Journal of Cell Science ucino ysai scnevdi utpeseisincluding species multiple in This conserved 2005). through Wagner, is 2000; part myostatin al., 2003; in of et al., Thomas function least et 1997; at (McCroskery al., et cells growth, McPherron progenitor muscle muscle of in disinhibition results growth inhibition myostatin muscle pharmacological or of of loss Genetic inhibitor 1997). endogenous al., et an Myostatin (McPherron 1997). as al., recognized et first (McPherron was muscle TGF- skeletal in the exclusively of member in fibrosis pre-existing reverse muscle. to skeletal shown previously been has agent pharmacological that no is there for However, goal dystrophies. therapeutic muscular important an considered currently is fibrosis of 1 Li Bo Zhao fibrosis apoptosis muscle through reverses myostatin of Inhibition Article Research hog h ees fvrosctkns(lxkse l,2007; al., process et (Alexakis Mun self-perpetuating and cytokines Serrano 1992; and various al., the et Saitoh of progressive remodel release dysregulated. a the and is through to proliferate progress leading to upon repair ECM, continue chronic in apoptosis injury fibroblasts However, 2007). the Activated undergo al., disorders, et subsequently (Hinz muscle injury and reduced the and proliferate of In to resolution in activated 2011). ECM, are resulting Wagner, fibroblasts produce and injury, myocytes muscle (Moyer other acute regeneration and and contractility myofiber capillaries the of of isolation deposition from causes ECM fibroblasts. by of by accumulation replaced produced Excessive components gradually (ECM) are matrix these extracellular degeneration and In of regeneration dystrophies. rounds and successive muscular undergo the myofibers of disorders disorders, including degenerative chronic muscle, of hallmark skeletal major a is Fibrosis Introduction histological, reversed by pharmacologically determined be as can fibrosis fibrosis muscle muscle words: vitro. Key pre-existing skeletal of in that number of apoptosis the demonstrate reversal apoptosis. to increases fibroblast results the significantly fibroblasts of myostatin These dystrophy, to muscle induction of muscular criteria. through leads of of Inhibition radiographical resistance This model signaling. and a reduces apoptosis. MAPK biochemical mice, (ActRIIB.Fc) undergoing and mdx dystrophic receptor growth, Smad senescent fibroblasts of muscle IIB in through muscle proliferation ActRIIB.Fc of activin apoptosis the of modulator soluble regulates to administration endogenous also a fibroblasts Systemic known myostatin of with that fibroblasts, a resistance pathways demonstrate by myostatin, thus increases signaling we replaced and that Here, and self-perpetuating are shown proliferate. progressive, fibroblasts, myofibers previously to is contractile muscle have injury fibroblasts which We repetitive muscle in to irreversible. normal muscle dystrophies stimulates considered of muscular been response the has maladaptive of This far, feature matrix. defining extracellular and a adipocytes is fibrosis muscle Skeletal Summary 10.1242/jcs.090365 doi: 3957–3965 125, ß Science Cell of Journal 2012 April 23 Accepted ( correspondence for *Author 2 etrfrGntcMsl iodr,Hg .MsrRsac nttt tKneyKigrIsiue 0 ot rawy n eatet of Departments and Broadway, North 707 Institute, Krieger Kennedy at Institute Research Moser W. Hugo Disorders, Neurology, Muscle Genetic for Center 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. n euao fmsl irssi ysai,ahgl conserved highly a myostatin, is fibrosis muscle of regulator One 3 uce uclrdsrpy irss eeeain Myostatin Regeneration, Fibrosis, dystrophy, Muscular Muscle, ersineand Neuroscience 1,2 inyn Zhang Jiangyang , [email protected] b uefml hti xrse almost expressed is that superfamily 4 ailg,TeJhsHpisSho fMdcn,Blioe D225 USA 21205, MD Baltimore, Medicine, of School Hopkins Johns The Radiology, ˜oz-Ca 4 ´ oe,21) Treatment 2010). noves, n ahy .Wagner R. Kathryn and ) rmti td rvd h is xeietleiec htmuscle that pharmacological evidence experimental first results the a The provide apoptosis. study this undergo apoptosis. from with muscle to fibroblasts reverse to inducing mice partially via fibrosis can fibroblasts mdx (ActRIIB.mFc) muscle inhibitor senescent myostatin of of myostatin resistance Treatment that show the we study, present 2008; increases the al., In et 2002). Qiao al., signaling 2008; et al., myostatin Wagner et normal Nakatani with 2002; al., strength mice et and mdx (Bogdanovich than mass fibrosis muscle less increased and have reduction with in signaling mice al., myostatin mdx fibrosis that et in shown (Stedman progressive have muscle studies Previous diaphragm exhibits 1991). in normally especially muscle model skeletal less mouse 1987). al., Duchenne the et This of (Hoffman dystrophy, Dystrophy model Muscular muscular phenotypic Becker allelic a fatal severe, and genetic and a (DMD), are dystrophy common and muscular a dystrophin for of gene have the model mice Mdx in 2002). mutation al., et nonsense Wagner a 2008; al., Parsons et 2006; Qiao al., 2006; et al., (Bartoli Ohsawa et 2002; dystrophy al., et muscular Bogdanovich 2007; of al., disease models et mouse of several amelioration in in features results to mechanisms al., pharmacological et (Li fibroblasts collagen as stimulates myocyte such proteins inhibits it 2008). ECM myostatin express differentiation, and While proliferate 2008). and al., receptor proliferation et IIB fibroblasts activin muscle (Li the receptor, Skeletal (ActRIIB) putative 2008). its directly and al., also myostatin et myostatin express that (Li shown fibroblasts have we regulates Recently, was 2004). (Schuelke myostatin family al., for German et a gene in the muscularity in increased with mutation associated site splice a where humans, 1,2,3, euto fmottnsgaigb ait fgntcand genetic of variety a by signaling myostatin of Reduction * 3957 Journal of Cell Science ru.( group. fe el eetetdwt cRI.F (100 ActRIIB.mFc mice mdx with of treated muscle were limb cells from isolated after fibroblasts primary in control loading ( ad eaiet hto nrae ibmsl fibroblasts. muscle limb untreated of immunoreactive that D1 cyclin to of relative densities bands Mean hours. 24 for (MSTN) myostatin mice. niio cRI.F o 4hours, 24 myostatin. for ng/ml ActRIIB.mFc 0–300 inhibitor with treatment after mice n mdx of diaphragms from irbat rmdahamo d n d/ysai-ul(mdx/ mdx/myostatin-null and mdx of diaphragm from fibroblasts 4hus h itga hw endniiso muoeciebands. immunoreactive of densities mean shows histogram The hours. 24 n iprg uce fmxmc nrae ( untreated mice mdx of muscles diaphragm of a production ECM fibroblasts. and muscles proliferation dystrophic regulates Myostatin 1. Fig. muscle mdx diaphragm compared myostatin and with limb treatment after both showed increased marker, significantly from cycle cell fibroblasts a D1, that cyclin of Immunoblot expression hours. for 24 analysis mice for mdx myostatin recombinant adult with of treated muscle and diaphragm and Fibroblasts limb fibroblasts). from dystrophic muscle isolated dystrophic from were as and derived to fibroblasts (referred proliferation muscle muscle first induces of we study, production also current ECM myostatin the In whether 2008). produce al., investigated and et proliferate (Li to components tissue ECM muscle normal stimulates myostatin from that fibroblasts demonstrated have studies previous Our of fibroblasts production muscle ECM dystrophic and proliferation regulates Myostatin muscle Results reversing myopathies. progressive in chronic, helpful other and be stimulate DMD in to also fibrosis therapy may novel regeneration, and proposed animals muscle a adult inhibitors, in myostatin reversed that pharmacologically be can fibrosis 3958 D 5 5 tbln salaigcnrl fpiayfbolssfo iband limb from fibroblasts primary of control, loading a as -tubulin, muoltaayi fteepeso fcci 1and D1 cyclin of expression the of analysis Immunoblot ) tp r30n/lmottnpu nraigcnetain fmyostatin of concentrations increasing plus myostatin ng/ml 300 or (top) 6 iefrec ru.* group. each for mice 3 n 5 B e aapit ( point. data per 6 oprsno [ of Comparison ) ora fCl cec 2 (17) 125 Science Cell of Journal C 3 P )[ ]rln noprto ewe primary between incorporation H]proline , 3 ]rln noprto npiayfibroblasts primary in incorporation H]proline .5 ** 0.05, ( A muoltaayi fcci 1and D1 cyclin of analysis Immunoblot ) n 5 P 0(otm.I5 s10 is IC50 (bottom). 10 , .1 ro asrpeets.d. represent bars Error 0.01. 2 rtetd()wt 0 ng/ml 300 with (+) treated or ) m )o B scnrlfor control as PBS or M) n 5 b o each for 5 atna a as -actin 2 9.4 Mstn M. 2 / 2 ) iha IC an with ihPScnrl(i.1) irbat eeioae from isolated lacking genetically were mice mdx ( Fibroblasts and mice myostatin 1A). mdx (Fig. adult of control diaphragm PBS with ucefbolssta ntesm ubro ucefibroblasts muscle mdx/ of mdx number of same cultures from the in in greater than fibroblasts was muscle fibroblasts, in synthesis collagen niiino ysai nue ppoi fdystrophic of apoptosis induces myostatin of Inhibition tuoprn SA Fg F.Tkntgte,teeresults these together, and ET Taken CHX, 2F). to (Fig. stimuli, fibroblasts (STA) pro-apoptotic of staurosporine by resistance the activation increased caspase-3 fibroblasts plasmid muscle myostatin- Smad3 transfecting with by blocked Conversely, expression 2E). SB203580 Smad3 (Fig. increasing activation inhibitor inhibitor caspase-3 to MAPK Smad3 resistance with induced p38 al., et signaling or (Li myostatin signaling SIS3 of MAPK p38 Inhibition addition, 2008). in single and, pathway apoptosis: Smad of 2C,D). (Fig. markers DNA associated histone other and DNA two myostatin-inducedstranded alone were results block examining Similar CHX 2B). to (Fig. observed caspase-3 with able of cleavage treated to was resistance ActRIIB.mFc fibroblasts 2B). to (Fig. ( fibroblasts compared muscle decreased myostatin dystrophic significantly cultured of were extracts cleaved plus in of Levels myostatin caspase-3 treated CHX. or with next apoptosis myostatin induced We and with ActRIIB.mFc S1). fibroblasts Fig. muscle material dystrophic supplementary or and CHX 2A, cell of (Fig. pyknosis that 65–80% as ET of nuclear apoptosis such induced filaments, alone with ActRIIB.mFc Treatment actin morphology S1). key Fig. material of (supplementary in as blebs a membrane loss apoptosis, changes caspase-3, with signaling characteristic shrinkage cascade cleaved as of proteolytic 2A), level well the (Fig. of expression of DNA by component of determined stranded as hours 12 treatment single ET following or apoptosis CHX dystrophic underwent cultured all fibroblasts Nearly 2007). muscle and al., (Cossu et death Takata cell 1999; programmed Borello, inhibitor of stimuli II two topoisomerase (ET), the etoposide or (CHX) synthesis cycloheximide protein inhibitor the muscle ActRIIBm.Fc, Dystrophic with cultured production. to were fibroblasts ECM addition in and apoptosis proliferation 2009; to inducing al., resistance et fibroblast similarly myostatin Kulasekaran muscle whether investigated 2011; regulates We al., 2003). al., et et Cunnington Yasuda 2010; (Akasaka al., tissues other et in apoptosis fibroblast against protecting in TGF- fibroblasts muscle xrsino ylnD Fg D.Teerslssgetthat suggest results of production fibroblasts. These ECM muscle and dystrophic 1D). proliferation (Fig. the to D1 contributes myostatin cyclin of ( expression vitro significantly and in proliferation fibroblasts their muscle reduces dystrophic of treatment ActRIIB.mFc d ucefbolssa esrdby measured as We fibroblasts 2010). muscle of al., stimulation mdx myostatin et exogenous by block Sako induced can synthesis 2009; Pharma) vivo Acceleron collagen al., from in gift et (a and ActRIIB.mFc Morrison vitro that found 2005; in al., pathway et myostatin (Lee the of activation inhibit regulate may muscle. fibroblasts dystrophic in by synthesis collagen production myostatin endogenous ysai inln nfbolssicue h canonical the includes fibroblasts in signaling Myostatin h hmrcfso rti,AtIBmc a ensonto shown been has ActRIIB.mFc, protein, fusion chimeric The b aiymmeshv ensont lya motn role important an play to shown been have members family 50 Mstn Mstn fapoiaey1n Fg C.Furthermore, 1C). (Fig. nM 1 approximately of 2 2 / / 2 2 ). nml Fg B.Ti ugssthat suggests This 1B). (Fig. animals 3 -rln noprto,amre of marker a incorporation, H-proline P , .1 fe ramn with treatment after 0.01) 3 -rln incorporation H-proline P , .1 eue the reduces 0.01) Journal of Cell Science iprg uceadt esretn,lm ucewith of muscle replacement limb have extent, lesser to a known to are and muscle mice diaphragm mdx with Senescent induced is mouse mdx ActRIIB.mFc the in apoptosis muscle Fibroblast dystrophic of muscle apoptosis dystrophic induces apoptosis fibroblasts. induces to ActRIIB.mFc resistance only produces while also not but proliferation myostatin fibroblast that suggest sa fcevdcsae3(i.3.TNLasy niae that indicated assays TUNEL 3). (Fig. caspase-3 immunohistochemistry cleaved by of transferase and assay (TUNEL) muscle deoxynucleotidyl labeling between terminal end al., nick space by et dUTP interstitial identified Morrison al., the 2007; were et al., in fibers to et (Akpan cells Lachey determined Apoptotic mice 2010; 2009). previously al., wild-type mg/kg et dose in 10 Cadena 2009; a hypertrophy or weeks, muscle control induce 6 as mice for PBS mdx Two-year-old ActRIIB.mFc administered 1991). al., systemically et were (Stedman fat and fibrosis fttlfbolssprmsl rs-eto.** cross-section. proportion muscle a per as fibroblasts fibroblasts total DAPI. apoptotic of with of stained percentage were show on nuclei Histograms (B) Cellular 3 triceps. caspase of and cleaved cryosections (A) of (TUNEL) expression TdT- assay for by labeling immunostaining identified end were nick (arrows) dUTP fibroblasts mediated Apoptotic weeks. 6 for ihmottnihbtrAtIBmc( ActRIIB.mFc inhibitor myostatin with i.3 niiino ysai nrae ppoi irbat in fibroblasts mice. apoptotic mdx increases of myostatin muscle of Inhibition 3. Fig. ysai niiinrvre irss3959 fibrosis reverses inhibition Myostatin ( N ee sasydb LS epesda A as (expressed ELISA by assayed as level DNA nuto faotsswt . gm H,25 CHX, mg/ml 0.1 with apoptosis of induction transfect fibroblast in expression of ( induction CHX. by with apoptosis followed p-38 (SB80) and SB203580 SIS3 inhibitor inhibitor MAPK Smad3 MSTN, with treated fibroblasts eitnet ppoi nvitro. in fibroblast apoptosis muscle to dystrophic resistance increases Myostatin 2. Fig. odn oto nalimnbos en n ..o triplicate of * s.d. presented and are Means experiments immunoblots. all in control of loading Expression hours. 12 for (STA) staurosporine CN,1m ysai niio cRI.F AtIB,25 (ActRIIB), ActRIIB.mFc inhibitor myostatin mM 1 md (CON), of muscle limb from isolated xrse sasrac t45n.( nm. and 405 level at DNA absorbance hours. stranded as expressed single 12 by for determined (CHX) was cycloheximide Apoptosis mg/ml 0.1 or (ET) etoposide ooeae ftetdfbolss C igesrne N levels DNA A stranded as Single (expressed in (C) expression fibroblasts. caspase-3 treated of cleaved homogenates mg/ml of analysis 0.1 mM Immunoblot with (B) 1 apoptosis CHX. plus of MSTN induction or by (MSTN) followed myostatin ActRIIB.mFc ng/mg 300 with treated E muoltaayi fcevdcsae3epeso in expression caspase-3 cleaved of analysis Immunoblot ) 405 ( A olwn ramns D itn associated Histone (D) treatments. following ) , B w-erodmxmc eetreated were mice mdx Two-year-old ) F muoltaayi fcevdcaspase-3 cleaved of analysis Immunoblot ) P , .5 ** 0.05, dwt md lsi olwdby followed plasmid Smad3 with ed xmiceweretreatedwithPBS ( A n 5 rmr ucefibroblasts muscle Primary ) B )o B ( PBS or 6) – P D , ucefbolsswere fibroblasts Muscle ) 0.01. P , 0.01. b m atnwsue as used was -actin 405–490 METor0.5 n 5 )a control as 5) ). m m M M Journal of Cell Science akro ygnccmimn Fg D n embryonic and 5D) (Fig. commitment myogenic of marker vrg iprg yfbrCAws683.32 was CSA myofiber diaphragm average 510.68 S a 1782.57 myofiber areas triceps average was cross-sectional analysis the which myofiber in Morphometric 5C CSA larger Fig. in 5B). shown to (Fig. as increased (CSA) shift to muscles led a isolated but demonstrated 5A) of (Fig. controls body mass on to effect compared no when had ActRIIB.mFc Treatmentmass with 2005). al., mice et mdx (Wagner 2-year-old months of 18 in as enhanced old are as animals regeneration mdx muscle and mass from muscle myostatin, muscles 4C). multiple (Fig. in controls to reduced compared mice was mdx collagen, treated amino in modified found a hydroxyproline, to acid addition, compared In mice mdx 4B). treated (Fig. from controls muscle collagen in and reduced (HSP47) all 47 were protein III shock heat (SMA), including actin mice proteins muscle associated mdx muscle fibroblast homogenized to that of compared Immunoblots confirmed 4A). weeks replacement, (Fig. mice PBS 6 fatty with mdx fibrosis, for treated in of ActRIIB.mFc reduced muscle area significantly with were of treated the index and area lipid by index the indicated fibrosis that by showed indicated muscle interstitial diaphragm S2). these Fig. suggested material including (supplementary 1 fibroblasts markers collagen were cells fibroblast and ER-TR7 multiple vimentin, with caspase-3 cleaved cleaved ( of was assay ( caspase-3 immunohistochemistry cross-section mice in observed ActRIIB.mFc control muscle were with results PBS treated in mice to mdx cells in compared 4-fold apoptotic over by of increased number the 3960 1229.31 ehv rvosysonta ntegntcasneof absence genetic the in that shown previously have We and muscle limb both of analysis morphometric and Histologic 6 6 136.25 ora fCl cec 2 (17) 125 Science Cell of Journal 446.94 P , .1 Fg B.Clclzto fTNLor TUNEL of Colocalization 3B). (Fig. 0.01) m m m m 2 2 6 ( nutetdaias( animals untreated in P 190 5 .3.Mo xrsin nearly an expression, MyoD 0.03). m m 2 ntetdaiasversus animals treated in P , .0)(i.3) Similar 3A). (Fig. 0.001) 6 P 55.70 5 .2 n the and 0.02) m m a -smooth 2 versus irbat nsnsetmxmc aeteaiiyt epn to respond to regeneration. ability as and the growth well muscle have inducing treatment as mice ActRIIB.mFc mdx precursors senescent myogenic in 5E) that fibroblasts (Fig. ActRIIB.mFc. demonstrate fibers with results treated regenerating mice These of mdx senescent marker in increased a were chain, heavy myosin omntrdsrpi ein nmxmc Wle ta. 05.In 2005). al., et (Walter mice mdx in method lesions noninvasive dystrophic a monitor as to biomarkers and clinical 2012) (Maron, as fibrosis extensively myocardial of used been have MR, enhanced ffboi nhnlm uce ihsgiiatices nmuscle in increase significant T with muscles areas hindlimb in to fibrosis correlated of enhancement gadolinium mice, mdx untreated aeiefboi nec nml T animal. of each degree in the muscles determine fibrosis hind-limb to of ActRIIB.mFc baseline with (MRI) treatment imaging triceps) before resonance (left muscle magnetic forelimb a and of biopsy mdx muscle 2-year-old before, underwent Six mice ActRIIB.mFc. mice with mdx treatment after senescent and individual during within fibrosis changes muscle studied of we fibrosis, established reverse can ActRIIB.mFc whether determine conclusively ActRIIB.mFc be To might fibrosis. with inhibition pre-existing myostatin reversing treated systemic that briefly suggested PBS mice versus of populations senescent between stable, observed fibrosis fairly of area fibroblasts in muscle reduction apoptotic and of number in increase significant The animals ActRIIB.mFc individual with in treated fibrosis pre-existing of Reversal infcnl nrae nmxmc fe rae with treated after mice ( mdx 6A,B) (Fig. in statistically ActRIIB.mFc but increased slightly was hindlimb that significantly of and showed volume injection results muscle MRI of total analysis. weeks the eight for 8 sacrificed for and were ActRIIB.mFc 4 animals after the mg/kg performed 10 was with MRI injected imaging,weeks. MR were baseline mice and biopsy mdx muscle Following 2005). al., et 2 nfboi ein splmnaymtra i.SAB (Walter S3A,B) Fig. material (supplementary regions fibrotic in iprg Dah.Errbr ersn ..* s.d. represent and bars (Gast) Error gastrocnemius (Diaph). (Tric), diaphragm triceps (Quad), quadriceps including ibaddahammsls itgasso vrg este of densities average ( show bands. Histograms immunoreactive muscles. and diaphragm III) and (Coll limb III collagen (HSP47), 47 markers, fibroblast hwma irssidxadlpdidxi rcp Lm)and (Limb) triceps in index ( lipid (Diaph). and diaphragm index fibrosis mean show ise(le rOlrdOfrlpd(e) cl as 75 bars: Scale (red). connective lipid for for Oil-red-O trichrome or Masson (blue) H&E, tissue with stained diaphragm and w-erodmxmc eetetdwt cRI.F ( ActRIIB.mFc with controls. ( treated mdx were treated mice PBS inhibitor mdx than Two-year-old myostatin fibrosis with less treated have mice (ActRIIB.mFc) mdx Senescent 4. Fig. n 5 )fr6wes ( weeks. 6 for 5) a sot uceatn( actin muscle -smooth A B itlgclaayi fties(ibmuscle) (limb triceps of analysis Histological ) muoltaayi fteepeso of expression the of analysis Immunoblot ) C yrxpoiecneto utpemuscles multiple of content Hydroxyproline ) P , .5.Fboi ein were regions Fibrotic 0.05). b 2 atna odn oto in control loading a as -actin wihe n contrast- and -weighted a SA,ha hc protein shock heat -SMA), P , .5 ** 0.05, m .Histograms m. n P 5 , )o PBS or 6) 0.01. Journal of Cell Science eeeaigclsprcosscinties cl a:150 bar: * Scale s.d. triceps. represent cross-section of per number cells chain mean regenerating heavy of myosin Histogram anti-embryonic immunohistochemistry. with (EMyHC) identified mice mdx PBS-treated or ( control. endniiso muoeciebns( bands of immunoreactive Histograms of mice. mdx densities treated mean ActRIIB.mFc and PBS from diaphragms and ( (Diaph). diaphragm 1.39 38 to treatment to prior biopsied triceps n rcp Ti) ( (Tric). triceps and animals. bars) (grey ActRIIB.mFc-treated ( and bars) (white PBS-treated hs eut ocuieydmntaeta niiino myostatin of inhibition six that fibrosis. demonstrate all in conclusively reduction results 6E,F, a These 2.46 Fig. by ActRIIB in to responded from shown animals As senescent hydroxyproline 6F). (Fig. treatment of ActRIIB.mFc 6D,E). (Fig. level average reduction ActRIIB.mFc showed the fibrosis in of following 54 analysis biochemical (left). significant from Furthermore, triceps fibrosis triceps showed of contralateral area tissue biopsied average muscle pretreated, the in of the reversal analysis to (right) Histological triceps compared contralateral the from was were S3C). tissue animals muscle Fig. treatment, unchanged and ActRIIB.mFc sacrificed material of were weeks (supplementary measures eight Following treatment these PBS while following 6A,C) (Fig. treatment ( decreased significantly were regions dniidb aoiimehneetadma T mean and enhancement gadolinium by identified ( ActRIIB.mFc regeneration inhibitor and growth mice. muscle mdx increases senescent myostatin in of Inhibition 5. Fig. B egt fioae uce:qarcp Qa) atonmu (Gast) gastrocnemius (Quad), quadriceps muscles: isolated of Weights ) 6 0.81 E eeeaigfbr ntiesmsl rmActRIIB.mFc-treated from muscle triceps in fibers Regenerating ) m /g( g/mg P , .5 ** 0.05, C D ucefbrcosscinlaes(S)i rcand Tric in (CSA) areas cross-sectional fiber Muscle ) muolto yDepeso ntiesmuscles triceps in expression MyoD of Immunoblot ) n w-erodmxmc eetetdwt myostatin with treated were mice mdx Two-year-old 5 P P )o B ( PBS or 6) , , .1 ntetiesfollowing triceps the in 0.01) 0.01. n 5 )fr6wes ( weeks. 6 for 5) n 5 P , )nraie to normalized 3) .5 fe ActRIIB.mFc after 0.05) 6 %( 9% A P 2 oywihsof weights Body ) m , ausi these in values .Errbars Error m. 6 .1 nthe in 0.01) b %i the in 8% atnas -actin 6 .6to 0.86 eoeadatrtetetwti i d ie ro asrpeets.d. represent bars Error mice. mdx six within * treatment after and individual six before in ActRIIB.mFc ( with mice. treatment mdx after and before muscle triceps ffboi ra fteties eoe rmsm d os eoeand before mouse 75 mdx bar: same Scale from ActRIIB.mFc. removed with triceps, treatment the after of areas fibrotic of eeiili earo naueijr uigwihfibrosis which initially during is injury acute process an this of muscle, repair skeletal in In beneficial 2011). Wagner, chronic many proliferate, to of activated express feature are fibroblasts end-stage injury, common Following diseases. a is fibrosis Tissue Discussion individual within fibrosis mice. muscle mdx established reverses partially n ek ftetetwt cRI.F.( 4 ActRIIB.mFc. 0, with treatment at of regions weeks muscle ( gadolinium-enhanced 8 data. the and MRI in the measured from time calculated relaxation treatment post- after the and in before signal ( enhanced images. of T1-weighted of regions gadolinium images indicate ( MRI Arrows ActRIIB.mFc. T1-weighted hindlimbs. inhibitor post-gadolinium mouse myostatin and with pre- weeks and 8 T2-weighted before for hindlimb of treatment (MRI) after imaging and resonance magnetic in and fibrosis (triceps) forelimb muscle pre-existing reverses mice. mdx myostatin senescent of Inhibition 6. 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Billerica, was MRI vivo In MRI and muscles mdx/ skeletal and hindlimb mdx including from mice isolated 6-month-old were from diaphragm fibroblasts muscle Primary cultures fibroblast muscle Primary ito cRI.F sdi hs tde,RP01 eg Warsing, Leigh RAP-031. studies, these kind in the used for ActRIIB.mFc Inc. of Pharma, gift Acceleron acknowledge to wish authors The Acknowledgements response. versus log(inhibitor) of analysis for when significant Student’s considered Two-tailed were errors. standard their and means t sample as presented are Data analysis Statistical mg/ml 0.1 with treating by ELISA vitro MA), Billerica, detection (Chemicon, in kit Death ELISA apoptosis Cell ssDNA apoptosis using analyzed was to Apoptosis 25 induced cycloheximide, were al., Fibroblasts et was assays (Woessner Apoptosis muscles colleagues 2008). and al., various et Woessner of (Li of described content as modification 1961) using Hydroxyproline a determined was (NIH). using band each performed software of density Image previously relative as The Scio performed 2008). were al., homogenates et (Li tissue described and lysates cell of Immunoblots analysis hydroxyproline and Immunoblot and was 1200– area section index total Fibrosis muscle measured by section. limb divided diaphragm We area each each fibrotic for for (NIH). as fields fields defined visual areas Software two visual cross-sectional in Image different fibers fiber of 500–800 four Scio Muscle among analysis using fashion. fibers morphometric 1500 blinded by a All determined in were immunohistochemistry. performed for was processed muscle or red-O analysis 10 morphometric and immunohistochemistry Histochemisty, T post-contrast The processing Image ie(E f3 s cotanlnt f4 inlaeae,adarslto of resolution a and echo averages, an signal ms, 4 4000 4, of of (TR) length time train echo repetition mm ms, a 0.11 30 with of sequence (TE) echo time spin fast a using oueci a sda h ai rqec rnmte n eevr High receiver. and transmitter frequency radio the diameter as mm T 20 used axial A resolution was concentration. anesthetic coil the volume adjusting by breaths/min 50–80 24/8m,4sga vrgs eouino .6mm 0.16 of resolution a 8/16/24/ of averages, TEs T multiple signal ms, Pre-contrast 2000 Coregistered of 4 TR a ms, with 32/40/48 sequence echo spin echo multiple L ta. 2008). al., et (Li ucecyscin eedrcl dniidb sn AS2d-lo nSitu in 2TdT-Fluor for immunocytochemistry TACS or using caspase-3. 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Danvers, muscle Signaling, (Cell caspase-3 cleaved of expression aoettt ielmn,Bre aoaois an,N) otcnrs T post-contrast of NJ), resolution a Wayne, and averages, Laboratories, signal 8 Berlex ms, dimeglumine, 8 of gadopentetate TE a ms, mm 300 0.16 of TR a with sequence ehiuspeiul ecie L ta. 08.I ir olgnpouto was production collagen vitro In 4 2008). of incorporation al., by et estimated (Li described previously techniques ahanimal. each wwmitdoog,adteaeaeT ROIEditor average using outlined the manually were and subtracting(www.mristudio.org), enhancement no By with segmentation. region neighboring manual and Magnevist via by enhanced The obtained areas 1998b). images, T1-weighted post-contrast was al., and pre- coregistered et volume Woods muscle 1998a; calf al., et entire (Woods package Automated (AIR) the Registration in provided Image transformation affine linear parameter 12 the using images acltdb o-ierftigo utpeT multiple of fitting Log-linear by calculated egtdiae eeaqie ihtesm aaeesa r-otatT pre-contrast as parameters same the with acquired were images weighted egtdiae.Tettliaigtm a prxmtl hour. 1 approximately was time imaging total The images. weighted ts a sdfrcmaio ftemasbtenaytogop.Differences groups. two any between means the of comparison for used was -test m ucecyscin eesandwt &,Mso rcrm,adOil- and trichrome, Masson H&E, with stained were cryosections muscle m 6 6 .1mm 0.11 .6mm 0.16 ysai niiinrvre irss3963 fibrosis reverses inhibition Myostatin 2 m 1 6 6 wihe mgso os idibmslswr acquired were muscles hindlimb mouse of images -weighted wihe mgswr lge otepecnrs T pre-contrast the to aligned were images -weighted tpsd r0.5 or etoposide M . m oeitrdailT axial Coregistered mm. 0.5 m fe ..ijcino anvs 1m f1:100 of ml (1 Magnevist of injection i.p. 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