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• W4280490566 abstract "•Fasting induces a highly resilient deep quiescent (DQ) state in MuSCs•DQ is characterized by delayed cell-cycle entry but heightened stress resistance•The ketone body β-hydroxybutyrate (BHB) can directly promote DQ in MuSCs•The effects of BHB are due to its role as an HDAC inhibitor and are mediated by p53 Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically β-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that β-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting. Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically β-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that β-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting. Dietary restriction (DR) robustly improves healthspan and lifespan in multiple model systems (Lee et al., 2006Lee G.D. Wilson M.A. Zhu M. Wolkow C.A. de Cabo R. Ingram D.K. Zou S. Dietary deprivation extends lifespan in Caenorhabditis elegans.Aging Cell. 2006; 5: 515-524Crossref PubMed Scopus (220) Google Scholar; McCay et al., 1935McCay C.M. Crowell M.F. Maynard L.A. The effect of retarded growth upon the length of life span and upon the ultimate body size.J. Nutr. 1935; 10: 63-79Crossref Google Scholar, McCay et al., 1989McCay C.M. Crowell M.F. Maynard L.A. The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935.Nutrition. 1989; 5 (discussion 172): 155-171PubMed Google Scholar; Weindruch, and Walford, 1982Weindruch R. Walford R.L. Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence.Science. 1982; 215: 1415-1418Crossref PubMed Scopus (615) Google Scholar). Beneficial effects of DR have been observed at the organismal level, from simple eukaryotes to humans, and also at the single-cell level in adult stem cell populations (Cheng et al., 2014Cheng C.-W. Adams G.B. Perin L. Wei M. Zhou X. Lam B.S. Da Sacco S. Mirisola M. Quinn D.I. Dorff T.B. et al.Prolonged fasting reduces IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and reverse immunosuppression.Cell Stem Cell. 2014; 14: 810-823Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar; Jiang et al., 2000Jiang J.C. Jaruga E. Repnevskaya M.V. Jazwinski S.M. An intervention resembling caloric restriction prolongs life span and retards aging in yeast.FASEB J. 2000; 14: 2135-2137Crossref PubMed Scopus (293) Google Scholar; Ravussin et al., 2015Ravussin E. Redman L.M. Rochon J. Das S.K. Fontana L. Kraus W.E. Romashkan S. Williamson D.A. Meydani S.N. Villareal D.T. et al.A 2-year randomized controlled trial of human caloric restriction: feasibility and effects on predictors of health span and longevity.J. Gerontol. A Biol. Sci. Med. Sci. 2015; 70: 1097-1104Crossref PubMed Scopus (287) Google Scholar). These improvements are associated with cell and tissue functional maintenance and a delay or even prevention of age-related pathologies such as cognitive decline, inflammation, and cancer (Catterson et al., 2018Catterson J.H. Khericha M. Dyson M.C. Vincent A.J. Callard R. Haveron S.M. Rajasingam A. Ahmad M. Partridge L. Short-term, intermittent fasting induces long-lasting gut health and TOR-independent lifespan extension.Curr. Biol. 2018; 28: 1714-1724.e4Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Colman et al., 2014Colman R.J. Beasley T.M. Kemnitz J.W. Johnson S.C. Weindruch R. Anderson R.M. Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys.Nat. Commun. 2014; 5: 3557Crossref PubMed Scopus (505) Google Scholar; Weindruch et al., 1982Weindruch R. Gottesman S.R. Walford R.L. Modification of age-related immune decline in mice dietarily restricted from or after midadulthood.Proc. Natl. Acad. Sci. USA. 1982; 79: 898-902Crossref PubMed Scopus (109) Google Scholar). Commonly studied paradigms of DR are fasting and caloric restriction (CR). Fasting can lead cells and tissues to enter a protected state in which they become highly resistant to environmental stresses and toxicity (Mitchell et al., 2010Mitchell J.R. Verweij M. Brand K. van de Ven M. Goemaere N. van den Engel S. Chu T. Forrer F. Müller C. de Jong M. et al.Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice.Aging Cell. 2010; 9: 40-53Crossref PubMed Scopus (182) Google Scholar; Raffaghello et al., 2008Raffaghello L. Lee C. Safdie F.M. Wei M. Madia F. Bianchi G. Longo V.D. Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy.Proc. Natl. Acad. Sci. USA. 2008; 105: 8215-8220Crossref PubMed Scopus (390) Google Scholar). For example, preoperative fasting lessens hepatic damage resulting from ischemia/reperfusion injury (Mitchell et al., 2010Mitchell J.R. Verweij M. Brand K. van de Ven M. Goemaere N. van den Engel S. Chu T. Forrer F. Müller C. de Jong M. et al.Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice.Aging Cell. 2010; 9: 40-53Crossref PubMed Scopus (182) Google Scholar). In some contexts, fasting or CR has been shown to accelerate tissue regeneration. For example, intestinal epithelial and hepatic tissue regeneration after acute damage have been reported to improve after intense CR (Ramaiah et al., 1998Ramaiah S.K. Bucci T.J. Warbritton A. Soni M.G. Mehendale H.M. Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide.Toxicol. Sci. 1998; 45: 233-241Crossref PubMed Google Scholar; Yousefi et al., 2018Yousefi M. Nakauka-Ddamba A. Berry C.T. Li N. Schoenberger J. Simeonov K.P. Cedeno R.J. Yu Z. Lengner C.J. Calorie restriction governs intestinal epithelial regeneration through cell-autonomous regulation of mTORC1 in reserve stem cells.Stem Cell Rep. 2018; 10: 703-711Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Fasting affects a diverse population of stem and progenitor cells, frequently ameliorating various stem cell aging phenotypes (Mana et al., 2017Mana M.D. Kuo E.Y.-S. Yilmaz Ö.H. Dietary regulation of adult stem cells.Curr. Stem Cell Rep. 2017; 3: 1-8Crossref PubMed Scopus (30) Google Scholar). For example, fasting increases the ability of intestinal stem cells (ISCs) from young and aged mice to form intestinal organoids (Mihaylova et al., 2018Mihaylova M.M. Cheng C.-W. Cao A.Q. Tripathi S. Mana M.D. Bauer-Rowe K.E. Abu-Remaileh M. Clavain L. Erdemir A. Lewis C.A. et al.Fasting activates fatty acid oxidation to enhance intestinal stem cell function during homeostasis and aging.Cell Stem Cell. 2018; 22: 769-778.e4Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The extent of this functional improvement positively correlates with the duration of the fast and is dependent on ISC fatty acid oxidation. Similarly, the age-associated decrease in expression of the muscle stem cell (MuSC)-specific transcription factor Pax7 can be restored to youthful levels after multiple rounds of a short-term calorically restrictive diet designed to mimic fasting (Brandhorst et al., 2015Brandhorst S. Choi I.Y. Wei M. Cheng C.W. Sedrakyan S. Navarrete G. Dubeau L. Yap L.P. Park R. Vinciguerra M. et al.A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan.Cell Metab. 2015; 22: 86-99Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). This same fasting-mimicking diet (FMD) was also shown to rescue the decline in mesenchymal stem and progenitor cell number within bone marrow that is observed with aging (Brandhorst et al., 2015Brandhorst S. Choi I.Y. Wei M. Cheng C.W. Sedrakyan S. Navarrete G. Dubeau L. Yap L.P. Park R. Vinciguerra M. et al.A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan.Cell Metab. 2015; 22: 86-99Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Likewise, fasting or FMD promotes self-renewal of hematopoietic stem cells (HSCs), increases HSC number, and protects HSCs against cytotoxic agents (Brandhorst et al., 2015Brandhorst S. Choi I.Y. Wei M. Cheng C.W. Sedrakyan S. Navarrete G. Dubeau L. Yap L.P. Park R. Vinciguerra M. et al.A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan.Cell Metab. 2015; 22: 86-99Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar; Cheng et al., 2014Cheng C.-W. Adams G.B. Perin L. Wei M. Zhou X. Lam B.S. Da Sacco S. Mirisola M. Quinn D.I. Dorff T.B. et al.Prolonged fasting reduces IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and reverse immunosuppression.Cell Stem Cell. 2014; 14: 810-823Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In some instances, the affected stem cells may even rejuvenate their associated tissue in response to DR. For example, periodic fasting corrects the myeloid bias observed in aged hematopoiesis (Cheng et al., 2014Cheng C.-W. Adams G.B. Perin L. Wei M. Zhou X. Lam B.S. Da Sacco S. Mirisola M. Quinn D.I. Dorff T.B. et al.Prolonged fasting reduces IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and reverse immunosuppression.Cell Stem Cell. 2014; 14: 810-823Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Adult stem cells are critically important for maintaining tissue integrity and for regenerating damaged tissue after injury. MuSCs are indispensable for muscle tissue repair (Lepper et al., 2011Lepper C. Partridge T.A. Fan C.-M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.Development. 2011; 138: 3639-3646Crossref PubMed Scopus (726) Google Scholar; Wang et al., 2014Wang Y.X. Dumont N.A. Rudnicki M.A. Muscle stem cells at a glance.J. Cell Sci. 2014; 127: 4543-4548Crossref PubMed Scopus (80) Google Scholar). Long-term regenerative capacity of muscle is dependent on the ability of MuSCs to remain quiescent in the absence of injury and inability to maintain quiescence results in defective muscle repair (Yue et al., 2017Yue F. Bi P. Wang C. Shan T. Nie Y. Ratliff T.L. Gavin T.P. Kuang S. Pten is necessary for the quiescence and maintenance of adult muscle stem cells.Nat. Commun. 2017; 8: 14328Crossref PubMed Scopus (69) Google Scholar). In response to muscle injury, MuSCs enter the cell cycle, proliferate as myoblasts, and either self-renew to replenish the stem cell pool or fuse into nascent fibers to repair the injury (Lepper et al., 2011Lepper C. Partridge T.A. Fan C.-M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.Development. 2011; 138: 3639-3646Crossref PubMed Scopus (726) Google Scholar; Wang et al., 2014Wang Y.X. Dumont N.A. Rudnicki M.A. Muscle stem cells at a glance.J. Cell Sci. 2014; 127: 4543-4548Crossref PubMed Scopus (80) Google Scholar). In the absence of injury, quiescence is actively maintained and tightly regulated by a combination of signaling factors from the surrounding microenvironment and cell-intrinsic gene regulation, namely expression of cell cycle inhibitors and repression of cyclins, cyclin-dependent kinases, and checkpoint kinases (Cheung, and Rando, 2013Cheung T.H. Rando T.A. Molecular regulation of stem cell quiescence.Nat. Rev. Mol. Cell Biol. 2013; 14: 329-340Crossref PubMed Scopus (735) Google Scholar; Fukada et al., 2007Fukada S. Uezumi A. Ikemoto M. Masuda S. Segawa M. Tanimura N. Yamamoto H. Miyagoe-Suzuki Y. Takeda S. Molecular signature of quiescent satellite cells in adult skeletal muscle.Stem Cells. 2007; 25: 2448-2459Crossref PubMed Scopus (334) Google Scholar). For example, Notch signaling from the MuSC niche is known to be important in both maintenance of the quiescent state as well as self-renewal and reestablishment of quiescence following MuSC activation (Bjornson et al., 2012Bjornson C.R.R. Cheung T.H. Liu L. Tripathi P.V. Steeper K.M. Rando T.A. Notch signaling is necessary to maintain quiescence in adult muscle stem cells.Stem Cells. 2012; 30: 232-242Crossref PubMed Scopus (368) Google Scholar; Conboy et al., 2003Conboy I.M. Conboy M.J. Smythe G.M. Rando T.A. Notch-mediated restoration of regenerative potential to aged muscle.Science. 2003; 302: 1575-1577Crossref PubMed Scopus (845) Google Scholar; Mourikis et al., 2012Mourikis P. Sambasivan R. Castel D. Rocheteau P. Bizzarro V. Tajbakhsh S. A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state.Stem Cells. 2012; 30: 243-252Crossref PubMed Scopus (333) Google Scholar; Wen et al., 2012Wen Y. Bi P. Liu W. Asakura A. Keller C. Kuang S. Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells.Mol. Cell. Biol. 2012; 32: 2300-2311Crossref PubMed Scopus (189) Google Scholar). Quiescent MuSCs, like other quiescent stem cell populations, are characterized by stress resistance and low metabolic activity (Cheung, and Rando, 2013Cheung T.H. Rando T.A. Molecular regulation of stem cell quiescence.Nat. Rev. Mol. Cell Biol. 2013; 14: 329-340Crossref PubMed Scopus (735) Google Scholar). The effect of fasting on maintenance of this quiescent state, and on MuSC function in general, is unknown. The goal of this study was to comprehensively characterize the effect of fasting on muscle tissue repair and MuSC function. In this study, we show that fasting causes MuSCs to enter a state of deep quiescence (DQ) that is characterized by delayed activation and enhanced resilience to nutrient, cytotoxic, and proliferative stress. In this state, MuSCs are functionally and transcriptionally less committed to a myogenic program and more stem-like as assessed by muscle regeneration and transplantation assays as well as RNA sequencing (RNA-seq) analysis. Furthermore, increased MuSC resilience and delayed activation also results from feeding mice a ketogenic diet or injecting them with exogenous ketone bodies. Mechanistically, this deep quiescent state results from the inhibition of HDAC1 activity by increased levels of the primary circulating ketone body, β-hydroxybutyrate (BHB). We show, using pharmacological and genetic tools, that p53 activation downstream of HDAC1 inhibition is both necessary and sufficient to drive ketosis-induced DQ. Altogether, this report highlights the novel finding that a metabolite produced endogenously during DR elicits a protective state in a stem cell population. To comprehensively characterize the effects of fasting on muscle regeneration, we injured the tibialis anterior (TA) muscle of the lower hindlimb in mice fasted for 0, 1, 2, or 2.5 days (Figure 1A ). After 7 days of recovery, we isolated the muscles and examined regeneration histologically. We found that recovery, as measured by regenerating myofiber cross-sectional area (CSA), progressively declined as a function of duration of fasting prior to injury (Figures 1B and 1C). To explore the reversibility of this regenerative delay, we measured the extent of muscle repair in mice that had been fasted for 2.5 days and subsequently refed for 1, 2, 3, or 7 days prior to injury (Figure 1D). Intriguingly, we found that an impairment of regeneration persisted up to 3 days after refeeding, despite a complete return of body weight (Figures 1E–1G). Importantly, 1 week of refeeding was able to restore muscle regeneration back to baseline (Figure 1F). These findings indicate that fasting causes a transient state of impaired regenerative activity that persists days after refeeding. Because MuSCs are critically important for muscle regeneration after injury (Lepper et al., 2011Lepper C. Partridge T.A. Fan C.-M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.Development. 2011; 138: 3639-3646Crossref PubMed Scopus (726) Google Scholar; Wang et al., 2014Wang Y.X. Dumont N.A. Rudnicki M.A. Muscle stem cells at a glance.J. Cell Sci. 2014; 127: 4543-4548Crossref PubMed Scopus (80) Google Scholar), we next wanted to examine how short-term fasting might be affecting the function of the MuSCs themselves. After fasting a cohort of mice, we isolated quiescent MuSCs from the hindlimbs by fluorescence-activated cell sorting (FACS) purification as previously described (Liu et al., 2016Liu L. Cheung T.H. Charville G.W. Rando T.A. Isolation of skeletal muscle stem cells by fluorescence- activated cell sorting.Nat. Protoc. 2016; 10: 1612-1624Crossref Scopus (200) Google Scholar). We found that MuSCs from fasted mice were smaller than their control counterparts at the time of isolation (Figure 2A ). Additionally, MuSCs from fasted mice exhibited a significant reduction in mitochondrial content, RNA content, and basal oxygen consumption compared with control MuSCs (Figures 2B, S1A, and S1B). These cells were also significantly delayed in their time to enter S phase both after ex vivo activation as well as in response to in vivo injury (Figures 2C and S1C). Therefore, MuSCs from fasted mice exhibit properties that are characteristic of cells in a state of DQ (Fujimaki et al., 2019Fujimaki K. Li R. Chen H. Della Croce K. Zhang H.H. Xing J. Bai F. Yao G. Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch.Proc. Natl. Acad. Sci. USA. 2019; 116: 22624-22634Crossref PubMed Scopus (49) Google Scholar; Kwon et al., 2017Kwon J.S. Everetts N.J. Wang X. Wang W. Della Croce K. Xing J. Yao G. Controlling depth of cellular quiescence by an Rb-E2F network switch.Cell Rep. 2017; 20: 3223-3235Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In agreement with our muscle regeneration data, we found that this state of DQ persists up to 2 days after refeeding (Figures S1D–S1F), despite the return to baseline of body weight (Figure 1G). These data suggest that DQ represents a stem cell state that can perdure for multiple days after the conclusion of fasting and that this perdurance may explain the delay in muscle regeneration that we observed after fasting, even following 72 h of refeeding. Recent work has suggested that many of the beneficial effects of fasting (especially longer term fasting and CR) may be the result of the ketosis that accompanies the period of fasting (Veech et al., 2017Veech R.L. Bradshaw P.C. Clarke K. Curtis W. Pawlosky R. King M.T. Ketone bodies mimic the life span extending properties of caloric restriction.IUBMB Life. 2017; 69: 305-314Crossref PubMed Scopus (102) Google Scholar). In order to assess whether ketosis, absence of the stark energy imbalance seen with fasting, might be responsible for promoting this state of DQ, we fed mice an ad libitum ketogenic diet for 3 weeks to induce endogenous ketosis (Figure S2A) and asked whether that might also induce a similar state of DQ in the MuSCs. We found that the MuSCs isolated from mice on the ketogenic diet exhibited the same hallmarks of the DQ state that we observed with fasting (Figures S2B–S2E). Similarly, we detected a significant delay in muscle regeneration in mice that were fed a ketogenic diet (Figures S2F and S2G). Given that the ketogenic diet and fasting both involve drastic hormonal changes that accompany carbohydrate restriction, we next asked whether supplementation of an ad libitum chow diet with exogenous ketone bodies could also promote MuSC DQ. Mice were injected intraperitoneally with the two primary ketone bodies (BHB and acetoacetate) three times per day for 7 days to mimic the endogenous ketosis brought about by fasting and the ketogenic diet (Figures S2H–S2K). Unlike fasting or the ketogenic diet, exogenous ketone body supplementation resulted in no change in blood glucose levels or body weight (Figures S2L and S2M). Similar to fasting and the ketogenic diet, exogenous ketone bodies also induced all of the hallmarks of DQ in MuSCs, including smaller size, less mitochondrial content, less oxygen consumption, less RNA content, and a slower rate of S phase entry (Figures 2D–2H and S2N). We also found that the MuSCs from ketone-treated mice had a decreased propensity to break quiescence and enter S phase under homeostatic conditions in vivo (Figure 2I). Collectively, these data suggest that ketosis itself, and ketone bodies in particular, might be a primary driver of MuSC DQ. To probe the molecular changes underlying this ketone-induced DQ (KIDQ), we sequenced the transcriptomes of freshly isolated MuSCs from mice that were injected with ketone bodies or vehicle control (Figure 3A ). Consistent with our evidence that MuSCs from ketone body-treated mice exist in a deeper state of quiescence, gene ontology analysis revealed significant and robust changes in cell cycle genes, including downregulation of well-established proliferation genes such as the E2F family members, CDK2, and multiple members of the cyclin family of proteins (Figures 3B and 3C). We also observed an upregulation of genes involved in the inhibition of cell proliferation and growth, including CDKN1A (p21) and CDKN2D (p19) (Figure 3C). This signature is consistent with the transcriptional signature of DQ recently described in fibroblasts in response to altered lysosomal activity in vitro (Fujimaki et al., 2019Fujimaki K. Li R. Chen H. Della Croce K. Zhang H.H. Xing J. Bai F. Yao G. Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch.Proc. Natl. Acad. Sci. USA. 2019; 116: 22624-22634Crossref PubMed Scopus (49) Google Scholar). Notably, we found a significantly increased ratio of p21 to cyclin D1 in MuSCs from ketone body-injected mice (Figure S3A), which is consistent with a higher E2F switching threshold that has previously been shown to be a critical driver of quiescence depth (Kwon et al., 2017Kwon J.S. Everetts N.J. Wang X. Wang W. Della Croce K. Xing J. Yao G. Controlling depth of cellular quiescence by an Rb-E2F network switch.Cell Rep. 2017; 20: 3223-3235Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). No change was observed in the senescence marker p16 in response to ketone bodies, consistent with the absence of any features of senescence in MuSCs in response to fasting, ketogenic diet, or ketone body injections (Figure S3B). In addition to cell-cycle changes, our transcriptomic analysis revealed that freshly isolated MuSCs from ketone-injected mice exhibited lower expression of myogenic genes such as MYF5 and MYOD1 (Figures 3D and S3C). They also displayed increased expression of CD34, a MuSC stemness marker (Ieronimakis et al., 2010Ieronimakis N. Balasundaram G. Rainey S. Srirangam K. Yablonka-Reuveni Z. Reyes M. Absence of CD34 on murine skeletal muscle satellite cells marks a reversible state of activation during acute injury.PLoS One. 2010; 5: e10920Crossref PubMed Scopus (54) Google Scholar) (Figure 3D). Previous work from our lab and others has shown that quiescent MuSCs contain MYOD1 transcripts (de Morrée et al., 2017de Morrée A. van Velthoven C.T.J. Gan Q. Salvi J.S. Klein J.D.D. Akimenko I. Quarta M. Biressi S. Rando T.A. Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence.Proc. Natl. Acad. Sci. USA. 2017; 114: E8996-E9005Crossref PubMed Scopus (50) Google Scholar), perhaps allowing quiescent MuSCs to be poised for myogenesis upon activation. Because DQ MuSCs contain less MYOD1 and higher levels of CD34, this may be indicative of MuSCs that are more stem-like and less poised for myogenic commitment. Although total Pax7 transcript levels were not significantly altered in the freshly isolated MuSCs from ketone-treated mice (Figure S3D), we found that freshly isolated DQ MuSCs had a larger percentage of cells with Pax7hi promoter activity (Figure 3E). Our work is consistent with previous findings that have found a strong correlation between Pax7 promoter activity and delayed exit from quiescence (Rocheteau et al., 2012Rocheteau P. Gayraud-Morel B. Siegl-Cachedenier I. Blasco M.A. Tajbakhsh S. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division.Cell. 2012; 148: 112-125Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Additionally, we found that Pax7 activity persisted much longer during the course of activation of MuSCs from ketone-treated mice (Figures 3F and S3E), consistent with a state of DQ. Previous work has shown that the Pax7hi subpopulation of MuSCs has a higher capacity to seed the MuSC niche than the Pax7lo population (Rocheteau et al., 2012Rocheteau P. Gayraud-Morel B. Siegl-Cachedenier I. Blasco M.A. Tajbakhsh S. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division.Cell. 2012; 148: 112-125Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). To assess the engraftment potential of DQ MuSCs compared with control MuSCs, we tested whether DQ cells could outcompete their control counterparts in a competitive transplantation assay. In this paradigm, we cotransplanted distinctly labeled DQ MuSCs and control MuSCs into the same injured recipient muscle. After 28 days, we isolated by FACS those transplanted DQ and control MuSCs that had engrafted. We found that DQ MuSCs consistently outcompeted their control counterparts and were present in higher numbers in the recipient muscle (Figure S3F). These results suggest that the state of MuSC DQ is also characterized by an enhanced capacity for long-term engraftment. In order to test for the propensity of DQ MuSCs to exit quiescence and enter the cell cycle, we plated MuSCs from ketotic and control mice in culture with the intention of studying in more detail their kinetics of activation. However, surprisingly, we observed that a greater number of MuSCs from ketotic mice were present in culture after 48 h of activation compared with their control counterparts (Figure S3G). Having already observed that DQ MuSCs take longer to break quiescence and enter the cell cycle, we surmised that the most likely explanation for this increase in cell number was not due to faster expansion but rather an enhanced ability to survive the stress of the activation process (Liu et al., 2018Liu L. Charville G.W. Cheung T.H. Yoo B. Santos P.J. Schroeder M. Rando T.A. Impaired notch signaling leads to a decrease in p53 activity and mitotic catastrophe in aged muscle stem cells.Cell Stem Cell. 2018; 23: 544-556.e4Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Consistent with this hypothesis, we found that DQ MuSCs exhibited a dramatic decrease in cell death in culture (Figures S3H–S3J), suggesting that the state of DQ is one of a marked increase in resilience. We next asked whether these DQ MuSCs could also resist other forms of stress as well. Indeed, we found that MuSCs isolated from ketone body-injected mice were much more resistant to oxidative stress as well as that of acute nutrient deprivation (Figures 4A and S3K). Together, these data highlight another key property of DQ cells and that is one of increased resilience and general stress resistance. We have recently shown that old MuSCs have an impaired ability to survive the transition between quiescence and activation (Liu et al., 2018Liu L. Charville G.W. Cheung T.H. Yoo B. Santos P.J. Schroeder M. Rando T.A. Impaired notch signaling leads to a decrease in p53 activity and mitotic catastrophe in aged muscle stem cells.Cell Stem Cell. 2018; 23: 544-556.e4Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Given the resistance of DQ MuSCs to activation-induced death, we wanted to know if we could rescue the known survival impairments of old MuSCs by promoting KIDQ. In order to address this, we injected a cohort of 24-month-old mice with either vehicle or ketone bodies for 1 week, at which point we sacrificed the mice and isolated MuSCs from the hindlimbs. After plating the cells for 48 h in culture, we again measured cell death. Quite strikingly, we found that ketone body treatment could indeed rescue the survival defects that are characteristic of old MuSC activation (Figure 4B). We next" @default.
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• W4280490566 date "2022-06-01" @default.
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• W4280490566 title "Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling" @default.
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