Regenerative decline of stem cells in sarcopenia

ARTICLE IN PRESS Molecular Aspects of Medicine ■■ (2016) ■■–■■

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Molecular Aspects of Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m a m

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Regenerative decline of stem cells in sarcopenia

Q1 Pedro Sousa-Victor a, Pura Muñoz-Cánoves b,c,* a

Buck Institute for Research on Aging, Novato, CA, USA Cell Biology Group, Department of Experimental and Health Sciences, CIBER on Neurodegenerative Diseases (CIBERNED), Pompeu Fabra University (UPF), Barcelona, Spain c ICREA, Barcelona, Spain b

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A R T I C L E

I N F O

Article history: Received 26 January 2016 Revised 27 January 2016 Accepted 19 February 2016 Available online Keywords: Sarcopenia Skeletal muscle regeneration Muscle stem cell (satellite cell) Aging Disease

A B S T R A C T

Skeletal muscle mass and function decline with aging, a process known as sarcopenia, which restrains posture maintenance, mobility and quality of life in the elderly. Sarcopenia is also linked to a progressive reduction in the regenerative capacity of the skeletal muscle stem cells (satellite cells), which are critical for myofiber formation in early life stages and for sustaining repair in response to muscle damage or trauma. Here we will review the most recent findings on the causes underlying satellite cell functional decline with aging, and will discuss the prevalent view whereby age-associated extrinsic factor alterations impact negatively on satellite cell-intrinsic mechanisms, resulting in deficient muscle regeneration with aging. Further understanding of the interplay between satellite cell extrinsic and intrinsic factors in sarcopenia will facilitate therapies aimed at improving muscle repair in the increasing aging population. © 2016 Published by Elsevier Ltd.

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Introduction .............................................................................................................................................................................................................................. 1.1. Background on sarcopenia and muscle stem cells ......................................................................................................................................... Stem cells and aging .............................................................................................................................................................................................................. Satellite cells in the sarcopenic muscle ........................................................................................................................................................................... 3.1. Regenerative functions of adult satellite cells ................................................................................................................................................. 3.2. Decline of satellite cell regenerative capacity with aging ........................................................................................................................... 3.2.1. Extrinsic changes and functional decline ......................................................................................................................................... 3.2.2. Intrinsic changes and numerical decline ......................................................................................................................................... Concluding remarks ............................................................................................................................................................................................................... Acknowledgements ................................................................................................................................................................................................................ References ..................................................................................................................................................................................................................................

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1. Introduction 51 1.1. Background on sarcopenia and muscle stem cells

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* Corresponding author. Cell Biology Group, Department of Experimental and Health Sciences, ICREA and CIBERNED, Universitat Pompeu Fabra, Dr. Aiguader, 88, E-08003 Barcelona, Spain. Tel.: +34 93 316 0891. E-mail address: [email protected] (P. Muñoz-Cánoves).

Clinically, sarcopenia is defined as a geriatric syndrome characterized by loss of skeletal muscle mass and decline in muscle strength that compromise health span (Delmonico et al., 2007). The sarcopenic muscles exhibit distinct features that include atrophy of type 2 (fast-twitch)

http://dx.doi.org/10.1016/j.mam.2016.02.002 0098-2997/© 2016 Published by Elsevier Ltd.

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myofibers and heterogeneity in fiber size, accumulation of intramuscular connective tissue and fat as well as decreased oxidative capacity. These cellular features are associated with defective mitochondrial energy metabolism, denervation, increased inflammation and protein catabolism, which distinguish the sarcopenic muscle from other features of normal aging (Barns et al., 2014; Ibebunjo et al., 2013). Motor neurons and mature myofibers are, thus, the major targets for clinical intervention to ameliorate sarcopenia (Grounds, 2014). Sarcopenia is also accompanied by a significant decline in satellite cell function and numbers, which strongly compromise regenerative capacity of the skeletal muscle (Sousa-Victor et al., 2014a). Satellite cell loss might not contribute to age-associated loss of muscle mass, as suggested by recent studies in which satellite cells were ablated in young animals, without affecting skeletal muscle myofiber size or composition (Fry et al., 2015); at variance, other studies proposed that satellite cells do contribute to myofibers in all adult muscles in sedentary mice, although the extent and timing differs (Keefe et al., 2015). Nevertheless, satellite cell loss contributes to agedependent muscle fibrosis (Fry et al., 2015) and agerelated decline in satellite cell function compromises the recovery capacity of sarcopenic muscles in response to injury (Sousa-Victor et al., 2014a). In this review we will discuss the state of the art on the age-associated decline of satellite cell function in the sarcopenic muscle, focusing on the consequences for muscle function and regenerative capacity. We will focus on the current knowledge on aging of murine satellite cells and will provide perspectives on the potential implications for human satellite cells. Further we will discuss recent findings on satellite cell rejuvenation strategies and their potential impact in counteracting age-related decline of regenerative capacity of the skeletal muscle in the elderly.

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2. Stem cells and aging Age is the major risk factor for chronic diseases (Kennedy et al., 2014), suggesting the existence of a mechanistic link between aging and disease. However, modern biogerontology supports the notion that aging itself is not a disease but a process that increases the chances of disease onset, leading to the emerging concept of age-related diseases when referring to conditions such as sarcopenia (Partridge, 2014). Although the consequences of aging on human health and tissue homeostasis are broadly apparent, the causes and drivers of the aging process are just beginning to be understood. Aging is a complex process that, in multicellular organisms, results from the interplay among cell intrinsic, inter-cellular communication and systemic dysregulations which coordinately compromise the homeostatic capacity of the organism. The aging community has now identified several hallmarks or pillars of aging and is starting to elucidate an integrated view of the basic mechanisms of the aging process (Kennedy et al., 2014; Lopez-Otin et al., 2013). Understanding the molecular mechanisms that underlie and link the different causes of aging is thus crucial to develop strategies that will impact the development and progression of age-related diseases.

Adult stem cells are specialized cell types responsible for sustaining homeostatic renewal and regenerative capacity in different tissues. Importantly, stem cells are a major target of the aging process where several of the aging pillars converge. Aging compromises stem cell maintenance and function which, in turn, contributes to a decline in regenerative capacity and tissue homeostasis throughout life. This causal link is the basis for the postulation of a stem-cell centered theory of aging (Sharpless and DePinho, 2007). Stem cells can exist in two different states – quiescent and activated. Quiescent stem cells are in a state of reversible cell cycle arrest and can become activated upon proliferative pressure imposed by homeostatic demand of a renewing tissue, or regenerative demand following an injury or damage. Upon activation, stem cells re-enter the cell cycle and proliferate. Activated stem cells need to coordinate two basic functions: self-renew a new stem cell by re-instating quiescence and thus ensuring the maintenance of the stem cell pool; and differentiate to give rise to a specialized mature progeny that can replenish the lost cells with specialized functions in the renewing or regenerating tissue. Stem cell quiescence, activation, self-renewal and differentiation are coordinated by the interplay between intrinsic programs and signals from the surrounding milieu, referred to as ‘niche’. These include growth factors, trophic factors and cytokines derived from the somatic and stromal cells in the niche as well as from the systemic environment that regulate stem cell function. Thus it is not surprising that the process of stem cell aging is a consequence of the combined effects of age-dependent alterations in the environment and ageassociated intrinsic dysregulations of the stem cell itself (Dumont et al., 2015). The relative impact of different aging stressors on a particular stem cell population depends on the proliferative pressure imposed by their host tissue. While genomic instability associated with repeated DNA replication is an aging hallmark of stem cells from high turnover tissues (e.g., intestine, hematopoietic system), genotoxic stress driven chronological aging contributes to stem cell dysfunction in quiescent stem cell populations of low turnover tissues (e.g., skeletal muscle and brain). These aging stressors can impact stem cell function at multiple levels, compromising selfrenewal capacity, activation and/or differentiation. Depending of the stem cell function(s) that are affected, aging can result in different outcomes that range from stem cell depletion to hyperplastic conditions that irrespectively compromise tissue maintenance and regenerative capacity (Adams et al., 2015; Burkhalter et al., 2015). 3. Satellite cells in the sarcopenic muscle 3.1. Regenerative functions of adult satellite cells The stem cells of the skeletal muscle are usually referred to as satellite cells due to their anatomical location peripheral to the myofiber and underneath the basal lamina. Muscle stem cells were first identified in the 1960s by electron microscopy (Mauro, 1961) and their chromatin and organelle characteristics suggested that they were mitotically and metabolically quiescent cells. Following studies showed that satellite cells are established early during

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development and are marked by the expression of the paired box transcription factor Pax7 (Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2006). During embryonic development and postnatal growth, satellite cells actively contribute to the increase in myonuclei in growing neonatal myofibers. However, by P21 (postnatal day 21) satellite cells enter into a quiescent state, remaining as dormant (or very slow cycling) stem cells throughout adult life. In adulthood, satellite cells contribute marginally to muscle growth and account only to about 2–10% of total myonuclei (White et al., 2010). During adulthood, satellite cells’ main function is to sustain skeletal muscle regenerative capacity. Upon activation by injury or pathological stimuli, satellite cells exit quiescence and actively proliferate. Subsequently, the active myogenic progenitor exits cell cycle and activates a differentiation program to generate new myofibers that will replenish the damaged tissue. A subpopulation of activated satellite cells reinstates quiescence to self-renew the satellite cell pool (Dumont et al., 2015). The ability of adult satellite cells to transit to a reversible quiescent state after providing a source of progeny is critical for homeostasis of tissue-resident stem cells and presumably for the maintenance of tissue function through numerous rounds of damage caused by various insults throughout life. Recent years have seen significant advances on the understanding of the intrinsic and extrinsic mechanisms that regulate satellite cell function (Dumont et al., 2015). It is also now apparent that it is the cumulative dysregulation of these intrinsic mechanisms and extrinsic regulatory cues that contributes the process of satellite cell aging (Sousa-Victor et al., 2015). 3.2. Decline of satellite cell regenerative capacity with aging The main consequence of satellite cell aging is the loss of skeletal muscle regenerative capacity which is particularly pronounced in the sarcopenic muscle of both humans and mice (Sousa-Victor et al., 2014a; Zwetsloot et al., 2013). Aging of the satellite cell is characterized by a decline in stem cell numbers and functionality, which has been attributed a combination of factors, including defects in self-renewing mechanisms, exhaustion by forced differentiation as well as apoptosis and senescence. The loss of functionality is also apparent at different levels including defects in activation and loss of capacity to produce the appropriate myogenic lineage (Sousa-Victor et al., 2015). Over the past decade, evidence has accumulated suggesting that there is a strong contribution of the environment to the satellite cell aging phenotype, including dysregulation of signals from the myofiber and from the circulatory system. Accordingly, exposure of old satellite cells to young environment and/or circulatory system has rejuvenating effects and can restore part of satellite cell function (Brack and Rando, 2007; Conboy et al., 2005; Villeda et al., 2011). However, recent studies suggest that in older, geriatric ages (28 months and beyond), when the sarcopenic phenotype is exacerbated, there are intrinsic alterations in the satellite cell, which irreversibly compromise its function, and cannot be reversed by exposure to youthful cues. Since aging is a progressive continuous process, it is likely that the aging environment lead to the

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accumulation of cellular stressors that culminate in irreversible changes to the satellite cell if no interventions take place (Sousa-Victor et al., 2014a). Interestingly, there are some phenotypes of patients with muscle dystrophies which resemble those of sarcopenia. This is the case of Myotonic Dystrophy Type 1 (DM1), a neuromuscular disease, characterized by muscle wasting and weakness among other symptoms. Also in this case, there is a dysregulation of the stem cells that resembles those found in geriatric satellite cells of sarcopenic muscles (Bigot et al., 2009; Sousa-Victor et al., 2014a), further supporting the notion that there is a link between the myofiber and satellite cell phenotypes in old muscle. Current efforts in the aging field focus in the discovery of rejuvenation strategies that can delay or slow the progression of age-related diseases. It is interesting to note that several of such interventions can affect both the phenotypes of the myofiber and of the satellite cell. A case in point are the effects of caloric restriction which can improve muscle regenerative capacity by improving satellite cell function in the old skeletal muscle (Cerletti et al., 2012) in parallel to improving preservation of muscle mass and strength with aging (Colman et al., 2008; Lee et al., 1998).

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3.2.1. Extrinsic changes and functional decline The idea that extrinsic factors from the niche and the circulatory environment impact satellite cell functionality is based on heterochronic tissue transplant studies (Carlson and Faulkner, 1983; Harrison, 1983), and heterochronic parabiosis studies (Brack and Rando, 2007; Conboy et al., 2005; Villeda et al., 2011). Early muscle transplantation studies showed that it is the age of the host animal that determines the success of the regenerative process associated with the transplant (Carlson and Faulkner, 1989; Gutmann and Carlson, 1976; Roberts et al., 1997). The heterochronic parabiosis experiments later showed that the regenerative capacity of the skeletal muscle from old animals can be significantly enhanced when its circulatory system is joined with that of a young counterpart (Brack and Rando, 2007; Conboy et al., 2005; Villeda et al., 2011), while the regenerative capacity of the satellite cells in the young animal declines in parallel. These studies stimulated the quest for the identification of factors derived from the old/young environment that could mediate the effects of age on satellite cells. Finding such factors would allow the development of specific strategies to ameliorate regenerative capacity in the sarcopenic skeletal muscle. The combined effort of several research groups has now generated a significant set of data that suggests that most of the signaling pathways known to regulate satellite cell function are dysregulated during aging and act synergistically to compromise function of muscle stem cells. Main age related changes in the niche and systemic environment identified so far include: increased levels of wnt ligands in the old serum, increased Q5 transforming growth factor β (TGFβ) and fibroblast growth factor 2 (FGF2) expression from the myofiber concomitant with defects in the expression of the Notch ligand Delta1. Notch signaling is a master regulator of satellite cell function and its balance controls satellite cell self-renewal and myogenic differentiation in a coordinated manner (Mourikis

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and Tajbakhsh, 2014). Not only myofibers but also old myogenic progenitors fail to appropriately up-regulate the Notch ligand Delta1 during myogenesis in aged skeletal muscle. Consistently, Notch signaling activation can partially compensate for the loss of regenerative capacity in the old skeletal muscle and is part of the mechanism through which young serum can restore satellite cell function in parabiosis studies (Conboy et al., 2003, 2005; Wagers and Conboy, 2005). Importantly, Notch signaling synergizes with Wnt and transforming growth factor (TGF)-β signaling in satellite cells to control muscle regeneration (Brack et al., 2008; Carlson et al., 2008). These signaling cascades are also dysregulated in aging, with important consequences for satellite cell function. TGF-β signals through the pSmad3 intracellular pathway and promotes the expression of cyclin-dependent kinase (CDK) inhibitors which contribute to the inhibition of satellite cell proliferation, while notch signaling can antagonize this function by removing pSmad3 from the promoters of these CDK inhibitor genes (Carlson et al., 2008). Therefore, the cumulative depletion of Notch signal and enhancement of TGF-β signaling in the aging muscle likely result in the enhanced expression of CDK inhibitors, associated with defective proliferative capacity and differentiation (Carlson et al., 2008; Conboy et al., 2003). Consistently, blockade of TGF-β signaling is also sufficient to alleviate some of the effects of aging on satellite cell function (Carlson et al., 2008). Dysregulation of Wnt signaling during aging has also been proposed to contribute to stem cell loss of function in the sarcopenic skeletal muscle. Increased Wnt signaling driven by systemically-derived ligand promotes a myogenic to fibrogenic fate conversion that contributes to increased tissue fibrosis and reduced regenerative capacity (Brack et al., 2007). Complement protein C1q was identified as the Wnt-activating ligand present in the old blood responsible for these phenotypes (Naito et al., 2012). Despite the negative impact of the age-related chronic activation of Wnt signaling on satellite cell function, a dynamic balance of the state of activation of Notch and Wnt pathways is thought to be required for effective muscle

regeneration. Myogenic commitment requires the Wntdependent inactivation of the glycogen synthase kinase 3 beta (GSK3β), which is activated in a Notch dependent manner (Brack et al., 2008). Therefore, synergistic effects of Notch loss of function and Wnt gain of function during aging may contribute to favor misdifferentiation at the expenses of self-renewal and contribute to the age-associated exhaustion of the stem cell pool. Although this model may hold true, a recent report has challenged this view and proposed that wnt/βcatenin signaling is dispensable for myogenesis during skeletal muscle regeneration and that Wnt silencing is the crucial step in the regulatory mechanism to prevent deleterious effects of prolonged regenerative response (Murphy et al., 2014). Finally, the age-associated increased expression of FGF2 in the myofiber compromises the maintenance of satellite cell quiescent state and can be reversed by neutralizing FGF2 activity or blocking FGF receptors. The effects are thought to be mediated by Sprouty1 protein, which intrinsically changes compromise satellite cell function. Sprouty is a negative regulator of FGF signaling and its genetic deletion or age-associated fluctuations are thought to compromise the ability of satellite cell to regulate the age-dependent increase of FGF signaling (Chakkalakal et al., 2012). Muscle precursor cells from adult and old humans have recently been shown to present increased DNA methylation at the Spry1 gene correlating with a decline in cells capable of returning to quiescence (Bigot et al., 2015) (see Fig. 1). These studies supported the view that there are signaling pathways dysregulated in the old environment and that targeting these pathways may yield viable rejuvenating strategies for the loss of regenerative capacity in the sarcopenic skeletal muscle. An independent line of research has focused, instead, on looking for factors in the young blood which may be responsible for the rejuvenating effects observed in parabiosis studies. Growth differentiation factor 11 (GDF11) (Sinha et al., 2014) and oxytocin (Elabd et al., 2014) were reported as such candidates, although the effects of GDF11 have been recently challenged (Egerman et al., 2015). Oxytocin levels are reduced in the plasma of old animals and the systemic administration of this compound to old animals

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Fig. 1. Determinants of muscle stem cells decline with aging.

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significantly rescued muscle regenerative capacity (Elabd et al., 2014). Similar results were reported for GDF11 (Sinha et al., 2014) while a more recent report shows instead that GDF11 tends to increase with age in the old plasma of rats and humans, and has a negative impact on muscle regeneration (Egerman et al., 2015). Indeed, the latter study suggests that GDF11 synergizes with myostatin (a closely related protein also known as GDF8), acting through a common SMAD2/3 pathway to inhibit myogenesis, resulting in a reduced regenerative capacity after exposure to exogenous GDF11 (Egerman et al., 2015). Myostatin is a known negative regulator of myofiber growth and myoblast differentiation (Rios et al., 2002; Trendelenburg et al., 2009) and a recent report has confirmed that the circulating levels of myostatin increase in older sarcopenic humans (Bergen et al., 2015) suggesting that it may contribute to sarcopenic phenotypes in man. Thus, it will be important to better and further understand the effects of pathways acting downstream of myostatin and GDF11 in muscle growth and satellite cell function to decide on appropriate coordinated strategies to counteract sarcopenia and its associated loss of skeletal muscle regenerative capacity. 3.2.2. Intrinsic changes and numerical decline The low rate of replacement of differentiated cells in the skeletal muscle means that satellite cells remain quiescent for most of the organism’s life. During this dormant state, the satellite cell is required to retain the capacity for rapid activation and engagement of the myogenic program in response to environmental cues, with a subset of progenitor cells returning to quiescence to reestablish the muscle stem cell pool. The recent development of reliable satellite cell markers, in combination with flow cytometry, have cleared the long standing controversy on whether satellite cell numbers decrease with age (Conboy et al., 2003; Hawke and Garry, 2001; Roth et al., 2000; Shefer et al., 2006, 2010; Wagers and Conboy, 2005; Zammit et al., 2002), showing that the muscle stem cell numbers are reduced by 50% in old mice (Brack et al., 2005; Cerletti et al., 2012; Chakkalakal et al., 2012). The underlying causes for the decline in satellite cell numbers with age are still largely unknown, but recent studies point to the combinatorial effect of age-associated changes in the niche and cell autonomous alterations that perturb the proper balance between cell quiescence and proliferation, having an impact in cell survival (Blau et al., 2015; Chang and Rudnicki, 2014; Garcia-Prat et al., 2013; Jung and Brack, 2014; Rezza et al., 2014; Sacco and Puri, 2015; Sousa-Victor et al., 2015). The observation that human satellite cells isolated from aged subjects display an increased susceptibility to nuclear apoptosis seems to support the idea that processes affecting cell survival are implicated at different levels in the age-associated depletion of muscle stem cell pools (Fulle et al., 2013). Consistently, mouse models with a satellite cell-specific deletion of Sprouty1 show persistent activation of the ERK/MAPK signaling pathway in proliferating muscle stem cells and impaired self-renewal. The inability of Sprouty1-null cycling satellite cells to return to quiescence was not due to changes in proliferative capacity or commitment to differentiation, but to an increased susceptibility to apoptosis (Shea et al., 2010). Furthermore, analysis of mice with reduced levels of AIF, a factor involved in the regulation of apoptotic processes in response to oxidative stress,

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displays signs of early sarcopenia and decreased satellite cell numbers that could be partly rescued by antioxidant treatment (Armand et al., 2011). Muscle injury in mouse models where Pax7 + cells are specifically targeted for elimination has confirmed the need of adult satellite cells for efficient skeletal muscle regeneration (Lepper et al., 2011; Sambasivan et al., 2011), pointing to the contribution of the numerical decrease in the satellite cell population to the overall decline in regenerative capacity of the old muscle. While apoptosis leads to a rapid elimination of dysfunctional cells, an alternative response of stressed or defective cells is the engagement of a senescence program. The senescent state is defined by an irreversible cell cycle arrest and characterized by the presence of markers such as senescence-associated β-galactosidase, HP-1 (heterochromatin protein-1) foci, and the expression of the CDK inhibitor p16INK4a. Although thought to have evolved alongside apoptosis as a cancer-protective mechanism, it is believed to be the driving force behind many age related pathologies (Campisi and Robert, 2014; van Deursen, 2014). Indeed, in muscles of geriatric mice, satellite cells were shown to be in a pre-senescent state associated with the upregulation of p16INK4a. Upon proliferative pressure, these cells undergo a quiescence-to-senescence conversion, causing an irreversible impairment in the activation/proliferation capacity required for self-renewal and progenitor expansion involved in the regenerative process. Preventing the conversion of satellite cells to a state of advance senescence through the silencing of the p16INK4a gene could restore the stem cell self-renewal capacity and improve the muscle regenerative potential (Sousa-Victor et al., 2014a). Further support for the notion that p16INK4a-induced senescence can drive satellite cell loss and is associated with sarcopenia is the analysis of mice with increased levels of p16INK4a due to the genetic loss of the polycomb repressor complex 1 (PRC1) protein Bmi1. Young adult Bmi1-deficient mice showed reduced numbers of satellite cells in comparison to agematched WT littermates and displayed a significant reduction in muscle size, suggesting premature muscle wasting (Sousa-Victor et al., 2014b). Interestingly, mutant mice carrying BubR1 hypomorphic alleles show early signs of sarcopenia accompanied by the expression of senescence markers (Baker et al., 2004, 2008). In this progeroid model, a gene targeting strategy for the elimination of p16INK4aexpressing cells was sufficient to attenuate the progression of sarcopenia, with late-life treated animals showing increased mean muscle fiber diameters and improved performance in treadmill exercise tests (Baker et al., 2011). A recent study showed that quiescent satellite cells, despite their dormant state, display continuous basal macroautophagy (from now on ‘autophagy’; i.e. the process for degradation of long-lived proteins and damaged intracellular organelles in lysosomes (Cuervo et al., 2005; He and Klionsky, 2009)) throughout life (Garcia-Prat et al., 2016), which constitutes a protective clean-up mechanism against intracellular waste accumulation. With aging, this protective mechanism is lost leading to the accumulation of damaged proteins and organelles. Furthermore, physiological failure of autophagy in aged satellite cells or genetic impairment of autophagy in young satellite cells causes senescence entry by loss of proteostasis and increased

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oxidative stress, resulting in numerical and functional muscle stem cell decline. Importantly, genetic and pharmacological regimes that reinstall basal autophagy in physiologically aged (geriatric) mice reversed muscle stem cell senescence and restored regeneration (Garcia-Prat et al., 2016). It is worth noting that ROS levels were uncovered as key epigenetic regulators of p16INK4a in aging stem cells, by impeding PRC1-mediated lysine119 H2A-ubiquitination. Consistent with this, treatment of geriatric mice with antioxidants not only restored PRC1-mediated INK4a locus repression and prevented satellite cell senescence, but also restored regenerative capacity (Garcia-Prat et al., 2016). Interestingly, a recent report showed cellular senescence during regeneration of skeletal muscle of adult mice (Le Roux et al., 2015), supporting that this terminal cellular destiny is not exclusive of aging muscle. Very recent studies also showed that muscle precursor cells from adult and aging humans do not undergo senescence (Bigot et al., 2015), overall suggesting the need of further investigating into the differences between age-associated satellite cell dysfunction between mice and humans. Senescence or apoptosis are cell fate decisions that result from overwhelming damage to the cell that cannot be resolved through repair mechanisms. Given the slow or nondividing nature of the quiescent state, satellite cells require a mechanism of double-strand break (DSB) repair that is independent from the cell cycle. Non-homologous endjoining (NHEJ) does not require a template strand for DNA repair, allowing the cell to restore DSBs while in G0/G1. However, the independence from a template makes NHEJ an error prone mechanism that increases the probability of mutagenesis (Burkhalter et al., 2015). A connection between DNA damage repair mechanisms and sarcopenia was provided by the analysis of mice with reduced levels of Ku80, a subunit essential to NHEJ. While complete loss of Ku80 leads to premature muscle wasting accompanied by a significant decrease in the number of satellite cells, loss of a single allele is sufficient to limit satellite cell self-renewal and to reduce muscle regenerative capacity (Didier et al., 2012). Although muscle stem cells appear to be more resistant to DNA damage than their committed progeny due to a higher repair accuracy and efficiency (Vahidi Ferdousi et al., 2014), satellite cells isolated from aged mice still show an increased number of foci containing the DNA damage marker γH2AX (Sinha et al., 2014), pointing to genomic instability as a potential contributor to muscle aging. Global transcriptome and epigenome analysis comparing young and aged satellite cells have pointed to changes in the transcriptional program that could underlie some of the observed age-associated defects in muscle stem cell function. Human satellite cells isolated from sarcopenic muscles of aged donors show a decreased expression of genes involved in DNA maintenance, changes in molecules involved in antioxidant activity, limited capacity to execute a complete differentiation program due to altered expression of myogenic differentiation-specific genes and activation of a FOXO-dependent atrophy program (Bortoli et al., 2003; Pietrangelo et al., 2009). Some of these transcriptional changes are likely due to an altered epigenetic program found through the comparison of chromatin profiles of satellite cells from young and old mice (Liu et al., 2013).

Supporting this notion, specific analysis of the p16INK4a promoter in satellite cells isolated from geriatric muscles showed a significant loss of the chromatin repressive mark H2AUb in parallel with increased expression of the p16INK4a gene (Sousa-Victor et al., 2014a). The recent advances in satellite cell isolation protocols and the development of transplantation assays have allowed the identification of multiple cell-autonomous signaling pathways dysregulated in aged satellite cells (Bernet et al., 2014; Cosgrove et al., 2014; Price et al., 2014; Tierney et al., 2014). The p38αβ MAPK signaling pathway was found activated in aged muscle stem cells with an impaired self-renewal capacity that cannot be overcome by exposure to a young environment. Importantly, pharmacological inhibition or RNAimediated downregulation of p38α and p38β was able to restore the aged satellite cells capacity for efficient regeneration following serial transplantation (Bernet et al., 2014; Cosgrove et al., 2014). The beneficial effects of p38 MAPK inhibition were strongly enhanced by the synergistic action of biophysical cues in the form of hydrogel cell culture systems. Strikingly, this approach allowed for the rejuvenation of old satellite cells capable of significantly improving the force generation of aged muscle following injury and transplantation, providing a potential strategy for treating severe muscular pathologies and for muscle regeneration in aging individuals (Cosgrove et al., 2014). Analysis of transcriptional profiles of old satellite cells also allowed for the identification of an age-related increase in JAK/STAT signaling. Activation of the JAK/STAT axis inhibits the capacity of satellite cells to undergo symmetric stem cell expansion, negatively impacting the regeneration potential of aged satellite cells (Price et al., 2014). Moreover, Stat3 activation mediated by the extracellular interleukins IL-6 affects the expression of MyoD (Tierney et al., 2014), promoting myogenic commitment at the expense satellite cell expansion. Also in this case, pharmacological or RNAi-mediated inhibition of the over activated JAK/STAT signaling pathway improved the regenerative capacity of aged satellite cells and enhanced muscle repair and force generation following their transplantation (Price et al., 2014). Interventions that act at the organismal level, such as dietary changes, were shown to be equally promising in addressing muscle aging. Caloric restriction (CR), a nutritional regime shown to delay the onset of age-related diseases and extend the lifespan of multiple species (Partridge, 2010), significantly enhanced satellite cell availability and function in the skeletal muscle of old mice (Cerletti et al., 2012) and attenuated the development of sarcopenia in aged rodents (Lee et al., 1998) and primates (Colman et al., 2008). The improved myogenic function and regenerative capacity of satellite cells in CR-treated mice were accompanied by increased expression of the conserved metabolic regulators Sirt1 and Foxo3a (Cerletti et al., 2012), also implicated in longevity extension of different animal models (Canto and Auwerx, 2009; van der Horst and Burgering, 2007). 4. Concluding remarks As a result from advances in medicine, the survival of individuals has increased in the last decades, resulting in a rising increment of the overall aging population. It is well known that organs and tissues deteriorate progressively with

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age. One of the most abundant tissues in mammals, skeletal muscle, also undergoes a profound loss of mass and function (sarcopenia) with aging, being maximal at very advanced, geriatric age. Sarcopenia dramatically restricts voluntary functions, including posture maintenance, mobility and autonomous actions, hence affecting the quality of life in the elderly. Findings over the last years have evidenced that, as organisms age, the resident stem cells of the distinct tissues and organs (including those of skeletal muscle – the satellite cells) deteriorate, as illustrated by their impaired capacity to respond to an insult or maintain homeostasis. For muscle stem cells, both extrinsic changes in the environment as well as cell-autonomous alterations with aging account for the age-associated deterioration of their functions. Since humans are living longer, advancing our understanding of the mechanisms underlying sarcopenia and age-associated muscle stem cell regenerative decline is critical to the development of preventive and therapeutic strategies to combat loss of muscle regenerative capacity. In particular, more studies will be required to identify the specific extrinsic factors associated with defective satellitedependent tissue repair in aged individuals and whether interference with their actions can rejuvenate old satellite cells and improve muscle regeneration. In parallel, future studies should identify how aging directly impacts on the satellite cell-autonomous functions and whether these can be linked to epigenetic changes of reversible or irreversible nature. Increasing our comprehension of these molecular networks operating in muscle stem cells during aging will pave the path to finding new strategies to attenuate or reverse the declining reparative capacity of skeletal muscle of aging individuals, particularly during the critical period of sarcopenia, which is tightly linked to their reduced quality of life.

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Acknowledgements Research in the authors’ laboratory is currently supported by MINECO-Spain (SAF2012-38547, SAF2015-67369-R), Q6 Duchenne PP-NL, E-Rare/ERANET, Fundació Marató-TV3, MDA, EU-FP7 (Myoage, Optistem and Endostem) and “María de Maeztu” Programme for Units of Excellence in R&D MDM-2014-0370.

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Please cite this article in press as: Pedro Sousa-Victor, Pura Muñoz-Cánoves, Regenerative decline of stem cells in sarcopenia, Molecular Aspects of Medicine (2016), doi: 10.1016/j.mam.2016.02.002

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