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REVIEWS–A PEER REVIEWED FORUM

Mechanisms of Muscle Growth and Atrophy inMammals and DrosophilaRosanna Piccirillo,1,2 Fabio Demontis,3,4* Norbert Perrimon,3,5 and Alfred L. Goldberg1

Background: The loss of skeletal muscle mass (atrophy) that accompanies disuse and systemic diseases ishighly debilitating. Although the pathogenesis of this condition has been primarily studied in mammals,Drosophila is emerging as an attractive system to investigate some of the mechanisms involved in musclegrowth and atrophy. Results: In this review, we highlight the outstanding unsolved questions that maybenefit from a combination of studies in both flies and mammals. In particular, we discuss how differentenvironmental stimuli and signaling pathways influence muscle mass and strength and how a variety ofdisease states can cause muscle wasting. Conclusions: Studies in Drosophila and mammals should helpidentify molecular targets for the treatment of muscle wasting in humans. Developmental Dynamics00:000–000, 2013. VC 2013 Wiley Periodicals, Inc.

Key words: skeletal muscle growth; muscle atrophy; animal models of muscle wasting; proteostasis

Key Findings:� Modulation of muscle mass by environmental stimuli and transcription factors during disease states� Role of protein synthesis and degradation pathways in muscle atrophy� Mammals and Drosophila are useful model organisms to identify the molecular and cellular mechanisms

responsible for muscle growth and atrophy in humans

Submitted 11 April 2013; First Decision 1 August 2013; Accepted 1 August 2013

INTRODUCTION

Skeletal muscle accounts for approxi-mately 40–50% of the body mass inhumans. Maintenance of muscle massand strength through proper nutri-tion and exercise is critical to main-tain full activity, prevent obesity, anddecrease the risk of heart disease, dia-betes, and cancer (Pate et al., 1995).Lack of contractile activity and a vari-

ety of systemic diseases, as well asinadequate nutrition, all lead to fiberatrophy (i.e., loss of cell proteins), andconsequently, a decrease in functionalcapacity. In addition, there is a pro-gressive loss of muscle mass andstrength in the aged, often termed“sarcopenia” (Nair, 2005; Demontis etal., 2013a), which is a major contribu-tor to the frailty in the elderly andincreases the risk of age-related dis-

eases, injury, and death (Demontis etal., 2013b). General wasting of allmuscles occurs with fasting and dis-use and is an integral feature of anumber of systemic diseases, includ-ing many cancers, cardiac failure,renal failure, sepsis, AIDS, as well asburns and traumatic injury, and mus-cle loss correlates with a poor progno-sis. In these conditions, the skeletalmuscles are inherently normal, and

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1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts2Department of Oncology, IRCCS, Mario Negri Institute for Pharmacological Research, Milano, Italy3Department of Genetics, Harvard Medical School, Boston, Massachusetts4Department of Developmental Neurobiology, Division of Developmental Biology, St. Jude Children’s Research Hospital, Memphis,Tennessee5Howard Hughes Medical Institute, Harvard Medical School, Boston, MassachusettsGrant sponsor: Muscular Dystrophy Association; Grant sponsor: NIH; Grant numbers: AR055255; R01AR057352; Grant sponsor: ItalianAssociation for Cancer Research AIRC-Start Up; Grant number: 11423; Grant sponsor: St. Jude Children’s Hospital/ALSAC; Grant spon-sor: The Ellison Medical Foundation - New Scholar in Aging Award; Grant sponsor: HHMI.*Correspondence to: Dr. Fabio Demontis, St. Jude Children’s Research Hospital, Memphis, TN 38105. E-mail:Fabio.Demontis@stjude.org

DOI: 10.1002/dvdy.24036Published online in Wiley Online Library (wileyonlinelibrary.com).

DEVELOPMENTAL DYNAMICS 00:000–000, 2013

VC 2013 Wiley Periodicals, Inc.

the loss of mass is a response to physi-ological or pathological stimuli (e.g.,fasting or cancer), rather than to anyintrinsic genetic defect. By contrast,there are a variety of primary musclediseases (termed myopathies) thatresult from inherent structural orenzymatic defects (e.g., muscular dys-trophies) or from inflammatory dis-eases (e.g., myosites) (Askanas andEngel, 2002). Many mutations thatperturb muscle function by affectingthe contractile apparatus, energymetabolism, or membrane integritycan lead to skeletal and cardiacmyopathies.

Extensive studies in vertebrateshave shown that skeletal muscle is ahighly adaptive tissue that responds toexercise, nutrient supply, and endocrine

factors with alterations in fiber compo-sition and size (Fig. 1). Similar observa-tions have been made in Drosophilawhere muscles account for 40 to 50% ofthe body mass, and muscle growthdepends on nutrient supply sensed viathe Insulin/Akt/TOR pathway (as inmammals). For example, under normalnutrient conditions, Drosophila musclesundergo a striking �50-fold increase infiber size in only 5 days during larvaldevelopment (Demontis and Perrimon,2009; Fig. 2A).

Similarities between vertebrateand Drosophila muscles are bothstructural and functional. Both arecomposed of tandem arrays of sarco-meres containing the thin and thickfilaments, which, in a typical muscletwitch, slide past each other in

response to calcium release from thesarcoplasmic reticulum (SR, the speci-alized endoplasmic reticulum [ER] ofmuscles), resulting in force genera-tion (Taylor, 2006). In addition, inboth insects and mammals, musclefibers can be either glycolytic or oxi-dative. For example, Drosophiladirect and indirect flight muscles,which promote wing motion indirectlyby compressing the thorax, can func-tion for extended periods during flightand are primarily oxidative. By con-trast, body wall muscles of the larvaand leg muscles of adult flies, whichare used only intermittently, relymainly on glycolysis (Taylor, 2006).These distinct patterns of energymetabolism resemble the differencesbetween type I and type IIb fibers in

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Fig. 1. Environmental stimuli and signaling pathways increasing proteolysis and depressing protein synthesis and leading to muscle atrophy.Representative fields of a transverse section of muscle fibers from fed mice or weight-matched mice deprived of food for 48 hr are shown. Nucleiand membranes are indicated in blue (Hoechst) and green, respectively. Scale bar ¼ 50 mm.

2 PICCIRILLO ET AL.

mammalian muscles. Type I slowfibers are non-fatiguing, primarilyburn fatty acids and glucose oxida-tively, and are dark in color becausethey are rich in mitochondria, myo-globin, and blood supply. By contrast,the easily fatigued, fast type IIb fibersare primarily glycolytic, and have alow mitochondrial content and capil-larity density (Taylor, 2006). Mostmammalian muscles, especially inhumans, are composed of mixtures offiber types that are recruited in an

ordered fashion, but overall fiber com-position is adapted to the specificfunctions of the muscle. For example,in rodents the antigravity soleus mus-cle, which is continually used instanding, is composed primarily ofoxidative fibers and is quite resistantto fatigue. In typical mixed muscles,the slower oxidative fibers are used inall contractions, but the easilyfatigued larger glycolytic fibers arerecruited only with maximal efforts(Brooke and Kaiser, 1970).

Here in this review, we discuss howDrosophila with its extensive genetictoolkit and short life cycle provides apowerful experimental system toaddress some of the outstandingunsolved questions about muscle atro-phy. Specifically, we review the mecha-nisms of skeletal muscle atrophy andhypertrophy that may be similar inDrosophila and mammals, and discussemerging insights and outstandingquestions that may benefit from stud-ies in both species (see Tables 1 and 2).

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Fig. 2. Developmental growth of skeletal muscles in Drosophila larvae is inhibited by FOXO overexpression. A: Skeletal muscle size dramaticallyincreases by 50-fold in the larval stage of development, which lasts 5 days. Muscle growth results from enhanced protein synthesis without anyaddition of muscle nuclei. Larvae expressing a GFP-tagged Myosin Heavy Chain (Mhc) protein in body wall muscles are shown. B: The sizeincrease of body wall muscles is inhibited by overexpressing the transcription factor FOXO in muscles (C: Dmef2-Gal4 UAS-foxo vs. Dmef2-Gal4in B). Ventral Longitudinal muscles 3 and 4 (VL3 and VL4) from animals at the end of larval development are outlined for comparison in B and C.The dramatic increase in muscle mass observed during Drosophila larval development provides a sensitive setup for the identification of evolutio-narily conserved genes regulating muscle mass. B0–B00, C0–C00: Micrographs outline sarcomeres and nuclei within muscle fibers (F-actin, red;Nuclei, blue). Scale bar ¼ 40 mm in B,C and 10 mm in B0,B00 and C0,C00. See Demontis and Perrimon (2009) for more information.

MECHANISMS OF MUSCLE GROWTH AND ATROPHY 3

MODULATION OF MUSCLE

MASS BY ENVIRONMENTAL

STIMULI AND

TRANSCRIPTION FACTORS

In mammals, muscles are the majorprotein reservoir in the body, and dur-ing fasting, amino acids generated bynet protein degradation are releasedinto the venous blood to provide sub-strates for hepatic gluconeogenesis.Thus, under starvation conditions,muscle proteolysis is critical for main-taining the supply of glucose, in par-ticular to the brain, and of aminoacids essential for continued proteinsynthesis. Myofibrillar proteins com-prise about two-thirds of muscle dryweight, and changes in muscle sizeare due primarily to changes in thecontent of the contractile apparatus(Cohen et al., 2009; Solomon andGoldberg, 1996). In most types ofmuscle atrophy, the loss of mass isdriven by an increase in protein deg-radation and to a lesser extent by adecrease in protein synthesis (Fig. 1).Protein degradation occurs via boththe ubiquitin proteasome system(UPS) and the autophagy/lysosomepathway, while protein synthesis isregulated primarily by Insulin/IGF-1(Insulin-like Growth Factor 1) actingthrough the Akt/TOR/FoxO pathway(Glass, 2010; Fig. 3). During atrophy,both UPS and autophagy/lysosomedegradative systems are activated,and key components are induced bytranscription factors of the FoxO fam-

ily (Sandri et al., 2004; Zhao et al.,2007) and NF-kB (Cai et al., 2004;Hunter et al., 2002).

In mammals, systemic muscle wast-ing is induced in response to fastingor diseases (i.e., untreated diabetes,AIDS, cancer, and heart failure) andby various circulating hormones, cyto-kines, and excess of glucocorticoids(Fig. 1). In addition, atrophy of spe-cific muscles occurs upon decreasedusage (e.g., with nerve injury orimmobilization in a cast). These verydifferent physiological stimuli mayinvolve some common signaling mech-anisms. Contractile activity causesrelease of IGF-1 (Insulin-like GrowthFactor 1), which functions as animportant autocrine growth factor.IGF-1 is also a circulating hormonereleased from the liver in response topituitary growth hormone. The auto-crine production of IGF-1 by musclefalls with disuse and following loss ofinnervation (Zeman et al., 2009), inanimals lacking pituitary growth hor-mone or during chronic heart failure(Schulze and Spate, 2005). So the lossof IGF-1, through both endocrine andautocrine mechanisms, contributes tothe atrophy process mainly by attenu-ating signaling by the IGF-1/Aktpathway in both systemic catabolicstates and locally with disuse.Reduced Akt signaling decreases pro-tein synthesis in muscle as in othercells and leads to enhanced muscleproteolysis via activation of FoxOtranscription factors (Glass, 2010;

Fig. 3). Similarly, upon fasting whencirculating insulin and IGF-1 levelsfall, autocrine production of IGF-1 bymuscle also decreases. As a result, theIGF-1/Akt pathway is markedlydepressed leading to rapid proteinloss. Furthermore, in fasting andother catabolic states, the release ofglucocorticoids by the adrenal glandsincreases and further promotes prote-olysis and inhibits muscle proteinsynthesis.

In addition to FoxOs, other tran-scription factors (i.e., Smad 2 and 3,NF-kB, JunB, Runx1, and Myogenin)have been shown to influence musclemass. For example, the Smad tran-scription factors mediate the cataboliceffects of myostatin, a circulatingTGF-family member that inhibits nor-mal muscle growth (Lee, 2004). Myo-statin binds to the Activin RIIB(ActRIIB) receptors and activatestranscription by Smad 2 and 3. Con-versely, inhibition of ActRIIB recep-tors or of Smad 2 and 3 causes musclehypertrophy (Sartori et al., 2009; Fig.4). NF-kB has also been shown to bean important factor in muscle atrophy(Bonetto et al., 2011; Cai et al., 2004),although its exact mechanismappears unclear (Mourkioti et al.,2006). Endurance exercise inducesthe production of the transcriptionalco-activator PGC-1a, which promotesmitochondrial gene expression lead-ing to oxidative phosphorylation andsubsequently differentiation of oxida-tive muscle fibers (Handschin and

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TABLE 1. A List of Prominent Yet Unsolved Questions for Future Research on Muscle Atrophy in Drosophila and

Mammals

In Drosophila� Which are the mechanisms of developmental muscle growth, atrophy, and segmental apoptosis in Drosophila?� What determines the distinct sensitivity to histolysis of different insect muscles during pupal metamorphosis?� Are Drosophila muscle precursor cells similarly regulated as mammalian satellite cells?� Is there turnover of the contractile apparatus in normal muscles?

In mammals� What is the basis for the differential response of different mammalian fiber types to catabolic stimuli?� How are components of the myofibril assembled and degraded both normally and at increased rates during atrophy,while preserving contractile function?� In atrophying muscles, how are the increases in proteolysis by the ubiquitin proteasome pathway and by autophagy andthe decreases in protein synthesis coordinated?� Do syncytial apoptosis and necroptosis contribute to muscle wasting?� What is the precise contribution, if any, of satellite stem cells to muscle atrophy and hypertrophy?

In both� How do various signaling pathways and transcription factors (NF-kB, JunB, etc.) interact with FoxO transcription fac-tors to cause (or combat) muscle atrophy in mammals and Drosophila?

4 PICCIRILLO ET AL.

Spiegelman, 2008). In addition, JunB,which has long been known as arapid-response gene that triggers cellproliferation, is also important indetermining the size and growth rate

of muscle fibers in adult mammals,which are postmitotic cells. JunBoverproduction can both promotehypertrophy and inhibit muscle atro-phy (Raffaello et al., 2010; Sandri

et al., 2006) by inhibiting FoxO3, andperhaps other catabolic processes.Interestingly, the transcription factorRunx1 is induced by denervation andlimits denervation-induced musclewasting by regulating the expressionof muscle structural proteins and ionchannels (Wang et al., 2005).

A number of other transcriptionfactors (e.g., Myogenin, MyoD andMEF2) are important in embryonicdifferentiation of muscle, but are notcritical in post-natal muscle growth.However, in the adult Myogenin par-ticipates in the induction of atrophyfollowing denervation (Moresi et al.,2010). While multiple transcriptionfactors can promote or inhibit muscleatrophy, their specific roles, modes ofactivation, and functional interac-tions occurring during different typesof muscle atrophy remain unclear.This area represents an importantgap in our knowledge and possiblycould lead to the development ofrational interventions to combat mus-cle wasting.

The powerful genetic toolkit available inDrosophila may be of particular advantageto analyze the genetic interactions amongtranscription factors inducing muscle atro-phy or hypertrophy and to identify novelregulators of muscle mass. In Drosophila,

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Fig. 3. Signaling pathways increasing proteolysis, depressing protein synthesis, and leading tomuscle atrophy. A comparison of the molecular players in Drosophila (indicated in italics) andmammals is shown.

TABLE 2. Similarities and Differences in Muscle Atrophy and Hypertrophy in Insects and Mammals.

Similarities Major differences

� Fiber composition differs in muscles with distinctphysiological roles (continually active oxidativevs. glycolytic muscles)

� Fiber atrophy and hypertrophy occur in adult mammalsbut muscle growth appears to occur only during developmentin Drosophila

� Distinct muscles differ in sensitivity toatrophic/histolytic stimuli (i.e. steroids)

� Muscle stem cells have not beenobserved in flies

� FoxOs cause muscle atrophy in mammals andinhibit growth of larval muscles in flies

� Drosophila lacks a major ubiquitin ligase implicatedin myofibrillar turnover (MuRF1)

� The UPS plays a role in muscle atrophy in mammals aswell as in muscle mass loss during insect metamorphosis

Advantages for Studying Muscle Atrophy and Hypertrophy

In Drosophila In mammals

� Flies have a shorter life cycle than mice � Rodents loose muscle mass similar to humans duringdifferent kinds of atrophy

� Flies are easily amenable to various geneticmanipulations (e.g. genome-wide RNAi screens)

� Muscles are accessible for physiological andbiochemical studies

� Large groups of flies can be analyzed for anygiven intervention

� Endocrine signals regulating atrophy have beencharacterized in mammals

� The 50-fold increase in muscle mass observed duringlarval development is valuable for identifying genesinvolved in myofibrillar assembly and muscle growth

� Energy metabolism and physiological regulation ofmuscle mass are well defined

MECHANISMS OF MUSCLE GROWTH AND ATROPHY 5

muscles undergo dramatic size increaseduring larval development, where fibergrowth also relies heavily on nutrient sens-ing via the Insulin/Akt/TOR pathway(Demontis and Perrimon, 2009). In thelarval muscles, the activities of the tran-scription factors FOXO, Myc and Mnt areintegrated to achieve normal musclegrowth. In particular, FOXO inhibits mus-cle growth at least in part by decreasingMyc (diminutive) gene expression and itstranscriptional activity (Demontis and Per-rimon, 2009). Conversely, overexpression ofthe Insulin receptor results in increasedMyc gene expression, which promotesnucleolar biogenesis and primes musclegrowth. In addition, RNAi-mediated knock-down of Myc inhibits muscle growth, asdoes overexpression of Mnt (Demontis andPerrimon, 2009), which antagonizes Mycactivity by binding to the common interac-tion partner Max (Bellosta and Gallant,2010). Finally, genome-wide RNAi analy-ses, as recently applied to the study of mus-cle morphogenesis (Schnorrer et al., 2010),may uncover new regulators of fiber size inDrosophila. Orthologs of these genes mayalso be important for muscle atrophy andhypertrophy in human and thus representnew therapeutic targets.

DENERVATION

Like nutrient deprivation, loss of mus-cle stimulation by nerves, as occursduring inactivity, immobilization,paralysis, nerve injury, and neuromus-

cular diseases, such as amyotrophic lat-eral sclerosis (ALS), cause profoundmuscle atrophy in mammals (Fig. 1).Different muscle fiber types have dis-tinct propensities to atrophy upondenervation, with type I oxidativefibers being more sensitive todenervation-induced atrophy than typeII (Herbison et al., 1979), even thoughtype II fibers are lost preferentially invarious systemic wasting states, e.g.,glucocorticoids treatment (Goldbergand Goodman, 1969), fasting (Li andGoldberg, 1976), sepsis (Tiao et al.,1997) or cancer (Acharyya et al., 2004;Baracos et al., 1995). Importantly, acti-vation of the Insulin/Akt pathway issufficient to counteract the loss of mus-cle mass associated with denervation(Bodine et al., 2001b). Acutely, theweight loss and transcriptionalresponse to denervation and pure dis-use (e.g., with spinal isolation) are verysimilar, but with time, denervatedmuscles show a more profound atrophy(Sacheck et al., 2007). So although thepost-natal maintenance of muscle massis clearly dependent on continual neu-ronal activity, which causes contractileactivity, it remains possible that trophicfactors released by innervating motorneurons may also be important indetermining muscle properties. Inter-estingly, some muscles, such as theextraocular muscles of primates, areextremely resistant to denervationatrophy when compared to limb

muscles (Porter et al., 1989). Differen-ces in fiber-type composition and inner-vation pattern may explain theresistance of extraocular muscles towasting following denervation.

Whether denervation influencesmuscle mass in the adult fly, andwhether it can be counteracted byInsulin/Akt signaling has not yet beenstudied, probably because of the diffi-culties in altering physiological loadand measuring the sizes of specificmuscles in this organism. However,muscle-nerve interactions have beenthoroughly examined during thedevelopmental phase of metamorpho-sis in Drosophila, when these interac-tions are important to define propermuscle size and patterning. In partic-ular, surgical severing of the meso-thoracic nerve with microbeam lasersleads to denervation that can retardthe formation of some muscle fibers,like the dorso-longitudinal flightmuscles, by regulating the rate of pro-liferation of the pool of myoblasts, themuscle stem cells that are precursorsof the adult musculature (Fernandesand Keshishian, 1998). Similarly,nerve–muscle interactions are neces-sary for achieving proper muscle sizein the moth Manduca sexta, wheredenervation during development alsodecreases the number of proliferatingmyoblasts (Bayline et al., 2001). Inaddition, denervation results in mus-cle patterning defects in Drosophila,with a failure to form dorsal ventralmuscles, indicating that the differen-tiation of specific muscle fibers is per-turbed (Fernandes and Keshishian,1998). The interconnection betweenneuronal activity and muscle pattern-ing has also been extensively studiedin adult mammals, where motoneur-ons influence fiber type differentiation(Grinnell, 1995) even in the adult.Moreover, prolonged electrical stimu-lation of type II fibers causes the mus-cle to acquire many characteristics oftype I fibers (Goldspink, 1985).Despite these observations, the tran-scriptional mechanisms underlyingmuscle–nerve interactions remain tobe clarified. Knowledge gained fromstudies in Drosophila should helpdefine the nature of muscle–nerveinteractions, which are presumablyrelated to the mechanisms function-ing in mammalian adult differenti-ated muscles.

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Fig. 4. Myostatin signaling pathway as a basis to counteract muscle wasting. Molecules toinhibit this pathway are currently in human clinical trials to cure the muscle wasting associatedwith Duchenne Muscular Dystrophy.

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GLUCOCORTICOIDS,

INFLAMMATION, AND

OXIDATIVE STRESS

Among the major stimuli that cantrigger muscle atrophy in mammalsare the glucocorticoids (e.g., cortisol)or its synthetic analogs (e.g., dexa-methasone), which at high pharmaco-logical doses cause atrophypreferentially of type IIb glycolyticfibers (Dahlmann et al., 1986; Gold-berg and Goodman, 1969). These ste-roids are released from the adrenalgland in stressful states and areessential for muscle wasting duringfasting, renal failure, diabetes, andsepsis (Menconi et al., 2007; Schak-man et al., 2008). Glucocorticoids actto reduce muscle size by inhibitingamino acid transport into muscle, pro-tein synthesis, and enhancing proteol-ysis. Consequently, they promote therelease from muscles of amino acidsthat can be burned directly or serveas substrates in the liver for glucoseproduction. Although not normallycatabolic, normal or slightly increasedlevels of glucocorticoids can inducemuscle wasting when there is a fall inInsulin/IGF-1 signaling, as occursduring fasting, diabetes, and insulin-resistant states (most diseases; Huet al., 2009). Because of their abilityto also reduce inflammation, prolifer-ation of white cells, and immuneresponses, glucocorticoids are widelyused to treat rheumatoid arthritis,allergic reactions, asthma, transplantrejection, lupus, and some forms ofcancer. The induction of muscle (andbone) wasting is a serious adverseeffect of high doses of these drugs andoften limits their use in the clinic.Interestingly, lack of contractile activ-ity enhances the tendency of musclesto atrophy in response to glucocorti-coids (Goldberg and Goodman, 1969).For example, although the dark oxi-dative soleus is relatively resistant tothese agents, the denervated soleus ishighly susceptible to cortisone-induced atrophy, which helps explainthe marked loss of body mass in thebed-ridden patients. Thus, an out-standing question is how the sensitiv-ity of different muscles toglucocorticoids or other catabolicstimuli is modulated at the molecularlevel by contractile work or other ana-bolic stimuli.

Although no hormones are knownthat function similarly to glucocorti-coids upon stress in insects, a class ofsteroid hormones called ecdysteroids(including ecdysone) has been impli-cated in the complete breakdown anddeath of muscle cells (also termed his-tolysis) during pupal metamorphosis.Throughout this process, most larvalmuscles degenerate completely inresponse to ecdysone and presumablyprovide amino acids for the develop-ment of the adult tissues. Althoughglucocorticoids do not cause death ofmuscles in mammals, the rapid loss ofmuscle mass and breakdown of myofi-brils in response to structurallyrelated molecules (glucocorticoids andecdysteroids) suggests some similarmechanisms of action. Accordingly,larval muscles undergo marked atro-phy before apoptosis is evident inresponse to ecdysone (Bayline et al.,1998; Hegstrom and Truman, 1996).However, not all insect musclesundergo histolysis in response toecdysteroids. An example of suchdiverse responses to ecdysteroidscomes from pioneering studies in themoth Manduca sexta. During pupalmetamorphosis, the larval musclesare either maintained, modified, ordegenerate (Bayline et al., 1998; Heg-strom and Truman, 1996). In particu-lar, the large intersegmental muscles(ISMs) from Manduca initially atro-phy and lose approximately 40% oftheir mass starting 3 days beforeadult eclosion, while at the sametime, the flight muscles of the adultmusculature are actively growing inmass. Subsequently, ISMs undergocell death and are completely lostwithin 30 hr of adult life, whilenewly-formed flight muscles do not(Schwartz, 1992).

Studies in Manduca have benefitedfrom the large sizes of these muscles,but the genetic analysis of this pro-cess is easier in Drosophila, wheredifferent muscles also respond in dis-tinct fashion to ecdysone. By 8 hrafter puparium formation, mostmuscles in the head and thoracic seg-ments undergo histolysis while theabdominal muscles are preserved(Fernandes et al., 1991). Among thethoracic muscles, the larval obliquemuscles escape histolysis and serve astemplate for the formation of the dor-sal longitudinal muscles (DLMs), a

group of indirect flight muscles(IFMs) of the adult (Farrell et al.,1996; Roy and VijayRaghavan, 1998;Dutta et al., 2004). Subsequently,most larval muscles in the abdomenundergo histolysis. For example, thedorsal external oblique muscles(DEOMs) degenerate in the late pre-pupal stage (Wasser et al., 2007).However, some other abdominalmuscles initially persist, undergoatrophy (day 1 and 2 of pupal stage),and subsequently increase in mass(hypertrophy; late pupal stage) toform the temporary dorsal internaloblique muscles (DIOMs) of the adult.DIOMs are needed for eclosion anddegenerate only after the adult hasemerged (Kimura and Truman, 1990).Interestingly, the nuclear proteinsEAST and Chromator have antagonis-tic functions and, respectively, accel-erate and delay the atrophy of DIOMsduring pupal metamorphosis. In addi-tion, Chromator partially blocks thebreakdown of DEOMs muscles(Wasser et al., 2007).

The mechanisms by which distinctinsect muscles respond differently toecdysone are still not understood, andthus this may represent a valuablemodel to better understand the speci-alized responses of distinct muscles tocatabolic stimuli. Algorithms recentlybecame available to facilitate theanalysis of images from high-throughput RNAi screens for musclehistolysis (Chinta et al., 2012). Inmammals, there are several sexuallydimorphic muscles (e.g., the levatoranus that is also involved in the con-trol of the penis) that show muchgreater sensitivity to the anabolicactions of testosterone than typicalmuscles (Herbst and Bhasin, 2004).The precise basis for their greatersensitivity to these steroids is largelyunknown.

In addition to glucocorticoids, pro-inflammatory cytokines includingTNF-a (Tumor Necrosis Factor a), IL-1 (Interleukin 1), IL-6, activin, andmyostatin have all been reported tocontribute to muscle wasting(cachexia) in mammals during sepsisand in cancer-bearing animals atleast in part by activating the NF-kB,STAT3, and/or the Smad transcriptionfactors (Bonetto et al., 2011). Some ofthese cytokines and the pathwaysthey activate are conserved in

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MECHANISMS OF MUSCLE GROWTH AND ATROPHY 7

Drosophila. For example, Drosophilaeiger (CG12919) is a TNF superfamilyligand that activates a canonical TNFsignaling cascade (Moreno et al.,2002). In addition, the JAK/STATpathway, which is activated in mam-mals by various members of the Inter-leukin family, is conserved inDrosophila where it is activated bythe Unpaired family of cytokine-likeligands (outstretched, unpaired 2, andunpaired 3). Studies on the effects ofthese signaling pathways on Drosoph-ila muscle growth and remodelingmay provide useful models ofinflammation-induced atrophy.

Another type of circulating factorthat can increase muscle size or coun-teract atrophy are cathecolaminesand their synthetic analogs, clenbu-terol or b2 agonists, which activate b2adrenergic receptors (whose Drosoph-ila homolog is octopamine receptor 2).These agents cause production ofcyclic-AMP and activation of PKAthat can ultimately cause cardiac andskeletal muscle hypertrophy (Nave-gantes et al., 2001). In fact, clenbu-terol has often been used illegally toinduce growth of cattle and enhancefood production but, because residuesof cathecolamines are retained in themeat, this practice is dangerous andis banned. Also, because of the sideeffects of cathecolamines (e.g., bloodpressure and cardiac function), theirability to inhibit atrophy has not beenexploited as a therapy for musclewasting.

Increased production of reactiveoxygen species (ROS) has been pro-posed to play a role in the muscledegeneration of the queen fire ant,Solenopsis spp., where histolysis fol-lows the mating flight and insemina-tion (Davis et al., 1993). Similarly, inmammals there are multiple observa-tions suggesting a possible role of oxi-dative stress in triggering muscleatrophy. First, the transcription factorATF4, which promotes the expressionof oxidative stress responsive genes,is upregulated in most types of atro-phy (Lecker et al., 2004) as are themetallothioneins, heavy metal-binding components that can serveantioxidant roles (Sacheck et al.,2007). In addition, during disuse,ROS have been proposed to activateseveral transcription factors involvedin muscle atrophy in mammals,

including NF-kB and FoxO (Doddet al., 2010), but other modes of acti-vation have also been demonstrated.Genetic manipulations that enhancemuscle ROS levels, such as knock-outof the cytoplasmic anti-oxidantenzyme Sod1 (superoxide dismutase1) in mice leads to oxidative damageto proteins and muscle wasting (Jangand Van Remmen, 2011; Muller et al.,2006). On the other hand, mice devoidof the mitochondrial superoxide dis-mutase 2 in muscle have elevated oxi-dative stress, but do not exhibitmuscle wasting (Kuwahara et al.,2010; Lustgarten et al., 2009). Thus,oxidative stress does not appear to besufficient to induce atrophy, and itremains unclear if these signs of oxi-dative stress are a key factor in theatrophy process or an incidentalfactor.

PHYSICAL ACTIVITY AND

INACTIVITY

Muscle wasting in adult mammals isgenerally viewed as a reversible pro-cess, but this reversibility has notbeen rigorously studied, e.g., at differ-ent ages. The loss of muscle on fastingis rapidly reversed by refeeding andcan even lead to “overshoot” growth.Similarly, the loss of body mass withacute illness is rapidly reversed uponrecovery, especially in children whereit has been termed “catch-up” growth.In the elderly, this capacity to reversewasting seems to be more limited butthis impression and its possible cellu-lar basis has not been systematicallystudied. The extent of muscle wastinginduced by food deprivation in mam-mals can be counteracted by physicalactivity, especially isometric exercise,which stimulates the IGF-1/Akt path-way and builds muscle mass andstrength. In fact, in starving ratswhere there is rapid muscle wasting,generally increased activity of the sol-eus (e.g., induced by loss of a syner-gist) can induce hypertrophy(Goldberg, 1968). Both strength andendurance exercise increase the activ-ity of the transcriptional co-activatorPGC-1a (Gibala et al., 2009; Teradaet al., 2002), which in turn stimulatesmitochondrial biogenesis and oxida-tive metabolism characteristic of typeI fibers but not hypertrophy (Hand-schin and Spiegelman, 2008). Type I

aerobic fibers are relatively resistantto atrophy induced by various circu-lating catabolic factors, includingfasting, glucocorticoids, cancer, andsepsis, apparently due to the presenceof PGC-1a. Its expression is decreasedin many, perhaps all, types of atrophy(Sacheck et al., 2007; Sandri et al.,2006), and PGC-1a and its homologPGC-1b, directly antagonize theinduction of the atrophy gene pro-gram by both FoxO3 (Sandri et al.,2006) and NF-kB, and thus suppressthe acceleration of protein degrada-tion (Brault et al., 2010; Fig. 3). Pre-sumably, these effects help accountfor the ability of exercise to retardatrophy. Following disuse and uponrestoration of activity, the increasedPGC-1a levels and IGF-1/PI3K-Aktsignaling inactivate FoxO leading todecreased expression of atrogenes.These genes are induced or sup-pressed coordinately during varioustypes of atrophy due to uremia, diabe-tes, cancer, denervation, disuse, andfasting and include components ofboth the UPS and autophagy/lyso-some pathways (Zhao et al., 2007).

Among these atrogenes, twomuscle-specific ubiquitin ligases,MuRF1 and atrogin-1, are dramati-cally induced by FoxO3 (Sandri et al.,2004) and are essential for muscleatrophy (Bodine et al., 2001a; Gomeset al., 2001). In addition to a role forPGC-1a in repressing FoxO activity,PGC-1a4 (a PGC-1a splice variantpreferentially produced after resist-ance exercise) protects muscles fromatrophy by inducing IGF-1 whilerepressing myostatin expression(Ruas et al., 2012).

The mechanisms linking contractileactivity to the action of these tran-scription factors in atrophy is still notclear even though this area is criticaland determines whether a cell growsor atrophies. One well-studied excep-tion is NFAT, a key transcription fac-tor that is activated by increases inCa2þ levels. In skeletal muscle, it isinvolved in fiber type specificationand influences PGC-1a expression(McCullagh et al., 2004) and in car-diac muscle it can trigger hypertro-phy (van Rooij et al., 2002). Anadditional potential link betweenphysical activity and protein turnoveris the dihydropyridine receptor(DHPR) a1S subunit, which can serve

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as a molecular sensor of muscle activ-ity (Pi�etri-Rouxel et al., 2010). DHPRa1S knock-down induces muscle atro-phy via FoxO3A activation, whichleads to upregulation of autophagy-related genes and the induction ofautophagy. However, the loss ofDHPR presumably represents a formof disuse since it should reduce con-tractile activity (i.e., disruptexcitation-contraction coupling).

Thus far, studies on the effects ofmuscular contractions have beenlacking in insects. Although geneticstudies in Drosophila in principlemight help elucidate the interconnec-tions between exercise and musclemass, experimental approaches forexercising this organism and methodsfor measuring changes in musclemass or metabolic adaptations havenot been described, in contrast to theextensive literature on mammals.Recently, a mechanized platform forthe physical training of flies has beendescribed (Piazza et al., 2009), inwhich a repeated tapping of the con-tainer where the flies are housed acti-vates the innate instinct of the flies toclimb (negative geotaxis). This prom-ising system may be useful to dissectthe genetic basis underlying the effectof physical exercise on muscles. Forexample, an interaction betweenphysical exercise and spargel, thePGC-1a homolog that promotes mito-chondrial activity in Drosophila (Tie-fenbock et al., 2010), has been foundwith this system, highlighting thatspargel is required for full exercisecapacity in Drosophila and the induc-tion of physiological effects derivingfrom exercise (Tinkerhess et al.,2012).

While endurance exercise clearlydecreases the loss of muscle mass inmice and in humans, no morphologi-cal signs of muscle deterioration havebeen observed in flightless Drosophilamutants or in response to transientlocal paralysis of adult flies inducedwith the temperature-sensitive dyna-min mutant shibire (which aredepleted of synaptic vesicles; Deak,1976). Such studies did not identifydecreases in muscle mass of the kindsseen with disuse in small mammalswhere fiber diameters decrease by10–40% within 1–2 weeks withoutany clear structural deterioration.This apparent lack of clear responses

to disuse is important to define morerigorously since Drosophila musclesmay differ radically from mammalsand lack the capability to alter musclemass in response to changes in con-tractile activity. While such an appa-rent difference may be due toinsufficient studies, it is also possiblethat because of their brief lifespanand very different lifestyle, Drosoph-ila may not have evolved similar reg-ulatory mechanisms as mammals formobilizing amino acids from muscleproteins. So disuse may not result inobvious muscle atrophy in flies.

ROLE OF PROTEIN

SYNTHESIS AND

DEGRADATION PATHWAYS

IN MUSCLE ATROPHY

Despite the greater diversity of possi-ble regulatory factors in mammals(ranging from hormones to cytokines),the final cellular mechanisms regulat-ing muscle size appear similar in Dro-sophila and mammals (Figs. 1–3). InDrosophila, the Insulin/Akt-respon-sive transcription factor FOXO medi-ates most of the gene expressionchanges induced by nutrient starva-tion in larval muscles (Teleman et al.,2008) and its activation is sufficient tostunt developmental muscle growth(Demontis and Perrimon, 2009; Fig.2B and C). Similarly, in mammals,various types of muscle atrophy sharea common transcriptional programmainly driven by FoxO3 (Leckeret al., 2004; Sandri et al., 2004) andpossibly other FoxO family members,like FoxO1. MuRF1 and atrogin-1 arekey atrogenes induced by FoxO3(Sandri et al., 2004) that are neces-sary for rapid muscle atrophy (Bodineet al., 2001a; Gomes et al., 2001).While proteins comprising the thickfilaments of the myofibrils have beenclearly identified as MuRF1 sub-strates (Cohen et al., 2009), howatrogin-1 causes muscle loss is lessclear, but probably involves the degra-dation of growth-related proteinsincluding the transcription factorMyoD, and the translation initiationfactor eIF3-f, which in turn reducesprotein synthesis (Lagirand-Canta-loube et al., 2008; Fig. 3). AdditionalE3s are also important in muscle sizecontrol. These include TRAF6, respon-

sible for activating many atrophy-related signaling cascades (Paul et al.,2010); TRIM32, involved in the degra-dation of thin filaments and the des-min cytoskeleton (Cohen et al., 2012;Kudryashova et al., 2005); and Cbl-b,which inhibits IGF-1 signaling by pro-moting the degradation of the InsulinReceptor Substrate IRS-1 (Nakaoet al., 2009). Homologs of atrogin-1(Drosophila CG11658), TRIM32 (Dro-sophila abba), TRAF6 (DrosophilaCG10961), and Cbl-b (Drosophila Cbl)are encoded in the Drosophila genome,suggesting possible similarities in thepathways governing proteolysis andmuscle atrophy in fruit flies and mam-mals. For example, CG11658 expres-sion levels increase in muscle wasted(mute) mutants, lacking a componentof the histone locus body and charac-terized by a severe loss of muscle massand integrity during development(Bulchand et al., 2010). Moreover, Dro-sophila TRIM32 (abba) is required forintegrity of the costamere, a structurethat connects the sarcomeres to theoverlying sarcolemma providing stabil-ity during muscle contraction, and itsmutation results in unbundling ofmyofibrils and progressive musclewasting (LaBeau-DiMenna et al.,2012).

However, no homologs of MuRF1, aE3 ligase that ubiquitinates myofibril-lar proteins, are found in flies. Veryrecently, the p97/VCP ATPase com-plex, which forms distinct complexesinvolving different adaptors (e.g., p47and Ufd-1) and distinct components ofthe thick (i.e., Myosin Light chains)and thin (i.e., actin) filaments, hasbeen shown to have a major role inmultiple types of atrophy, where itseems to catalyze the extraction ofubiquitinated proteins from the myo-fibrils prior to proteasomal degrada-tion (Piccirillo and Goldberg, 2012).Thus, Drosophila homologs of p97(Drosophila TER94) and Ufd-1 (Dro-sophila CG6233) could participate ina conserved mechanism for muscleprotein degradation, although p97also functions in other disassociationand degradative processes (e.g.,destruction of misfolded proteins inthe ER). Interestingly, human muta-tions in p97 cause an inclusion bodymyopathy and all amino acid residuesaltered in the disease are perfectlyconserved in Drosophila TER94

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(Ritson et al., 2010). It will clearly beof interest to define the biochemicalmechanisms for disassembly and deg-radation of the sarcomeric apparatusduring pupal metamorphosis and ifthey involve the p97 complex.

Studies in Manduca have high-lighted the involvement of the UPS inecdysone-induced muscle histolysis. Inparticular, levels of ubiquitin conju-gates dramatically increase at eclosion,during which loss of muscle proteins ismaximal. Histolysis is also character-ized by the coordinated induction ofubiquitin-activating enzymes (E1), sev-eral ubiquitin-conjugating enzymes(E2s), ubiquitin ligases (E3s), and ubiq-uitin itself (Haas et al., 1995; Schwartzet al., 1990) together with heightenedexpression in the abdominal interseg-mental muscles (ISMs) of MS73/Rpt3and S10b/Rpt4, which encode ATPasesubunits of the 26S proteasome (Daw-son et al., 1995; Hastings et al., 1999;Jones et al., 1995; Low et al., 1997).Thus, activation of the UPS seems toplay a fundamental role in the loss ofskeletal muscle mass during insectmetamorphosis similar to that in atro-phy in mammals. These insect musclesshrink in size markedly (atrophy)before the onset of apoptosis, presum-ably due to increased proteolysis. Itshould be noted that the UPS is alsorequired to maintain muscle mass andintegrity during larval development(Haas et al., 2007) and for viability inmammals, due to its many critical rolesin cellular quality control and regulat-ing metabolism. In other words, differ-ent degrees of protein breakdown havebeneficial or detrimental effects onmuscle mass, depending on what pro-teins are digested.

In addition to the UPS, autophagycontributes to the histolysis of severaltissues during metamorphosis in Dro-sophila (Baehrecke, 2003). Althougha recent study indicated that autoph-agy is not involved in muscle histoly-sis in response to ecdysone duringmetamorphosis (Zirin et al., 2013),other catabolic stimuli may activateautophagy to induce muscle atrophyin other contexts in insects.

Many atrogenes induced by FoxO3encode for proteins of the autophagy/lysosome system (e.g., Cathepsin L,LC3, GabarapL1; Lecker et al., 2004;Zhao et al., 2007), which contributes tomuscle atrophy in mammals especially

through the disposal of mitochondria(Romanello et al., 2010), as well assoluble cell proteins. Importantly,Mul1 is an E3 ubiquitin ligase recentlyimplicated in FoxO induced-mitophagy(Lokireddy et al., 2012), the autophagicdestruction of mitochondria, while theubiquitin ligase Parkin is needed formitophagy of damaged mitochondria(Yang and Yang, 2011). Both Mul1 andParkin are conserved in Drosophila(CG1134 and parkin, respectively) andmay play a role in muscle wasting inthis organism.

The loss of mitochondria must con-tribute importantly to the decreasedendurance during atrophy. Neverthe-less, the changes in mitochondrialnumbers and functional capacity dur-ing different types of atrophy havesurprisingly not been studied exten-sively. Because ubiquitination can alsotarget larger structures to autophagicvacuoles, several ubiquitin-bindingproteins (including p97, p62, NBR1,NDP52, and parkin) exist in cells thatbind insoluble or organellar ubiquiti-nated proteins and facilitate theirdocking to the autophagic vacuole viaLC3. Their functions in muscle meritfurther study to better understand thecoordination between different proteindegradation pathways during atrophyand their roles in clearing differentcellular constituents (reviewed in Kor-olchuk et al., 2010), especially sincecertain components (p97 cofactors) areessential in proteolysis by both degra-dative systems.

In addition to the activation of pro-tein degradation pathways, musclewasting upon fasting, glucocorticoidtreatment, and other disease statesresults from an inhibition of proteinsynthesis that is triggered bydecreased IGF-1/Akt/TOR signalingand inhibited by glucocorticoids (Huet al., 2009). Interestingly, ecdyste-roids inhibit Insulin signaling in Dro-sophila (Colombani et al., 2005),suggesting that muscle histolysis dur-ing metamorphosis may also rely oninhibition of Akt signaling and theactivation of FOXO. However, mutantflies lacking the only Drosophila foxogene are viable and have only minordevelopmental defects (Junger et al.,2003), suggesting that FOXO is notnecessary for overall muscle histolysisduring metamorphosis. By contrast,mammalian muscle contains three

closely related FoxO genes and theirrespective roles are still uncertain.FoxO1, 3, and 4 are coordinately acti-vated upon nutrient deprivation, butmay have distinct roles in specific cat-abolic states and may regulate dis-tinct genes (Moylan et al., 2008),although FoxO1 and 3 both induceatrogin-1, MuRF1, and Mul1. FoxO3aand FoxO4 knock-out mice (Hosakaet al., 2004) are viable and analysis ofFoxOs-deficient mice (Paik et al.,2007) may clarify whether specificFoxO transcription factors are neces-sary for the induction of atrophy inresponse to different stimuli. Thusfar, it is only known that overexpres-sion of FoxO-dominant negativemutants, which block all FoxOs inadult muscles, can prevent multipletypes of atrophy (Sandri et al., 2004).It would be interesting to testwhether FOXO is able to alter theexpression of the Drosophila homologof atrogin-1 as in mammals.

SYNCYTIAL APOPTOSIS

Additional mechanisms responsiblefor modifying muscle mass duringdevelopment and adulthood includethe poorly defined pathway of syncy-tial apoptosis. In this process, loss ofindividual nuclei within a fiber ismore commonly observed than thedeath of the entire fiber, possibly viacaspase-independent mechanisms act-ing via endonuclease-G (Primeauet al., 2002; Sandri and Carraro,1999) or nucleophagy, the selectiveautophagic degradation of nuclearcomponents (Park et al., 2009). Inaddition, segmental necrosis, thedeath of a portion of a fiber withoutthe overall loss of the fiber, is a char-acteristic feature of many myopathies,especially Duchenne muscular dystro-phy (Wallace and McNally, 2009).

The genetic regulation of syncytialapoptosis, and whether it differs fromsegmental necrosis, is unclear andmay benefit from genetic studies toelucidate the underlying regulatorypathways. In insects, syncytial apopto-sis occurs during metamorphosis,when some nuclei within the samefiber degenerate and are lost, whilethose in proximity to the site of inner-vation are spared (Bayline et al., 1998;Hegstrom and Truman, 1996).

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Therefore, in insects the local induc-tion of syncytial apoptosis may varyeven within a single fiber dependingon the proximity to the site ofinnervation.

While apoptosis occurs upon musclehistolysis during insect metamorpho-sis, fiber cell death has not beenobserved upon inhibition of larval mus-cle growth in Drosophila (Demontisand Perrimon, 2009) and during rapidatrophy in mammals (Bruusgaard andGundersen, 2008). In animal models ofuremia, fasting, diabetes, and cancer,the pro-apoptotic gene Bnip3 isinduced but it also functions in mitoph-agy (Mammucari et al., 2007; Leckeret al., 2004). However, segmental apo-ptosis of fibers has been reported dur-ing aging in mammals and Drosophila(Marzetti et al., 2012; Zheng et al.2005). Future studies in Drosophilaand mammals should elucidate thepathways governing muscle syncytialapoptosis, and how this poorly under-stood process impacts on the loss ofmuscle mass and strength.

Importantly, other pathways of pro-grammed cell death including necrop-tosis, which depends on the activity ofthe serine/threonine kinase RIP1,have not been examined in muscles.Notably, TNF-a, which helps triggermuscle wasting in inflammatorystates (Glass, 2010), although per-haps indirectly, is able to induce nec-roptosis in other cell types (Galluzziand Kroemer, 2008). The possiblerelevance of necroptosis duringmuscle wasting awaits furtherinvestigation.

ROLE OF MUSCLE STEM

CELLS

In vertebrates, satellite cells are thesmall mononucleated muscle stemcells localized between the sarco-lemma and the basal lamina of mus-cle fibers. In the adult, most stemcells are in a quiescent state, but canbecome dramatically activated uponinjury or specific stimuli like IGF-1,which promotes their proliferationand differentiation during hypertro-phy (Biressi and Rando, 2010). Forexample, after damage, satellite cellsfuse to one another or to an undam-aged fiber, promoting muscle regener-ation, through a process that partially

recapitulates myoblast fusion duringdevelopment. Interestingly, apoptosisof satellite cells but not pre-existingfibers has been observed during rapidatrophy (Bruusgaard and Gundersen,2008). Unfortunately, since satellitecells are heterogeneous, few in num-ber (less than 2% of the nuclear con-tent of muscle), and much lesssusceptible to in vivo electroporationthan muscle fibers, their specific rolein atrophy is still largely unknown.Although satellite cells are not neededfor muscle re-growth following disuse-induced atrophy (Jackson et al.,2012), further studies are necessaryto test whether satellite cells can pos-sibly reverse or prevent muscle atro-phy in other contexts, as has beenrecently suggested (Thornell, 2011).

Important roles in hypertrophyhave often been postulated, especiallyin providing additional nuclei to pre-vent a fall in concentration of nucleiwhen fibers enlarge. However, itremains controversial whether stemcell proliferation is essential duringhypertrophy as it clearly is uponregeneration (Zammit et al., 2002).New techniques have recently becomeavailable to dissect their possible con-tributions to muscle adaptations.These include the ex vivo transfectionof muscle stem cells followed by intra-muscular injection and the in vivoadministration of adenoviral vectorswhere transgene expression is drivenunder muscle stem cell–specific pro-moters of the Pax3 and Pax7 genes(Biressi and Rando, 2010).

Currently, there is no evidence ofsatellite cells in any Drosophila adultmuscles, and it is not clear whetherthey do not exist or simply have notyet been identified. If present theymay be related to the twist-expressingadult muscle precursors (AMPs),muscle progenitor cells that escapehistolysis and form all adult musclesin Drosophila (Figeac et al., 2007).Interestingly, even if the Drosophilahomologs of the mammalian satellitecells markers Pax3 and Pax7, pairedand gooseberry, respectively, are notexpressed in AMPs, these muscle-committed stem-like cells, like quies-cent satellite cells, have high levels ofNotch activation (Figeac et al., 2011).This finding suggests that studies ontwist-expressing muscle progenitorsin Drosophila may provide useful

information on the regulation of satel-lite cell function in mammals.

EFFECTS OF MUSCLE MASS

ON BODY METABOLISM IN

DROSOPHILA AND

MAMMALS

It is now clear that the skeletal mus-cle has major effects on the metabo-lism of the organism, its growth,aging, and resistance to disease(Demontis and Perrimon, 2010;Demontis et al., 2013bb). Theseorganismal effects involve muscle-derived signaling factors but alsoarise from indirect effects of musclemass and its high metabolic demand.Skeletal muscle consumes a majorfraction of nutrients, and with intenseexercise, its energetic demandincreases dramatically. Consequently,multiple physiological mechanismsexist to provide glucose or fatty acidsto muscle. For example, when musclegrowth was induced in adult mice byincreasing the expression of the Akt1kinase in type IIb fibers, the trans-genic mice displayed resistance toboth diet-induced obesity and hepaticsteatosis, at least in part via a stimu-lation of fatty acid oxidation in theliver (Izumiya et al., 2008). In addi-tion, postnatal loss of the transcrip-tion factor myogenin in musclesresults in smaller body size of mice,suggesting that Myogenin acts toinfluence the post-natal growth ofmuscles and also other tissues via anunknown mechanism (Knapp et al.,2006). As in mammals, in developingDrosophila larvae the extent of mus-cle growth influences whole-organismgrowth and metabolism. In particular,muscle-specific inhibition of the Insu-lin receptor/Akt/TOR pathway and ofMyc activity decreases not only mus-cle growth, but also feeding behaviorand the growth of non-muscle tissues,while muscle-specific activation of theInsulin receptor has the oppositeeffects (Demontis and Perrimon,2009).

A remarkable and very promisingintervention to increase muscle massand reduce atrophy in mammals isthrough inhibition of the TGF-b fam-ily members, like Myostatin and Acti-vin, all of which signal via theActRIIB complex (Fig. 4). Activin is

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MECHANISMS OF MUSCLE GROWTH AND ATROPHY 11

produced normally in multiple celltypes and also in cancerous cells andplays an important role in reproduc-tion (Xia and Schneyer, 2009), whileMyostatin is expressed predomi-nantly in skeletal muscles (Zhouet al., 2010; Zimmers et al., 2002).Myostatin normally acts to limit pre-natal and postnatal muscle growthand mutations in the Myostatin geneor its receptor result in increasedmuscle mass in mice, cattle, dogs, andhumans (Lee, 2004). Developmentalinhibition of Myostatin results in bothmuscle hyperplasia and hypertrophy,while postnatal inhibition increasesmuscle mass via hypertrophy, with noaccompanying hyperplasia in severalspecies (McPherron 2010). Suppres-sion of the Myostatin-based pathway(as in Fig. 4) has been also proveneffective in counteracting musclewasting in mice with chronic kidneydisease (Zhou et al., 2010), followingtreatment with glucocorticoids (Gil-son et al., 2007), as well as in cancercachexia (Benny Klimek et al., 2010;Zhou et al., 2010). This effect on mus-cle size resulted at least in part fromthe inhibition of the FOXO-inducedactivation of atrogenes and ubiquitin-dependent proteolysis and perhapsalso through the subsequent stimula-tion of muscle stem cell proliferation.Remarkably, inhibition of Activin andMyostatin signaling via blockade ofthe ActRIIB receptors not only dra-matically reverses cancer-inducedweight loss in certain cancer models,but also improves the survival of micewithout slowing tumor growth (Zhouet al., 2010). Thus, improving musclemass aside from enhancing the qual-ity of life can strikingly influencecancer-related mortality, at least inmice.

PERSPECTIVES

In this review, we have highlighted anumber of important similarities anddifferences between developmentaland post-natal mechanisms regulat-ing muscle size in Drosophila andmammals. The emerging evidencethat multiple signaling pathwaysinfluence muscle size and are intri-cately interconnected should haveimportant implications in under-standing human diseases and applica-tions in combating muscle wasting.

Future studies in both Drosophilaand mammals should further dissectthe pathways governing musclegrowth and atrophy in health and dis-ease to discover novel drugs and ther-apeutic interventions.

ACKNOWLEDGMENTSWe are grateful to the Muscular Dys-trophy Association and the NIH(AR055255) (to A.L.G.), the ItalianAssociation for Cancer ResearchAIRC-Start Up (11423) (to R.P.), St.Jude Children’s Research Hospital/ALSAC and the Ellison Medical Foun-dation (to F.D.), and the NIH(R01AR057352) and HHMI (to N.P.).

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