013Mozzetta C. Consalvi S. Saccone V. Tierney M. Diamantini A. Mitchell K.J. Marazzi G. Borsellino G. Battistini L. Sassoon D. et al.Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice.EMBO Mol. Med. 2013; 5: 626-639Crossref PubMed Scopus (151) Google Scholar). Permissive changes in the micro-environment of diseased muscle and altered FAP apoptosis in the late phase of regeneration promotes excessive differentiation toward fat or fibrotic tissue (Heredia et al., 2013Heredia J.E. Mukundan L. Chen F.M. Mueller A.A. Deo R.C. Locksley R.M. Rando T.A. Chawla A. Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration.Cell. 2013; 153: 376-388Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, Lemos et al., 2015Lemos D.R. Babaeijandaghi F. Low M. Chang C.K. Lee S.T. Fiore D. Zhang R.H. Natarajan A. Nedospasov S.A. Rossi F.M. Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.Nat. Med. 2015; 21: 786-794Crossref PubMed Scopus (383) Google Scholar, Uezumi et al., 2010Uezumi A. Fukada S. Yamamoto N. Takeda S. Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.Nat. Cell Biol. 2010; 12: 143-152Crossref PubMed Scopus (809) Google Scholar). One of the signals involved in fibrotic fate decisions of FAPs is platelet-derived growth factor (PDGF), which can be dynamically regulated by alternative processing of the Pdgfra mRNA in FAPs during muscle regeneration (Mueller et al., 2016Mueller A.A. van Velthoven C.T. Fukumoto K.D. Cheung T.H. Rando T.A. Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis.Nature. 2016; 540: 276-279Crossref PubMed Scopus (86) Google Scholar). These observations demonstrate that FAPs orchestrate a plethora of processes involved in regenerative myogenesis and highlight the need for a better understanding of the signals controlling MuSC function. Notably, aging affects mesenchymal progenitors in multiple tissues (Raggi and Berardi, 2012Raggi C. Berardi A.C. Mesenchymal stem cells, aging and regenerative medicine.Muscles Ligaments Tendons J. 2012; 2: 239-242PubMed Google Scholar). Similarly, oxidative stress and other senescence-associated processes impair adipogenic progenitors in aged fat tissue (Tchkonia et al., 2010Tchkonia T. Morbeck D.E. Von Zglinicki T. Van Deursen J. Lustgarten J. Scrable H. Khosla S. Jensen M.D. Kirkland J.L. Fat tissue, aging, and cellular senescence.Aging Cell. 2010; 9: 667-684Crossref PubMed Scopus (693) Google Scholar). These observations suggest that FAPs and their support function for myogenesis could also be deregulated by the aging process. Here, we set out to test this hypothesis and demonstrate that FAP activity is severely impaired as a consequence of old age. We describe that aged FAPs fail to support MuSCs due to reduced secretion of the matricellular protein WNT1 Inducible Signaling Pathway Protein 1 (WISP1). FAP-secreted WISP1 controls asymmetric MuSC commitment and activates the Akt pathway. Similar to aging, genetic deletion of WISP1 in mice perturbs the MuSC pool and impairs myogenesis. Conversely, systemic treatment of aged mice with recombinant WISP1, or transplantation of young but not aged or WISP1 knockout FAPs, rescues MuSC function, and rejuvenates the regenerative capacity of aged skeletal muscle. In summary, we demonstrate that the regenerative failure inherent to aged muscle can be ameliorated by targeting matricellular communication between FAPs and MuSCs. Given the negative impact of aging on mesenchymal stem cells (Raggi and Berardi, 2012Raggi C. Berardi A.C. Mesenchymal stem cells, aging and regenerative medicine.Muscles Ligaments Tendons J. 2012; 2: 239-242PubMed Google Scholar) and the pivotal role of FAPs as support cells in the MuSC niche (Joe et al., 2010Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat. Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (1013) Google Scholar, Lemos et al., 2015Lemos D.R. Babaeijandaghi F. Low M. Chang C.K. Lee S.T. Fiore D. Zhang R.H. Natarajan A. Nedospasov S.A. Rossi F.M. Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.Nat. Med. 2015; 21: 786-794Crossref PubMed Scopus (383) Google Scholar, Uezumi et al., 2010Uezumi A. Fukada S. Yamamoto N. Takeda S. Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.Nat. Cell Biol. 2010; 12: 143-152Crossref PubMed Scopus (809) Google Scholar), we first asked whether FAP function is affected during aging. To address this question, we collected FAPs and MuSCs from muscles of 9- to 13-week-old young mice and 20- to 25-month-old pre-geriatric aged mice (Sousa-Victor et al., 2014Sousa-Victor P. Gutarra S. García-Prat L. Rodriguez-Ubreva J. Ortet L. Ruiz-Bonilla V. Jardí M. Ballestar E. González S. Serrano A.L. et al.Geriatric muscle stem cells switch reversible quiescence into senescence.Nature. 2014; 506: 316-321Crossref PubMed Scopus (621) Google Scholar) using fluorescence-activated cell sorting (FACS; Figure S1A). Ex vivo culture of MuSCs confirmed previously described aging defects that included impaired proliferation, reduced upregulation of the myogenic commitment factor MyoD, and inefficient differentiation of aged MuSCs (Figures S1B–S1E). Notably, we observed that aged FAPs also displayed a range of altered cellular phenotypes. In ex vivo culture, the number of FAPs isolated from aged mice was reduced, and they incorporated less 5-Ethynyl-2′-deoxyUridine (EdU) compared to young controls (Figures 1A–1C). Immunostaining for PDGFRα revealed lower numbers of FAPs in muscles of aged mice (Figures S1F and S1G). To investigate how aging affects FAP levels during regeneration, we analyzed muscles at different time points after injury. This revealed decreased numbers of aged FAPs at 4 days post-injury (dpi), that failed to be cleared from the tissue at 7 dpi (Figures S1H and S1I). Functional ex vivo analysis of aged FAPs demonstrated impaired growth factor induced (Figures 1D and 1E) and spontaneous (Figure S2A) adipogenesis. Clonal analysis of single aged FAPs showed that the capacity for expansion and the number of adipogenic clones are reduced compared to the young condition (Figure S2B). No difference in differentiation was observed between young and aged FAPs once the cells have taken a fate decision and an adipogenic clone had emerged (Figure S2C), indicating that aging affects fate decisions at the progenitor level. The impaired adipogenic potential of aged FAPs was reflected by reduced levels of oil red O positive intramuscular adipocytes at 14 dpi (Figures 1F, 1G, and S2D). This effect was also observed in H&E stainings (Figure S2E) and confirmed by the quantification of perilipin-positive adipocytes in cross-sections of aged muscles at 14 dpi (Figures S2F and S2G). In contrast, fibrogenic FAP differentiation to α-smooth muscle actin and collagenIα1 positive cells was higher in aged FAPs (Figures 1H, 1I, and S2H). In agreement with these findings, masson trichrome staining of muscle cross-sections of young and aged mice showed elevated fibrosis in aged muscle (Figures 1J and 1K). Gene expression profiling of young and aged FAPs isolated from injured muscles at 7 dpi further confirmed this finding and revealed increased mRNA expression of the gene ontology (GO) term “extracellular matrix” (Figures S2I and S2J). Fibrotic and adipogenic fate decisions in FAPs have been recently demonstrated to be mediated by alternative processing of the PDGFRα transcript (Mueller et al., 2016Mueller A.A. van Velthoven C.T. Fukumoto K.D. Cheung T.H. Rando T.A. Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis.Nature. 2016; 540: 276-279Crossref PubMed Scopus (86) Google Scholar). An intronic variant of PDGFRα coding for a protein isoform with a truncated kinase domain acts as a decoy receptor to inhibit PDGF signaling and inhibit FAP differentiation into fibrotic cells. Interestingly, we observed that the relative amounts of this PDGFRα intronic variant (PDGFRα-In) is reduced after injury in aged muscles as well as in aged FAPs following FACS isolation (Figures 1L and 1M). Collectively, these data demonstrate that the function of aged FAPs is perturbed and that aging uncouples adipogenic from fibrogenic fate decisions at the progenitor level. Following injury, FAPs initially expand to support MuSC function (Fiore et al., 2016Fiore D. Judson R.N. Low M. Lee S. Zhang E. Hopkins C. Xu P. Lenzi A. Rossi F.M. Lemos D.R. Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration.Stem Cell Res. (Amst.). 2016; 17: 161-169Crossref PubMed Scopus (90) Google Scholar, Joe et al., 2010Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat. Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (1013) Google Scholar, Mozzetta et al., 2013Mozzetta C. Consalvi S. Saccone V. Tierney M. Diamantini A. Mitchell K.J. Marazzi G. Borsellino G. Battistini L. Sassoon D. et al.Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice.EMBO Mol. Med. 2013; 5: 626-639Crossref PubMed Scopus (151) Google Scholar). In order to characterize the cellular cross-talk between FAPs and MuSCs in a system where the age of each cell type can be uncoupled, we isolated MuSCs from tdTomato (Td) mice (Prigge et al., 2013Prigge J.R. Wiley J.A. Talago E.A. Young E.M. Johns L.L. Kundert J.A. Sonsteng K.M. Halford W.P. Capecchi M.R. Schmidt E.E. Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei.Mamm. Genome. 2013; 24: 389-399Crossref Scopus (42) Google Scholar), which constitutively express a nuclear red fluorescent protein (Figure S3A). The cells were then tracked based on the reporter in co-cultures with wild-type (WT) FAPs (Figures 2A and S3B). Aged FAPs displayed a reduced ability to support MuSC expansion and differentiation (Figures 2B–2D). The positive effect of FAPs on MuSC expansion correlated with the amount of FAPs (Figure S3C) and was not due to effects on the viability of MuSCs (Figure S3D). We next tested whether FAPs support MuSCs through soluble factors. Using single myofiber cultures, we demonstrated that the proliferation of MuSCs was increased in the presence of medium conditioned by young FAPs but was unaffected by medium conditioned by aged FAPs (Figures 2E and 2F). FAP conditioned culture medium was also able to promote the proliferation and differentiation of MuSCs ex vivo (Figures S3E and S3F). Thus, aging impairs the secretion of a soluble myogenic support signal from FAPs. To uncover the molecular nature of the FAP-derived MuSC support signal lost during aging, we profiled the transcriptome of FAPs and MuSCs that were isolated in injured and uninjured muscles from young and aged mice. We first analyzed the GO terms enriched in FAPs compared to MuSCs and identified strong signatures of “organ development,” “cell adhesion,” and “extracellular matrix organization” in FAPs (Table S1). The fibrogenic nature of FAPs was highlighted by the higher expression of genes of the “extracellular matrix” GO term (Figure S4), and aging enhanced both molecular regulators of fibrosis in quiescent FAPs (Table S2) and ECM or fibrosis signatures at 7 dpi (Figures S2I and S2J). We next examined which transcripts coding for secreted proteins were upregulated in activated FAPs. Out of the 321 genes significantly upregulated with a fold change >2 during activation of young FAPs, we identified 20 secreted signaling proteins (Figure 3A). Genes upregulated in activated FAPs were then filtered for differential regulation with age and activation. The resulting 10 transcripts differentially regulated in aged activated FAPs contained a single secreted protein belonging to the Cyr61/CTGF/NOV (CCN) family of matricellular proteins and termed Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4) (Figure 3B). WISP1 was also the most downregulated gene when aged activated FAPs were directly compared to young activated FAPs (Table S3). qPCR analysis confirmed that WISP1 is upregulated in young FAPs following muscle injury, and that this induction is blunted more than 2-fold in FAPs from aged mice (Figure 3C). In agreement with these observations, the upregulation of WISP1 mRNA and protein was blunted in regenerating tibialis anterior muscles of aged mice (Figures 3D and 3E). To confirm that FAPs are the principal cell type in which aging affects WISP1 secretion, we interrogated different cell types found in quiescent and regenerating muscles. These included lineage positive cells (Lin+), comprising immune, endothelial, and hematopoietic cells, MuSCs, FAPs, and Sca1+/CD34+/PDGFRα– cells (hereafter called PDGFRα–). FAPs, MuSCs, and PDGFRα– cells upregulated WISP1 expression following injury, while very low expression was observed in Lin+ cells (Figure S5A). Notably, FAPs were the only cell type that displayed an age-dependent reduction in WISP1 expression. Since WISP1 is a secreted protein, RNA fluorescence in situ hybridization (FISH) was used to elucidate where WISP1 is produced in skeletal muscle. Consistent with the localization of FAPs, we observed an upregulation of WISP1 mRNA granules typical of RNA FISH experiments in the interstitial space of regenerating muscles (Figures S5B and S5C). No staining was detected in muscles of WISP1 knockout (WISP1−/−) mice. We also observed that WISP1 mRNA was not directly associated with MuSCs but frequently localized to the vicinity of these cells (Figure S5D). These results identify WISP1 as a FAP-derived MuSC support signal that is impaired during aging. We next asked whether the loss of WISP1 is sufficient to impair muscle regeneration independently of other age-induced perturbations by analyzing WISP1−/− mice (Maeda et al., 2015Maeda A. Ono M. Holmbeck K. Li L. Kilts T.M. Kram V. Noonan M.L. Yoshioka Y. McNerny E.M. Tantillo M.A. et al.WNT1-induced Secreted Protein-1 (WISP1), a novel regulator of bone turnover and Wnt signaling.J. Biol. Chem. 2015; 290: 14004-14018Crossref PubMed Scopus (60) Google Scholar). As expected, WISP1 mRNA was not detected in uninjured and regenerating WISP1−/− muscle (Figure S6A). We first assessed how loss of WISP1 affects the ex vivo phenotype of MuSCs and FAPs. While WISP1−/−" @default.
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