Role of satellitand maintenan

G. Pallafacchina a,b,c,

aVenetian Institute of MolecularbConsiglio Nazionale delle RicerccDepartment of Biomedical Scien

ived i2

Skeletal muscle;Satellite cells; flecting the balance between myonuclear accretion and myonuclear loss. Myonuclear accre-

atrophying muscle. An increase in myofiber size can also occur by changes in protein turnover

muscle adaptation and in the establishment of functional muscle hypertrophy remains to be

* Corresponding author. Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129 Padova, Italy. Tel.: 39 49 7923232;fax: 39 49 7923250.

E-mail address: stefano.schiaffino@unipd.it (S. Schiaffino).

0939-4753/$ - see front matter 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.numecd.2012.02.002

Available online at www.sciencedirect.com

journal homepage: www.elsevier .com/locate/nmcd

Nutrition, Metabolism & Cardiovascular Diseases (2013) 23, S12eS18established. The identification of the signaling pathways mediating satellite cell activationmay provide therapeutic targets for combating muscle wasting in a variety of pathologicalconditions, including cancer cachexia, renal and cardiac failure, neuromuscular diseases, aswell as aging sarcopenia. 2012 Elsevier B.V. All rights reserved.without satellite cell activation, e.g. in late phases of postnatal development or in somemodels of muscle hypertrophy. The relative role of protein turnover and cell turnover inMuscle hypertrophy;Muscle atrophy

tion, i.e. increase in the number of myonuclei within the muscle fibers, takes place viaproliferation and fusion of satellite cells, myogenic stem cells associated to skeletal musclefibers and involved in muscle regeneration. In developing muscle, satellite cells undergo exten-sive proliferation and most of them fuse with myofibers, thus contributing to the increase inmyonuclei during early postnatal stages. A similar process is induced in adult skeletal muscleby functional overload and exercise. In contrast, satellite cells and myonuclei may undergoapoptosis during muscle atrophy, although it is debated whether myonuclear loss occurs inReceived 16 July 2011; receAvailable online 22 May 201

KEYWORDSe cells in muscle growthce of muscle mass

B. Blaauw a,c, S. Schiaffino a,b,*

Medicine (VIMM), Padova, Italyhe (CNR) Institute of Neurosciences, Padova, Italyces, University of Padova, Padova, Italy

n revised form 1 February 2012; accepted 6 February 2012

Abstract Changes in muscle mass may result from changes in protein turnover, reflecting thebalance between protein synthesis and protein degradation, and changes in cell turnover, re-REVIEW

between protein synthesis and protein degradation.

and mouse muscles [4]. Satellite cells can be recognized by

developmental stages [10], although the issue remains

Satellite cells and muscle growth S13the presence of specific markers, including transcriptionfactors, such as Pax7, or surface membrane proteins, suchas N-CAM, M-cadherin and CD34 (see [5]). Quiescent satel-lite cells are readily activated by muscle damage andacquire a new gene expression profile [6], which includesthe up-regulation of myogenic regulatory factors, such asMyoD and myogenin. Following muscle injury, satellite cellsundergo asymmetric divisions leading to the formation ofundifferentiated cells, which return to quiescence and thusreplenish the satellite cell compartment, and differenti-ated myoblasts which form new myofibers. The study ofmuscle satellite cells is complicated by the existence ofa wide heterogeneity of satellite cell populations, bothwith respect to embryological origin (head vs. bodymuscles), muscle fiber type (fast vs. slow muscles) andpostnatal stage (neonatal vs. adult satellite cells) [5,7]. Inaddition, it has been suggested that the satellite cell poolmay contain both self-renewing stem cells and myogenicprecursors with limited replicative potential [8].

Satellite cells and postnatal muscle growth

Satellite cells are major players in muscle development.At birth, they represent a substantial proportion of muscleHowever, it is now clear that cell turnover is also involved inmuscle growth and maintenance of muscle mass: addition ofnewmyonuclei due to fusion of satellite cells, the stem cellsof skeletal muscle, can take place during myofiber hyper-trophy, while loss of myonuclei via apoptosis has been re-ported during muscle atrophy. One can also envisage anintermediate level of regulation represented by organelleturnover, such asmyofibril assembly (myofibrillogenesis) anddisassembly, or mitochondrial biogenesis and mitochondrialloss. In other words, it is now apparent that muscle growthshould be viewed with the eyes of a cell biologist rather thanof a pure biochemist. In this new perspective, we focus hereon satellite cells. While the central role of satellite cells inmuscle regeneration is well established and is not discussedhere (see [1] for a recent review and [2] for a collection ofessays), the role of these cells in muscle hypertrophy is lessclear. In this short overview we address the contribution ofsatellite cell tomuscle growth, both during development andin adult skeletal muscle.

Muscle satellite cells

Satellite cells are mononucleated stem cells with myogenicpotential located under the basal lamina of myofibers butpossessing their own plasma membrane, distinct from theplasma membrane of the myofibers (see [3]). In adultskeletal muscles satellite cell nuclei represent 3e6% of allmuscle nuclei (nuclei contained within the basal lamina)and are more frequent in slow compared to fast fibers in ratIntroduction

The regulation of muscle mass has been traditionallyconsidered in purely biochemical terms as a problem ofprotein turnover, namely as the result of the balancecontroversial [11]. Attempts at conditional satellite cellablation during the neonatal period using tamoxifeninducible expression of diphtheria toxin A-chain in Pax7expressing cells failed because the animals died withina few days, presumably due to expression of Pax7-drivenCre in other tissues (C-M Fan, personal communication).Satellite cell proliferation in developing skeletal muscle isdependent on innervation, as it is drastically reducedone day after denervation of neonatal (6-day-old) ratmuscles [12].

Nuclear turnover during postnatal muscle growth hasbeen recently reinvestigated in a mouse fast-twitch legmuscle, the extensor digitorum longus [13]. Two stageswere identified (Fig. 1): i) an initial stage, corresponding tothe first three postnatal weeks, characterized by a parallelincrease in number of myonuclei and amount of cytoplasm;and ii) a later stage after P21 with increase in cytoplasmand fiber size but without addition of new myonuclei. Theproportion of satellite cells steadily decreases from P6(about 12% of muscle nuclei) to P21 (about 2%), with nofurther change thereafter. Two important lessons can belearned from this study: first, a muscle fiber can grow intwo ways, either by myonuclear accretion or withoutincrease in number of myonuclei; second, in maturemuscle fibers the myonuclear domain, i.e. the ratio ofcytoplasm to nucleus, is not fixed but rather flexible.Actually, myonuclear domain increases throughout post-natal development.

Satellite cells in muscle hypertrophy

In adult skeletal muscle satellite cells are mitoticallyquiescent and there is no evidence for myonuclear turn-over, except during late stages of life (see below), or duringmuscle regeneration, hypertrophy or atrophy. Does prolif-eration/fusion of satellite cells, such as seen in developingmuscle, take place also during hypertrophy of adult skeletalmuscle? Functional overload-induced by tenotomy orelimination of synergistic muscles leads to rapid activationof satellite cells, which undergo proliferation and fusewith the associated myofibers [4,14a,14b]. Incorporation of3H-thymidine in satellite cell nuclei was detected in the ratsoleus muscle few days after tenotomy of the synergisticgastrocnemius muscle, before any evidence of myofibernuclei, up to 30% in neonatal rat and mouse muscles. Theclassical study of Moss and Leblond, based on 3H-thymi-dine incorporation and autoradiography, showed thatsatellite cells undergo extensive proliferation during thefirst weeks after birth in rat muscles and that most ofthese cells are subsequently incorporated into the growingmyofibers [9]. Myonuclear accretion by satellite cellproliferation/fusion is likely required for myofiber growth,in order to maintain a constant myonuclear domain, i.e.the ratio of cytoplasm to nucleus within the multinucle-ated muscle fibers (but see below). However, a formalproof that satellite cell proliferation/fusion is necessaryfor myofiber growth is lacking. It is known that Pax7 is anessential factor for maintaining the satellite cell pool inneonatal muscle, whereas Pax7 is no longer required forsatellite cell survival and muscle regeneration at later

hypertrophy (Fig. 2). This leads to a marked increase in thenumber of satellite cells in hypertrophying muscles (Table1). Accordingly, in a similar experimental model an

Figure 1 Postnatal growth of the mouse extensor digitorum longus muscle. A, myofiber cross-sectional area (CSA); B, number ofmyonuclei per myofiber. Note that the CSA increases throughout the postnatal period considered (up to 56 days after birth),whereas the number of myonuclei increases exponentially from P3 to P21, but shows a negligible increase after P21. Modifiedfrom [13].

S14 G. Pallafacchina et al.increase in myonuclei was detected during the first weekpost-surgery and preceded myofiber hypertrophy [15].Interestingly, when the hypertrophied muscle was subse-quently denervated, the increase in the number of myo-nuclei was maintained even after three months [15].Satellite cell proliferation is also induced by exercise, bothin animal models [16] and in humans [17a]. In humans, thesatellite cell pool can increase as early as 4 days aftera single bout of exercise and is maintained at higher levelsfollowing several weeks of training, while cessation oftraining leads to a gradual reduction of the satellite cellpool [17a]. Exercise in humans, like electrical stimulation inanimal models [17b], can induce muscle damage, which isespecially evident following eccentric exercise, such asdownhill running, especially in untrained individuals.Indeed, most studies showing satellite cell activation haveused a maximal eccentric exercise protocol, althougha significant satellite cell response can be observed also inthe absence of gross muscle damage and without inflam-matory cell infiltration [18].Figure 2 DNA synthesis in a satellite cell. Electron micro-scope autoradiography showing 3H-thymidine incorporation ina satellite cell of the rat soleus muscle 4 days after tenotomyof the synergistic gastrocnemius muscle. A single injection oflabeled thymidine was given 6 h before muscle removal. Thesatellite cell nucleus is overlaid with silver grains, indicatingthat it has taken up labeled thymidine during the premitoticDNA synthesis. From [4].Is satellite cell proliferation/fusion required for musclehypertrophy? This question was addressed some years ago ina Point/Counterpoint debate, however no definitiveconclusion could be reached (see [19,20] and relatedcomments). Gamma radiation experiments have shown thatoverload-dependent muscle hypertrophy [21] and activity-dependent satellite cell activation [16] are blocked by X-ray or gamma ray radiation, suggesting an obligatory role ofsatellite cells in muscle hypertrophy. However, interpreta-tion of this experiment is complicated by possible effects ofgamma radiation on myofiber protein synthesis [22]. Indirectevidence for a role of satellite cells in human skeletal musclewas provided by studies comparing individuals showingvariable degree of myofiber hypertrophy after several weeksof resistance training: individuals developing markedhypertrophy (responders) had a greater proportion of satel-lite cells at baseline and greater satellite cell-mediatedmyonuclear addition after exercise compared to individualswho did not exhibit any change in fiber size (non-responders)[23]. However, different genetic animal models support thenotion that satellite cell activation is not required for musclehypertrophy, as discussed in the following section.

Genetic models of muscle hypertrophy

In one genetic model, muscle hypertrophy was produced byan inducible Akt transgene [24]. Akt is a kinase thatTable 1 Increase in number of satellite cells in the ratsoleus muscle during early stages of compensatoryhypertrophy.a

Days after surgery % Satellite cells

0 4.92 12.75 17.88 15.1a Changes seen in adult rat soleus after tenotomy of the

gastrocnemius muscle. Seven animals were used, and a total of501 muscle nuclei were counted in electron micrographs from51 transverse sections, each cut from a different block. Satel-lite cell number is expressed as percent of total muscle nuclei(true myonuclei satellite cell nuclei). From [4].

Satellite cells and muscle growth S15mediates the effect of insulin-like growth factor 1 (IGF-1)and promotes protein synthesis, by activating mTOR andS6K, and inhibits protein degradation by repressing thetranscription factor FoxO [25]. Muscle hypertrophy causedby muscle-specific activation of an inducible Akt transgeneis not accompanied by satellite cell proliferation, asassessed by BrdU incorporation [26]. Over-expression of thetranscription factor JunB likewise causes muscle fiberhypertrophy without satellite cell proliferation [27]. Myo-statin, a transforming growth factor-b (TGF-b) familymember acting via activin receptors and Smad transcriptionfactors, is a negative regulator of muscle growth, asabsence or blockade of myostatin causes muscle hyper-trophy. The hypertrophic muscle fibers of the myostatinnull mice were reported to contain fewer myonuclei perfiber than their controls and satellite cell-independentmuscle hypertrophy was seen in adult muscle followingmyostatin blockade induced by local injection of vectorscoding for the myostatin propeptide, which binds non-covalently to myostatin and inhibits its activity [28].However, inhibition of myostatin signaling in adult miceusing soluble ActRIIB, an activin type 2 receptor specific formyostatin and a subset of TGF-b family ligands, was foundto induce an increased incorporation of BrdU, indicatingDNA synthesis, in muscle nuclei and increased proportion ofsatellite cells labeled by Pax7 and M-cadherin [29]. Thediscrepancy between these results might be due to the factthat signaling of multiple TGF-b ligands, and not only ofmyostatin, is inhibited using soluble ActRIIB [30]. Otherexperiments suggest that satellite cell activation contrib-utes to the increase in muscle mass induced by local viral-mediated gene transfer of IGF-1, since muscle hypertrophyin this model is partially prevented by gamma radiation todestroy the proliferative capacity of satellite cells [31]. Aspointed out before, radiation experiments are difficult tointerpret, and only a genetic approach to block satellitecell activation in an inducible way in adult muscle, couldprovide direct evidence for an obligatory role of satellitecells in muscle hypertrophy. Indeed, conditional ablation of>90% of satellite cells was recently obtained in matureskeletal muscle using tamoxifen inducible expression ofdiphtheria toxin A-chain in Pax7 expressing cells [32].Overload hypertrophy and increase in force of the plantarismuscle after removal of the synergist gastrocnemius wasunchanged in these mice supporting the notion that satel-lite cells are not necessary for functional muscle hyper-trophy [32]. However, in a physiological context, satellitecells do undergo proliferation and fusion during overloadhypertrophy and it remains possible that they do contributeto functional hypertrophy: adaptive changes in the proteinsynthesis/protein degradation balance within the myofibersmight occur in the absence of satellite cells thus leading toan apparently normal hypertrophy process [11]. Indeed,a complex cross-talk between myofibers and satellite cellstake place during muscle hypertrophy, a shown by thefinding that deletion of serum response factor (SRF)specifically in myofibers and not in satellite cells bluntsoverload-induced hypertrophy and impairs satellite cellproliferation and recruitment to pre-existing fibers [33]. Itwill be important to establish whether functional hyper-trophy can be maintained for long periods without myonu-clear accretion.Signaling pathways involved in satellite cellactivation

Satellite cell activation is controlled by several growthfactors, intracellular signaling pathways and transcriptionfactors (see [34] for a review). In addition to systemic factors,e.g. hormones such as testosterone, local factors releasedby myofibers, fibroblasts or macrophages may act on satel-lite cells. Cytokines, such as interleukin-4 (IL-4) [35] andinterleukin-6 (IL-6) [36], aswell as prostaglandins [18,37] havebeen implicated in satellite cell proliferation. The Notch andWnt signaling pathways appear to have a major role incontrolling the balance between satellite cell proliferationand differentiation [38]. Notch, a membrane receptor acti-vatedby the ligandDelta, is involved in the initial proliferationof activated satellite cells. On the other hand, Wnt signaling(Wnt being the ligand of the membrane receptor Frizzled)decreases the proliferative capacity and promotes thedifferentiation of satellite cells to become fusion-competentmyoblasts. However, in aging muscle an increased Wntsignaling in the myogenic progenitors, possibly resulting fromincreased amounts of Wnt or Wnt-like molecules present inthe serum of aged animals and binding to Frizzled receptors,has been implicated in the conversion of satellite cells toa fibrogenic fate [39]. Other factors, including hepatocytegrowth factor (HGF), the ligand of the c-Met tyrosine kinasereceptor, promote satellite cells proliferation and inhibitdifferentiation [40]. Magic-Factor 1, an HGF-derived engi-neered protein that contains two Met-binding domains andactivates the Akt pathway, was recently shown to promotemyogenic precursor cell survival and differentiation andinduce muscle hypertrophy in vivo [41].

In contrast, as discussed above, satellite cell proliferationis impaired by inhibitory factors, such asmyostatin and otherTGF-b familymembers. Bonemorphogenetic proteins (BMPs)permit satellite cell proliferation but prevent their differ-entiation [42]. IGF-1 is unique, in that it promotes bothproliferation and differentiation of satellite cells. All thesefactors should be viewed as components of a complexsignaling network with multiple interactions between thevarious factors. For example,myostatin inhibits activation ofAkt, but IGF-1 can dominantly block the effects of myostatin[43,44]. Blockade of the Notch pathway likewise relievesmyostatin repression of myoblast proliferation and differ-entiation, and myostatin upregulates Notch downstreamtarget genes [45]. The intersections between the variouspathways and their relative role in satellite cells and inmyofibers have been incompletely characterized. A majoropen issue here is the nature of the interactions betweensatellite cells and associated myofibers, with particularreference to the signals that satellite cells may receive fromthe myofibers in an in vivo setting. IL-6 and IL-4 produced bymyofibers were recently shown to enhance satellite cellproliferation and fusion, respectively [33].

Satellite cells in muscle atrophy and aging

Satellite cells, like muscle interstitial cells, undergo tran-sient proliferation during the early stages after denervationof adult skeletal muscle, possibly as a result of the inflam-matory reaction induced by the degeneration of the distal

stump of the transected axons [4]. However,muscle satellitecells decrease in number at later stages after denervationprobably by apoptosis [46]. Satellite cell apoptosis has beenreported in different pathological conditions, includingunloading, muscular dystrophy, cancer cachexia andischemia, and can occur even in the absence of pathologicalchanges of the associated muscle fibers (Fig. 3). However,systematic quantitative studies on apoptosis of satellite cellsand myonuclei in muscle pathology are missing. It is evendebated whether myonuclear loss occurs in atrophyingmuscle, and recent studies indicate that denervation-induced atrophy is not accompanied by a reduction in thenumber of myonuclei per fiber except in old animals [47,48].Muscle atrophy occurs in a variety of very different condi-tions, including starvation, disuse, weightlessness in spaceflights, cancer cachexia, renal or cardiac failure, but theresponse of satellite cells and myonuclei to these conditionshas been poorly investigated.

A unique form of slowly progressive muscle atrophy,called sarcopenia, occurs during aging. It is controversialwhether satellite cells decrease in number in aging skeletalmuscle [48,49]. On the other hand, most studies indicatethat during aging satellite cells display reduced prolifera-tive response after damage and reduced regenerativecapacity. In aging muscle, satellite cells may also displaya tendency to adopt alternate lineages, showing fibrogenicpotential that could contribute to muscle fibrosis [39]. The

growth regulators, such as myostatin, or promoting or

S16 G. Pallafacchina et al.Figure 3 Apoptotic satellite cell in ischemic rat soleusmuscle. Serial, non consecutive sections of the same field. Thesatellite cell shows chromatin compaction, nuclear fragmen-tation and condensation of the cytoplasm typical of apoptoticcells. Note also vacuole formation (lower panel) in theapoptotic satellite cell. In contrast, the associated muscle fiberhas a normal ultrastructure. Hanzlikova & Schiaffino, unpub-lished observation (see [52] for the muscle ischemia modelused in this experiment).mimicking the effect of growth factors, such as IGF-1.A recent study supports the notion that addition of

myonuclei is a prerequisite for maintaining specific force inhypertrophic muscle fibers of the mouse fast-twitchextensor digitorum longus [53]. Myonuclear number andforce were analyzed in single fibers from two musclehypertrophy models, myostatin knockout and muscle-specific IGF-1 overexpression. In the IGF-1 overexpressionmodel, muscle fiber hypertrophy is accompanied by newmyonuclear incorporation, thus the myonuclear domainsize remains unchanged, and specific force is maintained.In contrast, a loss of specific force is seen in the fast fibersfrom myostatin knockout mice, in which fiber hypertrophyoccurs without addition of myonuclei, thus leading toexpansion of the myonuclear domains.

Two recent reviews deals with the muscle stem cellniche [54] and the role of satellite cells in muscle growthand in different muscle hypertrophy models [55]. Age-dependent changes in the stem cell niche, involving robustexpression of sprouty1 (Spry1), an inhibitor of fibroblastgrowth factor (FGF) signalling, were shown to influenceage-dependent decline in satellite cell activation is notthe result of an intrinsic deficiency, but is probably due tothe environment, as shown by muscle transplantation andparabiosis studies. Rat muscles, from either young or oldanimals, autografted in young rats regenerated significantlygreater mass and developed greater maximum contractileforce than muscles autografted in old rats, suggesting thatthe poor regeneration of muscles in old animals is a func-tion of the environment provided by the old host, which isnot appropriate to promote efficient muscle regeneration[50]. Parabiosis has been used to generate animals sharinga common blood circulation to test the presence of circu-lating factors that promote or impair satellite cell activa-tion. Heterochronic parabiosis experiments showed thatthe proliferation and differentiation capacity of agedsatellite cells is restored in old mice surgically joined toyoung partners [51]. Conversely, satellite cells in youngmice that had been paired with old mice showed a declinein functionality. Thus, satellite cell function appears to bepositively influenced by the young systemic environmentand negatively affected by the old systemic environment.

Conclusions

Muscle wasting is a serious complication of a variety ofdisorders, ranging from aging sarcopenia to cancer cachexia,renal failure, cardiac failure, and neuromuscular diseases.The development of interventions aimed at preventing theloss of muscle tissue requires a full understanding of themechanisms that control muscle growth and the mainte-nance of muscle mass. These mechanisms have been tradi-tionally investigated by biochemical techniques aimed atevaluating the relative role of anabolic and catabolic phasesof protein turnover. However, muscle mass regulation mayalso be controlled by cell turnover involving satellite cellsand changes in the number of myonuclei. Ongoing researchaims at exploring whether muscle growth can be promotedand muscle atrophy can be prevented by boosting satellitecell activation either by blocking the effect of negative

[13] White RB, Bierinx AS, Gnocchi VF, Zammit PS. Dynamics of

Satellite cells and muscle growth S17stem cell quiescence and function [56]. Aging has beenassociated with diminished muscle re-growth and satellitecell proliferation in the early recovery phase after immo-bility-induced atrophy in human skeletal muscle [57].Another study shows that satellite cells appear to play littleor no role in myostatin/activin A signaling in vivo, since i)myostatin/activin A inhibition can cause muscle hyper-trophy in mice lacking either syndecan4 or Pax7, both ofwhich are essential for satellite cell function, and ii) musclehypertrophy after pharmacological blockade of thispathway occurs without significant satellite cell prolifera-tion and fusion to myofibers and without an increase in thenumber of myonuclei per myofiber [58].

Conflict of interest

The authors have no conflict of interest to report.

Acknowledgments

This work was supported by grants from the EuropeanCommission (FP7 Integrated Project MYOAGE to S.S.) andthe Italian Space Agency (ASI, project OSMA to S.S.). Wethank Chen-Ming Fan and Peter Zammit for critical readingof the manuscript and Chen-Ming Fan for communicatingresults prior to publication. We apologize to all thoseauthors whose work could not be cited in this short over-view due to space restrictions.

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Role of satellite cells in muscle growth and maintenance of muscle massIntroductionMuscle satellite cellsSatellite cells and postnatal muscle growthSatellite cells in muscle hypertrophyGenetic models of muscle hypertrophySignaling pathways involved in satellite cell activationSatellite cells in muscle atrophy and agingConclusionsConflict of interestAcknowledgmentsReferences