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Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis… Pisconti, Addolorata; Banks, Glen B; Babaeijandaghi, Farshad; Betta, Nicole D; Rossi, Fabio M V; Chamberlain, Jeffrey S; Olwin, Bradley B Oct 4, 2016

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RESEARCHLoss of niche-satellite cellsdaacterized by progressive muscle loss, chronic inflamma- cell cycle regulators [9], and cell-intrinsic disruptions ofhomeostasis.ely decreasesh myofibers,and strengthoss of musclereased matrixinfiltration.aged musclePisconti et al. Skeletal Muscle  (2016) 6:34 DOI 10.1186/s13395-016-0104-8sarcopenia has been recently questioned, although aColorado at Boulder, Boulder, CO 80309, USAFull list of author information is available at the end of the articleand associated with cell-intrinsic deficits in satellite cellfunction [15–20]; however, satellite cell contribution to* Correspondence: pisconti@liverpool.ac.uk; bradley.olwin@colorado.edu1Department of Cellular, Molecular and Developmental Biology, University ofetic defect imposes a high demand for myofiber repair,which is sustained by muscle progenitors. The prolifera-tive potential of the resident muscle progenitors, namedsatellite cells, is presumed to be prematurely exhaustedin muscular dystrophy, abrogating muscle regenerationand leading to fibrosis [3–7]. Although a thorough un-derstanding of the molecular mechanisms regulatingplay critical roles in regulating satellite cellDuring aging, myofiber size progressivwith an accompanying loss of fast twitcleading to reduced overall muscle massthat, when severe, results in sarcopenia. Lmass and strength is accompanied by incdeposition (fibrosis) and increased fatSkeletal muscle regeneration is impaired intion and replacement of muscle tissue with fibrotictissue [1, 2]. In several types of muscular dystrophy, thecontinuous myofiber damage caused by the primary gen-self-renewal and cell division upon dystrophin loss fromsatellite cells [10] as well as non cell autonomous regula-tion, including the extracellular environment [11–14],Muscular dystrophy is a family of genetic disorders char-required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressedin satellite cells, supports communication with the niche, providing cell interactions and signals to maintainquiescent satellite cells.Results: Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophicmice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscleaging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulatePax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellitecell homeostasis and satellite cell homing to the niche.Conclusions: Manipulating syndecan-3 provides a promising target for development of therapies to enhancemuscle regeneration in muscular dystrophies and in aged muscle.Keywords: Satellite cells, Muscle regeneration, Muscular dystrophy, Niche, Cell adhesion, Cell migration,Syndecan-3, Pax7Background satellite cells in muscular dystrophy is incomplete, cell-intrinsic mechanisms, such as telomerase expression [8],Background: The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells,in syndecan-3 null mice aprogenitor cell homeostamuscle regenerationAddolorata Pisconti1,2*, Glen B. Banks3, Farshad BabaeijanJeffrey S. Chamberlain3 and Bradley B. Olwin1*Abstract© 2016 The Author(s). Open Access This articInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/zeOpen Accessinteractionslters muscleis improvingghi4, Nicole Dalla Betta1, Fabio M. V. Rossi4,le is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 2 of 14contribution of satellite cell loss to aging-associatedfibrosis is supported [21].Satellite cells in G0 phase reside within the muscula-ture and are poised to rapidly activate in response to in-jury [22–26]. Upon activation, satellite cells re-enter thecell cycle, migrate away from their niche, and proliferateas myoblasts, eventually undergoing terminal differenti-ation into myocytes that fuse into pre-existing damagedmuscle fibers or fuse to one another generating newmuscle fibers [27]. During regeneration, a portion ofsatellite cells returns to its niche, re-enters quiescence,and expresses Pax7 but no other myogenic transcriptionfactors [27–29]. The transmembrane heparan sulfateproteoglycan syndecan-3, a component of the satellitecell niche, controls satellite cell homeostasis by regulat-ing signaling pathways within the niche [12, 14, 30–32].Moreover, members of the Syndecan family regulatecell-cell adhesion and cell-matrix adhesion via inter-action with integrins and cadherins [33]. Following amuscle injury, syndecan-3 null (Sdc3−/−) satellite cellsfail to replenish the resident pool of quiescent satellitecells within the niche [14] and therefore syndecan-3appears to regulate satellite cell homeostasis [14].We show that syndecan-3 loss alters satellite cell adhe-sion to the myofiber, altering interactions with the nicheand (i) improves muscle regeneration upon repeatedacute muscle injuries, (ii) rescues muscle histopathologyand function in dystrophic muscle tissue, and (iii)improves muscle aging with a reduction in fibrosis. Thelifelong improvement in muscle regeneration observedin Sdc3−/− muscle arises in part by altered satellite cellhomeostasis and changes in satellite cell adhesiveness tothe myofiber.MethodsMiceMice were housed in a pathogen-free facility at the Universityof Colorado at Boulder, USA, or at the University ofLiverpool, UK. All injuries and other procedures were per-formed at the University of Colorado, and protocols were ap-proved by the IACUC at the University of Colorado. Animalshoused at the University of Liverpool were used in accord-ance with the Animals (Scientific Procedures) Act 1986 andthe EU Directive 2010/63/EU and after local ethical reviewand approval by Liverpool University’s Animal Welfareand Ethical Review Body (AWERB). Sdc3−/− mice weredonated by Dr. Heikki Rauvala, University of Helsinki,Finland. Mdx4cv mice were donated by Dr. JeffreyChamberlain, University of Washington, Seattle, USA.Generation of double mutant colonies is described indetails in Additional file 1. In all experiments, wild typeand mdx4cv;Sdc3+/+ controls were all siblings or closelyrelated, inbred, sex- and age-matched animals for alltransgenic lines.ImmunofluorescenceTissue samples were collected and either immediatelyfrozen in liquid nitrogen-cooled isopentane or fixed in10 % formalin. For all immunofluorescence staining ex-cept Myf5 and Pax7, sections were fixed with 4 % para-formaldehyde (PFA) in phosphate buffered saline (PBS)for 10 min at room temperature. For Myf5 staining, sec-tions were fixed for 10 min with acetone at −20 °C. ForPax7 staining, sections were either fixed and stainedusing an anti-Pax7 rabbit polyclonal antibody (Genetex)or non fixed, processed for antigen retrieval, and stainedwith an anti-Pax7 mouse monoclonal antibody (DSHB).The antibodies used were as follows: rabbit polyclonalanti-Pax7 (Genetex) at 1:250; rabbit polyclonal anti-laminin (Sigma) 1:150; rat polyclonal anti-laminin α2(Sigma) 1:100; rat anti-F4/80 (Genetex) 1:200; rat anti-BrdU (Serotec) 1:100; mouse anti-Pax7 monoclonal(DSHB) 1:200; rabbit anti-myogenin (SCBT) 1:50; rabbitanti Myf5 (SCBT) 1:200; rat anti-CD31 (BD Biosciences)1:100; rabbit anti-NG2 (Chemicon) 1:200; rabbit anti-Ki67 (Abcam) 1:400; rat anti-Sca1 (unconjugated, PE-conjugated, APC-Cy7-conjugated and FITC-conjugatedwere all from BD Biosciences), 1:100; rabbit anti-GFP(BD Biosciences), 1:400. Secondary antibodies conju-gated with Alexa594, Alexa555, Alexa488, or Alexa647(Molecular Probes) were used at 1:500 dilution. Vecta-shield with DAPI (Vector Laboratories) was used as amounting medium.Sirius red stainingFlash-frozen sections were fixed for 1 h at 56 °C inBouin’s fixative, washed in water, stained for 1 h in Mas-ter*Tech Picro Sirius Red, washed in 0.5 % acetic acid,dehydrated, equilibrated with xylene, and mounted usingPermount™.Trichrome stainingTrichrome staining was performed according to stand-ard protocols by Premier Laboratory LLC, Boulder, CO,on paraffin-embedded tissues fixed in 10 % formalin inneutral buffered saline and preserved in 70 % ethanol.Morphometric analysisMyofiber cross-sectional area and numbers in uninjuredand injured TA muscles were quantified as previouslydescribed [14]. The fibrotic index (% collagen + area inSirius Red staining relative to total section area) wasquantified by selecting red pixels in Adobe Photoshop,deleting all non-red pixels, converting the resultingimage to a binary image, and counting red pixels usingthe ImageJ Analyze Particles function. The necroticindex was calculated by counting the number of mIgG+myofibers and normalizing to total number of myofi-bers in the image. Capillary density was calculated byon alternate fibers in order to avoid overlapping scor-force-producing capacity of each muscle at its optimum(1 mM Na3VO4 + 1 mM NaF) using an UltraTurrex1:2000. Anti-rabbit conjugated secondary antibodiesPisconti et al. Skeletal Muscle  (2016) 6:34 Page 3 of 14(Santa Cruz) were used at 1:10,000, and HRP activitywas visualized using the ECL plus system (Amersham).RT-PCR primershomogenizer followed by incubation on ice for 20 minand then cleared by centrifugation at 13,000 rpm for10 min at 4 °C. Western blot was performed as previ-ously described [14]. The antibodies used were asfollows: rabbit polyclonal anti-dystrophin (Abcam) at1:1000; rabbit polyclonal anti-utrophin (kindly donatedby Dr. Froehner, University of Washington, Seattle)length according to maximal stimulation over 300 ms toelicit tetanic contraction. The peak force was then di-vided by the unit area of muscle to obtain specific force(kN/m2) using the equation: specific force = peak force ×muscle length × 0.6 × 1.04/muscle weight [34]. Next, wemeasured protection from contraction-induced injury.The force-producing capacity of the muscle was mea-sured immediately prior to increased length changesduring maximal stimulation at 20-s intervals. Lengthchanges were increased in 5 % increments from 5 to45 % of muscle fiber length to produce injury. The rateof length change was 2 lengths/s.Western blottingQuadriceps were homogenized in 20 mM HEPES,50 mM KCl, 1 mM DTT, 2 mM MgCl2, 0.5 mM EDTA,0.5 % NP40 supplemented with protease inhibitor cock-tail (Complete, Roche), and phosphatase inhibitorsings. Ten sections per mouse for three different micewere scored.Endurance trainingFemale and male mice of different genotypes were indi-vidually housed in cages equipped with a training wheelconnected to a bicycle computer (Schwinn) with adlibitum access to food and water for 3 weeks. Time anddistance run were recorded daily.Muscle physiologyMice were anesthetized with 2,2,2-tribromoethanol(Sigma) such that they were insensitive to tactile stimuli.Peak isometric force of the TA muscle was analyzed insitu via nerve stimulation. First, we found the maximummeasuring the numbers of capillary around each fiberDystrophin forward: CAGCTGCAGAACAGGAGTT.Dystrophin reverse: GCATCTACTGTGTGAGGACC.Mouse injuryMice were anesthetized with isofluorane and the rightTA muscle was injected with 50 μL of 1.2 % BaCl2 [35]in three places along the length of the muscle and thenboth the injured muscle and the contralateral uninjuredmuscle harvested at the indicated time points. For re-peated injuries, the same TA muscle was injured asabove for a total of three times with 3-week intervals be-tween injuries. The injured TA muscle and the contralat-eral uninjured TA muscle were harvested 3 weeks afterthe last injury.Fluorescence-activated cell sortingHindlimb muscles of 3–6-month-old Sdc3−/− and litter-mate wild type female mice were dissected, minced, anddigested in 400 U/mL collagenase type I in Ham’s F-12C(F12 + 0.4 mM CaCl2) at 3 °C for 1 h, gently vortexingevery 10 min. Collagenase was diluted 1:3 with F12C +15 % horse serum (HS) and tissue debris removed bycentrifugation at 30×g for 5 min (pellet contains largedebris) followed by straining of the supernatant (con-taining mononucleated cells and smaller debris) through40-μm cell strainers (BD Falcon). Flow through was thencentrifuged at 300×g, and the cell pellets were re-suspended in PBS + 5 % fetal bovine serum (FBS) and in-cubated for 45 min at 4 °C with 1:100 phycoerythrin(PE)- or fluorescein isothiocyanate (FITC)-directly con-jugated rat anti-Sca1 antibody (BD Biosciences) and1:500 chicken anti-Sdc4 [12] followed by an incubationfor 45 min at 4 °C with Alexa 647-conjugated anti-chicken IgY (Molecular Probes). Sca1+, Sca1+/Sdc4−,and Sca1−/Sdc4+ cells were sorted on a MoFlo XDPCell Sorter (Dako Cytomation) into Ham’s F12C + 15 %horse serum (HS) and cultured in a myogenic growthmedium (F12C + 15 % HS + 2 nM FGF2) or transplanted,see transplantation details below. To assess the expressionof Pax7, Pax3, and Myf5 and MyoD, fibro-adipogenic pro-genitors (FAPs) were sorted as Hoechstmid PIlo CD45−CD31− Sca1+ CD34+ cells and muscle progenitors (MPs)were sorted as Hoechstmid PIlo CD45− CD31− Sca1−CD34+ as previously described [36] directly into lysisbuffer (CellsDirect Resuspension & Lysis Buffer, LifeTechnologies).Droplet Digital PCRFollowing RNA isolation (CellsDirect Resuspension &Lysis Buffer, Life Technologies) and reverse transcription(High Capacity cDNA Reverse Transcription Kit, LifeTechnologies) according to the manufacturer’s instruc-tions, complementary DNA (cDNA) was diluted five timesin TE buffer and 5 μL were used in a reaction mix contain-ing Droplet Digital™ PCR Supermix (BioRad), 1× TaqManprobes from Life Technologies [Pax7 (Mm03053796-s1),Myf5 (Mm00435125-m1), Hprt (Mm00446968_m1), Pax3muscle histopathology and functionmdx mice due to lower numbers of revertant muscle fi-Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 4 of 14(Mm00435493_m1), and Myod1 (Mm00440387_m1)] andH2O. Droplets were generated with a QX100 droplet gen-erator (BioRad), after mixing 20 μL of reaction mix and70 μL of droplet generator oil (BioRad). The emulsifiedsamples were loaded onto 96-well plates, and endpointPCRs were performed in C1000 Touch thermal cycler(BioRad) at the following cycling conditions (95 °C for10 min, followed by 45 cycles of 94 °C for 30 s and 60 °Cfor 1 min, followed by 98 °C for 10 min). The dropletsfrom each sample were read through the QX100 dropletreader (BioRad). Resulting PCR-positive and PCR-negativedroplets were counted using QuantaSoft software(BioRad). Expression levels were normalized to Hprt.Cell transplantationSca1+ cells were FACS-isolated as described above fromSdc3+/+;β-actin-GFP and Sdc3−/−;β-actin-GFP mice, cen-trifuged, and washed twice with sterile 0.9 % NaCl to re-move the serum, re-suspended into 0.9 % NaCl at theconcentration of 2400 cells/μL and 30 μL (~70,000 cells)immediately injected into the right TA muscle of wildtype mice which had been injured 4 h before with an in-jection of 30 μL of 1.2 % BaCl2. Three weeks after ani-mals were sacrificed, the right (injured and transplanted)and left (uninjured, untransplanted) TA muscles weredissected and cryopreserved for subsequent histologicalanalysis.Myofiber isolation and cultureThe gastrocnemius muscles of wild type and Sdc3−/−mice were dissected and incubated with 400 U/mL colla-genase type I in F12C at 37 °C, with gentle mixing by in-version every 15 min for 1 h 30 min, after whichcollagenase was diluted 1:5 with F12C + 15 % HS andmuscles gently rocked at room temperature for 15 minto allow for myofiber release from the digested muscle.Individual myofibers were manually picked and trans-ferred to fresh F12C + 15 % HS using a sterile, flame-polished Pasteur pipette. Myofibers were cultured insuspension in F12C + 15 % HS + 2 nM FGF2 in non-coated sterile petri dishes unless otherwise specifiedand transferred every 24 h to fresh medium.Microscopy, image processing, and figure preparationMicrographs were taken with a Leica TCS SP2 AOBSconfocal microscope using dedicated Leica software, orwith a Nikon (Eclipse E800) epifluorescence microscopeusing Slidebook v4.1 acquisition software (IntelligentImaging Innovations Inc.) coupled to a Cooke Sensicamdigital camera or with an EVOS-FL inverted microscope(Life Technologies). Lenses used with the Leica confocalmicroscope were either HC PL APO 20×/0.70 IMMCORR CS or HCX PL APO 40×/1.25–0.75. Lenses usedwith the Nikon Eclipse microscope were Nikon Planbers [38]. As expected, a reduction in Pax7+ satellitecells was observed in mdx4cv;Sdc3−/− mice compared tomdx4cv;Sdc3+/+ mice (Fig. 1a, b), but, surprisingly, thehistopathology of mdx4cv;Sdc3−/− muscles was improvedcompared to mdx4cv;Sdc3+/+ muscles (Fig. 1c–f ). Fibrosiswas reduced in mdx4cv;Sdc3−/− muscles (Fig. 1c, d), ac-companied by a reduction in sarcolemmal permeability(Fig. 1e, f ) compared to mdx4cv;Sdc3+/+ littermate con-trols. These differences were not due to a strain effectsince (i) Sdc3−/− mice and mdx4cv mice share the samebackground (C57Bl/6); (ii) all experiments were carriedThe numbers of satellite cells per myofiber is increasedin Sdc3−/− mice [12]; however, the numbers of Pax7+satellite cells in uninjured Sdc3−/− muscles are similar tothose found in wild type muscles (Fig. 1a, top panels, b).Loss of Pax7+ satellite cells in Sdc3−/− muscle occursafter injury-induced regeneration [14] and thus we askedwhether chronic injury would also lead to loss of Pax7+satellite cells. Sdc3−/− mice were bred with dystrophicmdx4cv mice (Additional file 1: Figure S1A) to determineif syndecan-3 loss would exacerbate loss of Pax7+ satel-lite cells. We chose the mdx4cv strain [37] because it de-velops a more severe form of muscular dystrophy thanFluor either 40×/0.75 DIC M or 20×/0.50 Ph1 DLL.Lenses used with the EVOS microscope were PL FL, ei-ther 10× LWD PH, 0.25NA/9.2WD or 40× LWD PH,0.56NA/1.6WD. All digital microscopic images were ac-quired at room temperature. For figure preparation,images were exported in Adobe Photoshop, if necessarybrightness and contrast adjusted and the background re-moved for the entire image, the image cropped and indi-vidual color channels extracted (when required) withoutcolor correction or gamma adjustments.Statistical analysisTo assess statistical significance, two-tailed, unpairedStudent’s t test or one-way analysis of variance (ANOVA)were performed. p < 0.05 was considered significant. Atleast three different animals per genotype and per agegroup were used in all experiments. Cell culture experi-ments (both myofiber and myoblast cell cultures) were re-peated three independent times using three differentanimals per genotype group. For force measurements, fiveto seven animals per genotype were used. For musclefunction testing (voluntary wheel), three to seven animalsper genotype group were used.ResultsDystrophic mice lacking syndecan-3 show improvedout using the inbred progeny of Sdc3−/− and mdx4cvfounders (Additional file 1: Figure S1A).Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 5 of 14Improved muscle function in syndecan-3 null dys-trophic mice accompanied the improved muscle histo-pathology as compared to mdx4cv;Sdc3+/+ mice. Both maleand female mdx4cv;Sdc3−/− mice ran for longer distancesand for longer periods of time than mdx4cv;Sdc3+/+controls when assayed on a voluntary wheel, performingsimilar to the times and distances recorded for wild typeFig. 1 Loss of syndecan-3 improves dystrophic muscle histopathology andnumbers in wild type and Sdc3−/− muscle (top panels) but are reduced in mmuscles (lower panels). Interstitial Pax7 immunoreactive cells were occasionAverage numbers of Pax7+ sublaminar cells plotted in b as percentage of tot(4cv;S3+/+), and mdx4cv;Sdc3−/− (4cv;S3−/−) mice were stained to detect co(e, green), and laminin (e, red). Connective tissue quantified as area stainedwith increased sarcolemmal permeability quantified as number of myofiberpercentage of the total myofiber numbers. g–j Exercise performance in maage-matched mice measured as time run (g and i) or distance run (h and j) dare plotted. Non-dystrophic Sdc3+/+ and Sdc3−/− mice were averaged and plotrichrome staining. Error bars are S.E.M. ** = p < 0.01, * = p < 0.05. Scale bars aremice (Fig. 1g, j). This was not due to an intrinsically in-creased propensity of Sdc3−/− mice to perform better inendurance training tests as no significant differencesin time and distance run were recorded for Sdc3−/−non-dystrophic mice compared to wild type mice.The diaphragm muscle, which was severely affectedfollowing voluntary running in mdx4cv;Sdc3+/+ mice,muscle function. a, b Pax7+ satellite cells are present in equaldx4cv;Sdc3−/− (4cv;S3−/−) compared to mdx4cv;Sdc3+/+ (4cv;S3+/+)ally observed in mdx4cv;Sdc3−/− muscle; these were rare and not scored.al area. c–f Cross sections of wild type, Sdc3−/− (S3−/−), mdx4cv;Sdc3+/+llagen (c, red), muscle tissue (c, yellow), mouse immunoglobulinsin red (c) and plotted in d as a percentage of the total area. Myofiberss containing mouse IgG immunostaining in e and plotted in f asle (g, h) and female (i, j) mdx4cv;Sdc3−/− and mdx4cv;Sdc3+/+ sex- anduring 3 weeks of volunteer running. Daily averages for each genotypetted as control (g–j). k Diaphragm histology in exercised mice by100 μm in c, 50 μm in e, and 30 μm in aimproving myofiber integrity, then both contraction-Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 6 of 14induced injury and muscle force would be improved. In-stead, we find the opposite: neither contraction-inducedinjury nor muscle force are improved in mdx4cv;Sdc3−/−muscles. Therefore, loss of syndecan-3 in dystrophicmuscle does not prevent myofiber rupture in responseto stretch, yet the overall muscle histology is improvedand is associated with an overall improvement in exer-cise performance. These apparently conflicting resultscould be explained if muscle regeneration was improvedwas dramatically improved in mdx4cv;Sdc3−/− mice follow-ing voluntary exercise (Fig. 1k). Amelioration of the dys-trophic phenotype in mdx4cv;Sdc3−/− mice was likelymaintained throughout life as in 14-month-old (Additionalfile 1: Figure S1B, C) and 19-month-old (Additional file 1:Figure S1D, E) muscle, collagen deposition is reducedin mdx4cv;Sdc3−/− compared to mdx4cv;Sdc3+/+ mice(Additional file 1: Figure S1B-E).Regeneration is improved in dystrophic muscle lackingsyndecan-3An improvement in muscle histopathology and musclefunction in dystrophic mice lacking syndecan-3 could re-sult from reduced myofiber damage or from improvedmyofiber regeneration. Since syndecan-3 is not expressedin adult myofibers [32], reduced myofiber damage isunlikely to be responsible. In agreement with this, wefound that contraction-induced injury was indistinguish-able in syndecan-3 null dystrophic muscles compared todystrophic muscles expressing syndecan-3 (Fig. 2a). No re-version of dystrophin expression (Fig. 2b, c) or compensa-tory overexpression of utrophin (data not shown) inmdx4cv;Sdc3−/− mice compared to mdx4cv;Sdc3+/+ micewas observed. Consistently, peak force and specific forcein mdx4cv;Sdc3−/− TA muscles were only slightly greaterthan the peak and specific force elicited by mdx4cv;Sdc3+/+TA muscles (Fig. 2d, e). Compensatory muscle hyper-trophy is a hallmark of all mdx mouse strains, includingthe mdx4cv strain [39–43]. A whole-mouse examination ofmdx4cv;Sdc3−/− mice showed only a modest increase incompensatory hypertrophy compared to mdx4cv;Sdc3+/+mice (Fig. 2g), consistent with the finding that the peakforce is only modestly increased in mdx4cv;Sdc3−/− micecompared to mdx4cv;Sdc3+/+ mice. Moreover, the TAaverage wet weight of mdx4cv;Sdc3−/− mice was notstatistically different from the average wet TA weightof mdx4cv;Sdc3+/+ mice (Fig. 2f ).Contraction-induced injury and muscle force measure-ment are carried out on individual muscles and repre-sent a measure of muscle performance prior to damage.If syndecan-3 loss improved exercise performance byin mdx4cv;Sdc3−/− enhancing muscle function, improvingfatigue resistance during exercise and reducing fibrosis.We asked if enhanced regeneration in syndecan-3 nulldystrophic muscle ameliorates the dystrophic phenotype.We observed an increase in myofiber area (Fig. 2h)accompanied by increases in centrally located nuclei(Fig. 2i) and numbers of myofibers with two or morecentrally located nuclei (Fig. 2j) in mdx4cv;Sdc3−/− mus-cles compared to mdx4cv;Sdc3+/+ muscles. These obser-vations, together with our previous data that Sdc3−/−satellite cells generate larger myotubes ex vivo [14]support the hypothesis that syndecan-3 loss enhancesmyofiber regeneration in chronically injured muscles byincreasing muscle progenitor contribution to damagedmyofibers.Fibro-adipogenic progenitors (FAPs) can convert tomyogenic progenitors in a dystrophic environment [44].To test whether myogenic conversion of FAPs wasresponsible for increased muscle regeneration anddecreased fibrosis observed in mdx4cv;Sdc3−/− mice, weisolated FAPs from mdx4cv;Sdc3+/+ and mdx4cv;Sdc3−/−mice and profiled them by qPCR for expression of myo-genic markers. Although MyoD, Pax7, and Myf5 expres-sion was detected in prospective satellite cells isolatedfrom mdx4cv;Sdc3+/+ or mdx4cv;Sdc3−/− muscle, noMyoD, Pax7, Pax3, and Myf5 expression was detected inFAPs isolated from either mdx4cv;Sdc3+/+ or mdx4cv;Sdc3−/− muscle (Additional file 1: Figure S2). These resultssupport the conclusion that in vivo conversion of FAPsto myogenic progenitors is negligible or absent in themdx4cv dystrophic background and is not enhanced bysyndecan-3 loss.Syndecan-3 loss improves regeneration repeatedlyinjured muscle and muscle agingUpon injury, depletion of Pax7+ satellite cells occurs inSdc3−/− non-dystrophic muscle [14], similar to the Pax7+satellite cell depletion observed in syndecan-3 nulldystrophic muscle. To test whether the regenerativecapacity of non-dystrophic Sdc3−/− muscle is similarlyenhanced by syndecan-3 loss as is in dystrophic muscle,we repeatedly injured wild type and Sdc3−/− muscles andmeasured the extent of muscle regeneration. After threeconsecutive injuries, the median myofiber size of wildtype muscle decreased, as expected to a loss of regenera-tive capacity (Fig. 3a, b). In contrast, the median myofibersize of repeatedly injured Sdc3−/− muscle progressively in-creased without an apparent increase in extracellularmatrix deposition (Fig. 3a, b), similar to what was ob-served in syndecan-3 null dystrophic muscle (Fig. 2h).Moreover, the number of myofibers with more than twocentrally located nuclei was increased in Sdc3−/− musclesthat were injured either twice (Fig. 3c) or three times(Fig. 3d), a similar phenotype observed in mdx4cv;Sdc3−/−muscle compared to mdx4cv;Sdc3+/+ muscle (Fig. 2j).Thus, lack of syndecan-3 appears to confer enhancedPisconti et al. Skeletal Muscle  (2016) 6:34 Page 7 of 14regenerative capacity in dystrophic muscle and in non-dystrophic muscle, repeatedly injured skeletal muscle.During aging, a progressive loss of satellite cells occursvia loss of satellite cell self-renewal [15, 16, 45], which isthought to contribute to age-associated muscle fibrosisFig. 2 Syndecan-3 loss does not affect myofiber fragility in dystrophic musdystrophic Sdc3−/− mice (S3−/−), and dystrophic mice either wild type for sfor contraction-induced muscle injuries. b, c Dystrophin protein levels (b, wmice lacking syndecan-3. d, e Syndecan-3 loss modestly improves muscle f(d) elicited by mdx4cv;Sdc3−/− TA muscles is increased compared to mdx4cv;showed no significant difference in peak or specific force and were averagf, g Syndecan-3 loss does not prevent compensatory hypertrophy and incrg and indicated by white arrowheads. h Myofiber cross-sectional area in md(black bars). Inset numbers indicate the median myofiber cross-sectional armdx4cv;Sdc3−/− muscles compared to mdx4cv;Sdc3+/+ muscles. The fractionscalculated and plotted for mdx4cv;Sdc3−/− and mdx4cv;Sdc3+/+ muscles (i) antwo or more nuclei was increased (j). Error bars are S.E.M. ** = p < 0.01, * = p[21]. To determine if syndecan-3 loss affects fibrosis andmuscle aging, we measured the levels of extracellularmatrix deposition in 2-year-old wild type and Sdc3−/−muscles. Although an occasional accumulation of lipiddroplets was previously described in aged Sdc3−/−cle. a The TA muscles of non-dystrophic wild type mice (WT), non-yndecan-3 (4cv;S3+/+) or syndecan-3 null (4cv;S3−/−) were assessedestern blot) and gene expression (c, RT-PCR) are not restored in mdx4cvorce transduction in mdx4cv mice. Specific force (e) but not peak forceSdc3+/+ TA muscles. Non-dystrophic Sdc3+/+ and Sdc3−/− TA musclesed altogether and plotted as control (CTRL, white bar in d and e).eased muscle mass in mdx4cv mice. Glutei and calves are highlighted inx4cv;Sdc3−/− muscles (white bars) compared to mdx4cv;Sdc3+/+ musclesea in square micrometers. i, j Increased myonuclear accretion inof centrally nucleated and peripherally nucleated myofibers wered showed that the number of centrally nucleated myofibers containing< 0.05, # = p > 0.05Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 8 of 14muscle [12], a significant decrease in collagen was ob-served in aged Sdc3−/− muscle compared to aged wildtype muscle (Fig. 3e, f ). Reduced muscle fibrosis in oldSdc3−/− muscle was associated with increased numbersof myogenin + cells (Fig. 3g) and increased numbers ofcentrally nucleated fibers (Fig. 3h), suggesting that deple-tion of the pool of Pax7+ satellite cells upon activationin Sdc3−/− mice does not exhaust muscle regenerativecapacity. Instead, syndecan-3 loss is associated with im-proved muscle in aged mice and improved regenerationin repeatedly injured muscle and in dystrophic muscle.Muscle progenitors distinct from satellite cells contributeminimally to muscle regeneration in the absence ofsyndecan-3Muscle progenitor cells distinct from satellite cells mayparticipate in muscle regeneration as illustrated by trans-plantation of pericytes [46], myoendothelial cells [47],and side population cells [48] into injured muscle. Thesemuscle progenitor cells are associated with blood vessels[46–49]. Sdc3−/− muscle is more vascularized than wildtype muscle as assessed by increased capillary density(Fig. 4a, b) and increased numbers of endothelial cellsFig. 3 Syndecan-3 loss increases myofiber hypertrophy and myonuclear accrtype (WT) and Sdc3−/− (S3−/−) TA muscles harvested 3 weeks after three succb Myofiber cross-sectional area of Sdc3−/− (S3−/−, black bars) and wild type (Wtwo or three successive BaCl2 injections (insets indicate median cross-sectionalocated nuclei after two (c) and three (d) successive injuries. e Fibrosis indicatSdc3−/− mice compared to TA muscles from age- and sex-matched wild typecentrally nucleated myofibers (h) in TA muscles from 2 years old Sdc3−/− micetype mice. Scale bars are 30 μm in a and 50 μm in e. Error bars are S.E.M. ** =(Additional file 1: Figure S3A-E), consistent with the ob-servation that syndecan-3 inhibits VEGF signaling inblood vessel development [50]. Thus, increased musclevascularization associated with syndecan-3 loss may pro-vide increased numbers of vessel-associated myogenicprogenitors that could be responsible for the improvementin muscle maintenance and muscle regeneration occur-ring in Sdc3−/− muscle. Since these blood vessel-associatedmyogenic progenitors express Sca1, we assessed thepercentage of Sca1+ cells present in the population ofmononucleated cells in wild type and Sdc3−/− muscle. Anincrease in Sca1+ cells in Sdc3−/− muscle compared towild type muscle is evident (Fig. 4c). Fibro-adipogenic pro-genitors (FAPs), which are not myogenic but also expressSca1, were not increased in Sdc3−/− muscle compared towild type muscle (Fig. 4d, e). To test if any of theseinterstitial cell populations contributed to the enhancedregeneration we observe in Sdc3−/− muscle, we trans-planted Sca1+/eGFP cells isolated from transgenic ß-actin-eGFP;Sdc3+/+ and ß-actin-eGFP;Sdc3−/− muscle intoinjured wild type hosts (Additional file 1: Figure S3G).Sdc3−/− donor cells but not wild type donor cells engraftedthe satellite cell niche and engrafted into myofibersetion in repeatedly injured muscles and improves muscle aging. a Wildessive injuries and stained to detect laminin (red) and nuclei (blue).T, white bars) from uninjured TA muscles and TA muscle injured withl area in μm2). c, d Quantification of regenerating myofibers with centrallyed by collagen staining is reduced in TA muscles from aged (2 years old)mice. f Quantification of (e). g–h Myogenin + cells (g) and the percent ofare increased compared to TA muscles from age- and sex-matched wildp < 0.01; * = p < 0.05Pisconti et al. Skeletal Muscle  (2016) 6:34 Page 9 of 14(Fig. 4f). Although these data demonstrate that Sca1+ cellsin Sdc3−/− mice contain myogenic progenitors capable ofmuscle engraftment upon transplantation, the extent ofengraftment was minimal (observed only in two of fivetransplanted mice) and appears unlikely to account for theenhancement of muscle function and regeneration ob-served in dystrophic and non-dystrophic Sdc3−/− mice.In addition to interstitial, non-myogenic cells, Sca1 isalso expressed in a subpopulation of satellite cellsFig. 4 Vessel-associated progenitors unlikely contribute to myofibers in Sdcmuscles, immunostained to detect the endothelial cell marker CD31 (green) amuscles compared to wild type muscles. Images representative of three bioreveals increased numbers of Sca1+ cells in Sdc3−/− (S3−/−) muscles compfile 1: Figure S3A-D and F. d, e The numbers of fibro-adipogenic progenito(e) in uninjured muscle are comparable between wild type (WT) and synde(WT) mice and Sdc3−/− (S3−/−) mice transplanted into wild type recipients thdetect GFP (green) and laminin (red) and stained with DAPI to detect nuclei 3due to differences in tissue sectioning. Insets identify an interstitial WT donorcell niche (magnifications, right-hand side). g Wild type muscle cross sections(green) and stained with DAPI to detect nuclei, show that syndecan-4 (Sdc4) ih FACS-isolated Sca1+/Sdc4− cells from wild type (WT) and Sdc3−/− (S3−/−) m4 days and scored as myogenic (clones with myotubes) or non-myogenic cloindicate S.E.M. ** = p < 0.01marked by syndecan-4 [49, 51] and is induced upon sat-ellite cell activation in a subpopulation of satellite cellsthat self-renew [52, 53]. Therefore, we asked if differ-ences exist between wild type and Sdc3−/− muscle-derived Sca1+/Sdc4− cells (Fig. 4g). When Sca1+/Sdc4−cells from wild type and Sdc3−/− mice were isolated byFACS and cultured at clonal density, a higher percentageof myogenic Sca1+/Sdc4− clones was present in Sdc3−/−muscle as opposed to wild type muscle (Fig. 4h).3−/− muscles. a Uninjured wild type (WT) and Sdc3−/− (S3−/−) TAnd nuclei (DAPI, blue) show an increase in capillary density in Sdc3−/−logical replicates. b Quantification of a. c Flow cytometric analysisared to wild type (WT) muscles. Gating scheme shown in Additionalrs (FAPs) either in total (d) or expressed as percentage of all live cellscan-3 null (S3−/−) muscles. f FACS-isolated Sca1+ cells from wild typeat were injured with BaCl2 4 h prior to transplant immunostained toweeks post-transplantation. GFP staining is not entirely homogenouscell (magnification, left-hand side) and a Sdc3−/− donor cell in the satelliteimmunostained to detect laminin (white), syndecan-4 (red), and Sca1s expressed by satellite cells and that Sca1+/Sdc4− cells are interstitial.uscles were cultured in myoblast growth medium at clonal density fornes. Scale bars are 100 μm in a, 30 μm in f, and 10 μm in g. Error barsSatellite cell homeostasis is altered in mice lackingsyndecan-3It appears unlikely that the restoration of regenerativecapacity in syndecan-3 null dystrophic mice is due tonon-satellite progenitors, since (i) we did not detectmyogenic conversion of FAPs; and (ii) engraftment oftransplanted Sca1+ cells from Sdc3−/− mice into musclewas only modestly increased compared to engraftmentof wild type Sca1+ cells. Although the number of sublami-nar Pax7+ cells is reduced in Sdc3−/− muscle followinginjury, the number of Sdc4+ satellite cells is paradoxicallyincreased in Sdc3−/− muscle compared to wild type muscle.This could be explained by downregulation of Pax7 accom-panied by an increase in myoblasts that express low levelsof Pax7. To test these ideas, we first isolated myofibers anddetermined the relative immunreactivity for syndecan-4(Sdc4), Myf5, and Pax7 in Sdc3−/− cells and in wild typecells (Fig. 5a–c). Nearly all Sdc4+ satellite cells on isolatedmyofibers were Myf5+ in both genotypes (Fig. 5a), con-firming the validity of Sdc4 as a satellite cell marker.However, when we quantified Pax7+/Myf5+ cells andPax7−/My5+ cells, few if any wild type cells werePax7−/Myf5+ while nearly 25 % of Sdc3−/− Myf5+ cellswere Pax7− (Fig. 5a–c), suggesting that Pax7 protein levelsare lower in the absence of syndecan-3.Since Pax7 promotes satellite cell quiescence [54, 55]and Sdc3−/− satellite cells are more prone to activationthan wild type cells in uninjured muscle [14], it seemsreasonable that reduced Pax7 levels (Fig. 5a–c) and lossof self-renewal capacity in Sdc3−/− satellite cells [14] arelinked. If correct, then regenerated Sdc3−/− muscle,which is depleted of Pax7+ satellite cells, should possessactivated satellite cells. As Myf5 protein is a marker foractivated satellite cells, we assessed muscle sections ofregenerated wild type and Sdc3−/− muscles for Myf5+cells and found numerous Myf5+ sublaminar and inter-stitial cells in Sdc3−/− muscles that were not present inwild type muscles (Fig. 5d–f ). The increase in Myf5+cells was accompanied by an increase in Ki67+ cells(Fig. 5g), primarily localized to the interstitial spaced tdc7+ifyr cethsh MPisconti et al. Skeletal Muscle  (2016) 6:34 Page 10 of 14Fig. 5 Syndecan-3 regulates myoblast homeostasis and migration. a Wilcultured in suspension for 4 days, fixed, and immunostained to detect Ssatellite cell doublet on a Sdc3−/− myofiber where one cell is Myf5 + Paxcells (b) and Myf5 + Pax7+ cells (c) as in a. d Muscle cross sections ident3 months post-injury. Arrows are interstitial cells; arrowheads are sublamina(normalized to area) in Sdc3−/− (S3−/−) and wild type (WT) muscles 3 monarea) in wild type (WT) and Sdc3−/− (S3−/−) muscles 3 months post-injury.to gelatin-coated coverslips after 2.5 days culture in suspension than from wilmyofiber transfer as in g. Scale bars are 100 μm in a, 50 μm in e, and 20 μm iype (WT, top panels) and Sdc3−/− (S3−/−, bottom panels) myofibers4 (white), Pax7 (red), Myf5 (green), and nuclei (blue). Arrows indicate aand the other cell is Myf5 + Pax7−. b, c Quantification of Myf5 + Pax7−ing Myf5+ cells (green) in Sdc3−/− (S3−/−) and wild type (WT) muscleslls. e, f Quantification of sublaminar (e) and interstitial (f) Myf5+ cellspost-injury. g Quantification of the numbers of Ki67+ cells (normalized toore myoblasts migrate away from Sdc3−/− (S3−/−) myofibers transferredd type (WT) myofibers. i Quantification of adherent myoblasts 4 h aftern f. Error bars are S.E.M. and ** = p < 0.01; * = p < 0.05between myofibers. Remarkably, the total number ofmyogenic cells in regenerated Sdc3−/− muscle is twofoldgreater than the number of myogenic cells in wild typemuscle (Table 1) and is consistent with our observationthat a greater number of syndecan-4+ cells is present inuninjured Sdc3−/− muscle compared to wild type muscle[12]. Thus, after injury, satellite cells appear to redistrib-ute in Sdc3−/− muscle, accompanied by a reduction inPax7 protein levels and an expansion in Myf5+ cells,which are likely responsible for the increases in centrallylocated nuclei found in Sdc3−/− muscle and responsiblefor the enhanced regenerative capacity of Sdc3−/− musclePisconti et al. Skeletal Muscle  (2016) 6:34 Page 11 of 14and mdx4cv;Sdc3−/− muscle.Maintenance of interstitial Myf5+ cells in Sdc3−/− micemay reflect changes in Sdc3−/− cell adhesion since synde-cans are adhesion molecules. Sdc3−/− myofiber-associatedsatellite cells appear less adhesive than wild type satellitecells (Additional file 1: Figure S4A-B). When isolatedmyofibers from wild type and Sdc3−/− muscles were cul-tured in suspension and then transferred onto gelatin-coated dishes, twofold more Sdc3−/− myoblasts adhered tothe gelatin-coated surface than wild type myoblasts 4 hpost-transfer (Fig. 5h, i and Additional file 1: Figure S4C).The propensity of Sdc3−/− satellite cells to migrate awayfrom their native niche is consistent with the finding thatthe majority of Myf5+ and Ki67+ cells observed in regen-erated Sdc3−/− muscle are located in the interstitial space,and supports the idea that the My5+ myoblast populationobserved in regenerated Sdc3−/− muscle is derived fromsatellite cells that migrated away from their niche.DiscussionIn adult wild type muscle, Pax7+ satellite cells are quies-cent and indispensable for muscle regeneration [56, 57];Pax7 is necessary to maintain this population [58, 59]. Sat-ellite cell niche components including Notch, syndecan-4,integrin-α7, Wnt, FGFs, HGF, the calcitonin receptor, andfibronectin play critical roles in maintaining satellite cellsin their niche [12, 14, 15, 27, 60–64]. Syndecan-3, aTable 1 Total numbers of myogenic progenitors are increasedin uninjured Sdc3−/− muscle compared to wild type muscleWT S3−/− ReferencePax7 sublaminar (A) 3.62 ± 0.38 2.23 ± 0.14 Pisconti, JCB 2010Myf5 sublaminar (B) 0.12 ± 0.08 0.97 ± 0.36 Fig. 5d, eTotal sublaminar 3.74 3.20 A + BMyf5 interstitial (C) 0.5 ± 0.16 4.03 ± 0.57 Fig. 5d, fTotal myogenic cells 4.24 7.23 A + B + CThe numbers Pax7+ nuclei, sublaminar Myf5+ nuclei and interstitial Myf5+nuclei normalized to area in the respective sections scored for wild type TAmuscles (WT, column 2) and Sdc3−/− muscles (S3−/−, column 3) 3 months afteran induced muscle injury. References refer to the source for scoring wherestatistical analysis can be found (column 4). The final row is a summation ofeach column with the total numbers of myogenic cells in therespective sectionstransmembrane proteoglycan expressed in satellite cellsand involved in regulating satellite cell responses togrowth factors and to Notch [12, 14, 30], appears to pro-mote satellite cell identity, the association of satellite cellswith their niche and satellite cell quiescence.Upon muscle injury, wild type satellite cells activate,rapidly induce the myogenic transcription factors MyoDand Myf5, and abandon their anatomical niche to migrateto the site of injury. These cells proliferate as myoblasts,eventually differentiating and fusing into damaged myofi-bers or with each other to form new myofibers [27, 29].Compared to wild type satellite cells, Pax7 expression isreduced and Myf5 elevated in Sdc3−/− satellite cells at least3 months post-injury. Thus, Sdc3−/− mice maintainMyf5 + Pax7− cells long term, which likely continue toproliferate, consistent with their reduction in Notch sig-naling [14], which, in turn promotes Pax7 expression anda return to quiescence [54, 65, 66]. The hypersensitivity ofSyndecan-3 null satellite cells to HGF and FGF2 [12, 30]promotes satellite cell activation and proliferation[22, 67–69]. The appearance of an interstitial Myf5+ cellpopulation is consistent with the reduced myofiber adher-ence and enhanced migration of Sdc3−/− satellite cellsaway from the myofiber, where the reduction in Notch sig-naling prevents re-homing to the satellite cell niche [70].Thus, syndecan-3 loss leads to the sustained presence ofincreased numbers of proliferating myogenic progenitors,which provide for increased myofiber size and increasednumbers of centrally nucleated myofibers (Fig. 6).Since Sdc3−/− satellite cells proliferate slowly and showincreased rates of cell death due to a defect in Notchsignaling [14], the process of myonuclear accretion isslow and in the short lifespan of a mouse does not leadto an appreciable increase in muscle size. Nonetheless, asignificant increase in satellite cell contribution to myofi-bers, shown by the presence of centrally nucleated myo-fibers, accompanied by a significant reduction inmuscle fibrosis, is observed in wild type or dystrophicaged mice lacking syndecan-3. Thus, syndecan-3 lossappears to provide a lifelong benefit to muscle regen-erative capacity in mice.Although other potential myogenic progenitors, such aspericytes, myoendothelial cells, and side population cells,which are increased in Sdc3−/− muscle and show increasedmyogenicity in vitro, may contribute to interstitial and sub-laminar myoblasts, the relative contribution of these cellsappears low and may possibly be due to satellite cell con-tamination of the interstitial cell preparation. We cannotdirectly lineage trace the Myf5+ interstitial cells identifiedin regenerated Sdc3−/− muscle due to (i) the close proximityof Pax7 and syndecan-3 on the same chromosome, (ii) thelower levels of Pax7 in Sdc3−/− satellite cells, and (iii) theco-expression of MyoD and Myf5 by activated satellite cellsand the interstitial myoblasts in Sdc3−/− muscle.hoellallyrenthersstsartiPisconti et al. Skeletal Muscle  (2016) 6:34 Page 12 of 14Fig. 6 Syndecan-3 regulates satellite cell-niche interactions and satellite cellcells) activate in response to myofiber injury and self-renew via asymmetric cbasal lamina (yellow) or outside their niche, in the endomysium, and eventuto one another. In Sdc3−/− muscle (S3−/−, bottom drawing), satellite cell self-which proliferate mostly outside the niche, due to reduced adhesiveness toincreased numbers of differentiated myocytes that fuse to damaged myofibself-renewal is decreased, a population of activated and proliferating myoblapossibly derived from blood vessel-associated progenitors (gray cells) may pLoss of muscle regenerative capacity in the musculardystrophies is often attributed to satellite cell exhaustion[3–9, 71]; however, there are only few experiments dir-ectly supporting this hypothesis. We utilized mdx4cvmice [37, 72], which develop a more severe form ofmuscular dystrophy than mdx mice that is exacerbatedwhen challenged with exercise [42]. The dystrophy be-comes more severe as the mice age, presumably due tothe lower numbers of revertant fibers in mdx4cv micethan in mdx mice [38]. Loss of syndecan-3 in dystrophicmice reduces muscle fibrosis while improving exerciseperformance without ameliorating myofiber fragility orincreasing the specific force. Since myofiber damage ap-pears equivalent in dystrophic muscle with or withoutsyndecan-3, we postulate that muscle regeneration isenhanced, leading to improved exercise performance.This conclusion is supported by the finding thatmdx4cv;Sdc3−/− muscles contain more regenerating myo-fibers than mdx4cv;Sdc3+/+ muscles and enhanced myo-nuclear accretion, consistent with a role for syndecan-3in supporting Notch signals which promotes self-renewal while inhibiting myoblast fusion [14].The Sdc3−/− satellite cell phenotypes appear cell autono-mous as they occur in culture as well as in dystrophicmice lacking syndecan-3 and in aged Sdc3−/− mice. Over-all the mechanism responsible for the enhancement ofregeneration in double mutant mdx4cv;Sdc3−/− mice, theamelioration of the dystrophic phenotype, and themeostasis. In wild type muscle (WT, top drawing) satellite cells (light bluedivision (1) or proliferate as myoblasts (green cells) either underneath thedifferentiate (red mononucleated cells) to fuse to damaged myofibers orewal is decreased (2) leading to increased numbers of activated myoblasts,e myofiber. The increased number of proliferating myoblasts provides forleading to larger, hyperplastic regenerated myofibers. Since satellite cellpersists. Other myogenic progenitor cells distinct from satellite cells andcipate in muscle regeneration in Sdc3−/− musclesimprovement of muscle maintenance in aged mice ap-pears to be the failure of Sdc3−/− satellite cells to return toquiescence and re-home to their niche after activation,which maintains an expanding population of interstitialMyf5+ myoblasts. The numbers of Sdc3−/− myoblasts in-crease over time leading to an expanded muscle progeni-tor population in the muscle interstitium that eventuallygenerates large, centrally nucleated myofibers (Fig. 6).ConclusionsSdc3−/− mice maintain lifelong muscle regenerativecapacity and resist injury-induced loss of regenerativecapacity by maintaining a population of activated,Myf5+Pax7− satellite cells and a proliferating myo-blast population in the myofiber interstitium. Sdc3−/−satellite cells do not appear exhausted in either dystrophicmuscle or aged muscle apparently enhancing muscle re-generative capacity, identifying a new potential therapeutictarget for the treatment and management of musculardystrophies, repeated acute injuries and muscle aging.Additional fileAdditional file 1: Supplementary figures. (PDF 4815 kb)AcknowledgementsWe thank Dr. Jeffrey Chamberlain for the mdx4cv mice and Dr. Heikki Rauvalafor the Sdc3−/− mice. We also thank Dr. Michelle Doyle for critical reading ofPisconti et al. Skeletal Muscle  (2016) 6:34 Page 13 of 14the manuscript and insightful discussions; Dr. Malea Murphy for help withdevelopment of histology techniques and automated image quantificationand Ms. Tiffany Antwine for technical help with histology and flowcytometry. This work was supported by the MDA, The Ellison MedicalFoundation, and NIH Grants AR049446 and AG040074 to BBO, by aWellcome Trust ISSF and a Marie Curie IEF to AP, an MDA developmentgrant to GBB, CIHR grant MOP-97856 to FMVR, and a 4YF fellowship fromUBC to FB.Authors’ contributionsAP designed and performed all experiments except those shown in Figs. 2a, c,d–f, 4d, e, and 5a, Additional file 1: Figure S1B and D. AP also carried out dataanalysis and drafted the manuscript. GBB and JSC designed and performed theexperiments shown in Fig. 2a, d, e and contributed to the manuscriptpreparation. FB and FMVR designed and performed the experiments shown inFig. 4d, e and Additional file 1: Figure S2. BBO participated in experimentaldesign and in drafting of the manuscript. All authors read and approved thefinal manuscript.Competing interestsThe authors declare that they have no competing interests.Author details1Department of Cellular, Molecular and Developmental Biology, University ofColorado at Boulder, Boulder, CO 80309, USA. 2Department of Biochemistry,Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.3Department of Neurology, University of Washington, Mail Stop 357720,Seattle, WA 98195, USA. 4The Biomedical Research Centre, UBC, Vancouver,BC V6T 1Z, Canada.Received: 20 September 2015 Accepted: 26 August 2016References1. Cohn RD, Campbell KP. Molecular basis of muscular dystrophies. MuscleNerve. 2000;23:1456–71.2. Straub V, Rafael JA, Chamberlain JS, Campbell KP. Animal models formuscular dystrophy show different patterns of sarcolemmal disruption.J Cell Biol. 1997;139:375–85.3. Mouly V, Aamiri A, Bigot A, Cooper RN, Di Donna S, Furling D, Gidaro T,Jacquemin V, Mamchaoui K, Negroni E, Perie S, Renault V, Silva-Barbosa SD,Butler-Browne GS. The mitotic clock in skeletal muscle regeneration, diseaseand cell mediated gene therapy. Acta Physiol Scand. 2005;184:3–15.4. Luz MAM, Marques MJ, Santo Neto H. Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.Braz J Med Biol Res. 2002;35:691–5.5. Reimann J, Irintchev A, Wernig A. Regenerative capacity and the number ofsatellite cells in soleus muscles of normal and mdx mice. NeuromusculDisord. 2000;10:276–82.6. Maier F, Bornemann A. Comparison of the muscle fiber diameter andsatellite cell frequency in human muscle biopsies. Muscle Nerve. 1999;22:578–83.7. Decary S, Hamida CB, Mouly V, Barbet JP, Hentati F, Butler-Browne GS.Shorter telomeres in dystrophic muscle consistent with extensiveregeneration in young children. Neuromuscul Disord. 2000;10:113–20.8. Sacco A, Mourkioti F, Tran R, Choi J, Llewellyn M, Kraft P, Shkreli M, Delp S,Pomerantz JH, Artandi SE, Blau HM. Short telomeres and stem cellexhaustion model Duchenne muscular dystrophy in mdx/mTR mice. Cell.2010;143:1059–71.9. Di Donna S, Renault V, Forestier C, Piron-Hamelin G, Thiesson D, Cooper RN,Ponsot E, Decary S, Amouri R, Hentati F, Butler-Browne GS, Mouly V.Regenerative capacity of human satellite cells: the mitotic clock in celltransplantation. Neurol Sci. 2000;21:S943–51.10. Dumont NA, Wang YX, von Maltzahn J, Pasut A, Bentzinger CF, Brun CE,Rudnicki MA. Dystrophin expression in muscle stem cells regulates theirpolarity and asymmetric division. Nat Med. 2015;21:1455–63.11. Bentzinger CF, Wang YX, von Maltzahn J, Soleimani VD, Yin H, Rudnicki MA.Fibronectin regulates Wnt7a signaling and satellite cell expansion. Cell StemCell. 2013;12:75–87.12. Cornelison DDW, Wilcox-Adelman SA, Goetinck PF, Rauvala H, RapraegerAC, Olwin BB. Essential and separable roles for Syndecan-3 and Syndecan-4in skeletal muscle development and regeneration. Genes Dev. 2004;18:2231–6.13. Kuang S, Kuroda K, Le Grand F, Rudnicki MA. Asymmetric self-renewal andcommitment of satellite stem cells in muscle. Cell. 2007;129:999–1010.14. Pisconti A, Cornelison DDW, Olguín HC, Antwine TL, Olwin BB. Syndecan-3 andNotch cooperate in regulating adult myogenesis. J Cell Biol. 2010;190:427–41.15. Bernet JD, Doles JD, Hall JK, Kelly Tanaka K, Carter TA, Olwin BB. p38 MAPKsignaling underlies a cell-autonomous loss of stem cell self-renewal inskeletal muscle of aged mice. Nat Med. 2014;20:265–71.16. Cosgrove BD, Gilbert PM, Porpiglia E, Mourkioti F, Lee SP, Corbel SY, Llewellyn ME,Delp SL, Blau HM. Rejuvenation of the muscle stem cell population restoresstrength to injured aged muscles. Nat Med. 2014;20:255–64.17. Carlson ME, Hsu M, Conboy IM. Imbalance between pSmad3 and Notchinduces CDK inhibitors in old muscle stem cells. Nature. 200818. Sousa-Victor P, Gutarra S, García-Prat L, Rodriguez-Ubreva J, Ortet L,Ruiz-Bonilla V, Jardí M, Ballestar E, González S, Serrano AL, Perdiguero E,Muñoz-Cánoves P. Geriatric muscle stem cells switch reversiblequiescence into senescence. Nature. 2014;506:316–21.19. Price FD, von Maltzahn J, Bentzinger CF, Dumont NA, Yin H, Chang NC,Wilson DH, Frenette J, Rudnicki MA. Inhibition of JAK-STAT signalingstimulates adult satellite cell function. Nat Med. 2014;20:1174–81.20. García-Prat L, Martínez-Vicente M, Perdiguero E, Ortet L, Rodriguez-Ubreva J,Rebollo E, Ruiz-Bonilla V, Gutarra S, Ballestar E, Serrano AL, Sandri M, Muñoz-Cánoves P. Autophagy maintains stemness by preventing senescence.Nature. 2016;529:37–42.21. Fry CS, Lee JD, Mula J, Kirby TJ, Jackson JR, Liu F, Yang L, Mendias CL,Dupont-Versteegden EE, McCarthy JJ, Peterson CA. Inducible depletion ofsatellite cells in adult, sedentary mice impairs muscle regenerative capacitywithout affecting sarcopenia. Nat Med. 2015;21:76–80.22. Jones NC, Tyner KJ, Nibarger L, Stanley HM, Cornelison DDW, Fedorov YV,Olwin BB. The p38alpha/beta MAPK functions as a molecular switch toactivate the quiescent satellite cell. J Cell Biol. 2005;169:105–16.23. MAURO A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol.1961;9:493–5.24. Schultz E. Changes in the satellite cells of growing muscle followingdenervation. Anat Rec. 1978;190:299–311.25. Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V,Marquez VE, Valente S, Mai A, Forcales SV, Sartorelli V, Puri PL. TNF/p38α/polycomb signaling to Pax7 locus in satellite cells linksinflammation to the epigenetic control of muscle regeneration. CellStem Cell. 2010;7:455–69.26. Crist CG, Montarras D, Buckingham M. Muscle satellite cells are primed formyogenesis but maintain quiescence with sequestration of Myf5 mRNAtargeted by microRNA-31 in mRNP granules. Cell Stem Cell. 2012;11:118–26.27. Olguín HC, Pisconti A. Marking the tempo for myogenesis: Pax7 and theregulation of muscle stem cell fate decisions. J Cell Mol Med. 2012;16:1013–25.28. Shea KL, Xiang W, LaPorta VS, Licht JD, Keller C, Basson MA, Brack AS.Sprouty1 regulates reversible quiescence of a self-renewing adult musclestem cell pool during regeneration. Cell Stem Cell. 2010;6:117–29.29. Siegel AL, Atchison K, Fisher KE, Davis GE, Cornelison DDW. 3D timelapseanalysis of muscle satellite cell motility. Stem Cells. 2009;27:2527–38.30. Fuentealba L, Carey DJ, Brandan E. Antisense inhibition of syndecan-3expression during skeletal muscle differentiation accelerates myogenesisthrough a basic fibroblast growth factor-dependent mechanism. J BiolChem. 1999;274:37876–84.31. Casar JC, Cabello-Verrugio C, Olguin H, Aldunate R, Inestrosa NC, Brandan E.Heparan sulfate proteoglycans are increased during skeletal muscleregeneration: requirement of syndecan-3 for successful fiber formation.J Cell Sci. 2004;117:73–84.32. Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3and syndecan-4 specifically mark skeletal muscle satellite cells and areimplicated in satellite cell maintenance and muscle regeneration. Dev Biol.2001;239:79–94.33. Rapraeger AC. Syndecan-regulated receptor signaling. J Cell Biol. 2000;149:995–8.34. Banks GB, Gregorevic P, Allen JM, Finn EE, Chamberlain JS. Functionalcapacity of dystrophins carrying deletions in the N-terminal actin-bindingdomain. Hum Mol Genet. 2007;16:2105–13.35. Caldwell CJ, Mattey DL, Weller RO. Role of the basement membrane inthe regeneration of skeletal muscle. Neuropathol Appl Neurobiol. 1990;16:225–38.stem cells. Cell Stem Cell. 2009;4:535–47.63. Polesskaya A, Seale P, Rudnicki MA. Wnt signaling induces the myogenicspecification of resident CD45+ adult stem cells during muscleregeneration. Cell. 2003;113:841–52.64. Sheehan SM, Tatsumi R, Temm-Grove CJ, Allen RE. HGF is an autocrinegrowth factor for skeletal muscle satellite cells in vitro. Muscle Nerve. 2000;23:239–45.65. Fukada S-I, Yamaguchi M, Kokubo H, Ogawa R, Uezumi A, Yoneda T, Matev MM,Motohashi N, Ito T, Zolkiewska A, Johnson RL, Saga Y, Miyagoe-Suzuki Y,Tsujikawa K, Takeda S, Yamamoto H. Hesr1 and Hesr3 are essential toPisconti et al. Skeletal Muscle  (2016) 6:34 Page 14 of 1436. Joe AWB, Yi L, Natarajan A, Le Grand F, So L, Wang J, Rudnicki MA, Rossi FMV.Muscle injury activates resident fibro/adipogenic progenitors thatfacilitate myogenesis. Nat Cell Biol. 2010;12:153–63.37. Chapman VM, Miller DR, Armstrong D, Caskey CT. Recovery of inducedmutations for X chromosome-linked muscular dystrophy in mice. Proc NatlAcad Sci U S A. 1989;86:1292–6.38. Danko I, Chapman V, Wolff JA. The frequency of revertants in mdx mousegenetic models for Duchenne muscular dystrophy. Pediatr Res. 1992;32:128–31.39. Hakim CH, Duan D. A marginal level of dystrophin partially ameliorateshindlimb muscle passive mechanical properties in dystrophin-null mice.Muscle Nerve. 2012;46:948–50.40. Li D, Shin J-H, Duan D. iNOS ablation does not improve specific force of theextensor digitorum longus muscle in dystrophin-deficient mdx4cv mice.PLoS One. 2011;6:e21618.41. Hakim CH, Burkin DJ, Duan D. Alpha 7 integrin preserves the function of theextensor digitorum longus muscle in dystrophin-null mice. J Appl Physiol.2013;115:1388–92.42. Zhang Y, Yue Y, Li L, Hakim CH, Zhang K, Thomas GD, Duan D. Dual AAVtherapy ameliorates exercise-induced muscle injury and functional ischemiain murine models of Duchenne muscular dystrophy. Hum Mol Genet. 2013;22:3720–9.43. Banks GB, Combs AC, Odom GL, Bloch RJ, Chamberlain JS. Muscle structureinfluences utrophin expression in mdx mice. PLoS Genet. 2014;10:e1004431.44. Saccone V, Consalvi S, Giordani L, Mozzetta C, Barozzi I, Sandoná M, Ryan T,Rojas-Muñoz A, Madaro L, Fasanaro P, Borsellino G, De Bardi M, Frigè G,Termanini A, Sun X, Rossant J, Bruneau BG, Mercola M, Minucci S, Puri PL.HDAC-regulated myomiRs control BAF60 variant exchange and direct thefunctional phenotype of fibro-adipogenic progenitors in dystrophicmuscles. Genes Dev. 2014;28:841–57.45. Chakkalakal JV, Jones KM, Basson MA, Brack AS. The aged niche disruptsmuscle stem cell quiescence. Nature. 2012;490:355–60.46. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L,Innocenzi A, Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G,Chamberlain JS, Wright WE, Torrente Y, Ferrari S, Bianco P, Cossu G.Pericytes of human skeletal muscle are myogenic precursors distinct fromsatellite cells. Nat Cell Biol. 2007;9:255–67.47. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A, Yap S, Pollett JB, Drowley L,Cassino T, Gharaibeh B, Deasy BM, Huard J, Peault B. Prospectiveidentification of myogenic endothelial cells in human skeletal muscle.Nat Biotechnol. 2007;25:1025–34.48. Asakura A, Rudnicki MA. Side population cells from diverse adult tissues arecapable of in vitro hematopoietic differentiation. Exp Hematol. 2002;30:1339–45.49. Doyle MJ, Zhou S, Tanaka KK, Pisconti A, Farina NH, Sorrentino BP, Olwin BB.Abcg2 labels multiple cell types in skeletal muscle and participates inmuscle regeneration. J Cell Biol. 2011;195:147–63.50. De Rossi G, Whiteford JR. A novel role for syndecan-3 in angiogenesis.F1000Res. 2013;2:270.51. Tanaka KK, Hall JK, Troy AA, Cornelison DDW, Majka SM, Olwin BB.Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellitecells during muscle regeneration. Cell Stem Cell. 2009;4:217–25.52. Troy A, Cadwallader A, Fedorov Y, Tyner K, Tanaka K, Olwin B. Coordinationof satellite cell activation and self-renewal by par complex-dependentasymmetric activation of p38$\alpha$/$\beta$ MAPK. Cell Stem Cell. 2012.In press.53. Mitchell PO, Mills T, O'Connor RS, Kline ER, Graubert T, Dzierzak E, Pavlath GK.Sca-1 negatively regulates proliferation and differentiation of muscle cells.Dev Biol. 2005;283:240–52.54. Bjornson CRR, Cheung TH, Liu L, Tripathi PV, Steeper KM, Rando TA. Notchsignaling is necessary to maintain quiescence in adult muscle stem cells.Stem Cells. 2012;30:232–42.55. Olguín HC, Olwin BB. Pax-7 up-regulation inhibits myogenesis and cell cycleprogression in satellite cells: a potential mechanism for self-renewal. Dev Biol.2004;275:375–88.56. Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G. Satellitecells, connective tissue fibroblasts and their interactions are crucial formuscle regeneration. Development. 2011;138:3625–37.57. Sambasivan R, Yao R, Kissenpfennig A, Van Wittenberghe L, Paldi AAS,Gayraud-Morel B, Guenou H, Malissen B, Tajbakhsh S, Galy A. Pax7-expressing satellite cells are indispensable for adult skeletal muscleregeneration. Development. 2011;138:3647–56.generate undifferentiated quiescent satellite cells and to maintainsatellite cell numbers. Development. 2011;138:4609–19.66. Wen Y, Bi P, Liu W, Asakura A, Keller C, Kuang S. Constitutive Notchactivation upregulates Pax7 and promotes the self-renewal of skeletalmuscle satellite cells. Mol Cell Biol. 2012;32:2300–11.67. Jones NC, Fedorov YV, Rosenthal RS, Olwin BB. ERK1/2 is required formyoblast proliferation but is dispensable for muscle gene expression andcell fusion. J Cell Physiol. 2001;186:104–15.68. Tatsumi R, Yamada M, Katsuki Y, Okamoto S, Ishizaki J, Mizunoya W, Ikeuchi Y,Hattori A, Shimokawa H, Allen RE. Low-pH preparation of skeletalmuscle satellite cells can be used to study activation in vitro. Int J BiochemCell Biol. 2006;38:1678–85.69. Johnson SE, Allen RE. Activation of skeletal muscle satellite cells and therole of fibroblast growth factor receptors. Exp Cell Res. 1995;219:449–53.70. Bröhl D, Vasyutina E, Czajkowski MT, Griger J, Rassek C, Rahn H-P, Purfürst B,Wende H, Birchmeier C. Colonization of the satellite cell niche by skeletalmuscle progenitor cells depends on Notch signals. Dev Cell. 2012;23:469–81.71. Lund TC, Grange RW, Lowe DA. Telomere shortening in diaphragm andtibialis anterior muscles of aged mdx mice. Muscle Nerve. 2007;36:387–90.72. Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS.Differential expression of dystrophin isoforms in strains of mdx mice withdifferent mutations. Hum Mol Genet. 1996;5:1149–53.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services Submit your next manuscript to BioMed Central and we will help you at every step:58. Günther S, Kim J, Kostin S, Lepper C, Fan C-M, Braun T. Myf5-positivesatellite cells contribute to Pax7-dependent long-term maintenance of adultmuscle stem cells. Cell Stem Cell. 2013;13:590–601.59. von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA. Pax7 is critical for thenormal function of satellite cells in adult skeletal muscle. Proc Natl Acad SciU S A. 2013;110:16474–9.60. Abou-Khalil R, Brack AS. Muscle stem cells and reversible quiescence: Therole of sprouty. Cell Cycle. 2010;961. Kudla AJ, Jones NC, Rosenthal RS, Arthur K, Clase KL, Olwin BB. The FGFreceptor-1 tyrosine kinase domain regulates myogenesis but is notsufficient to stimulate proliferation. J Cell Biol. 1998;142:241–50.62. Le Grand F, Jones AE, Seale V, Scime A, Rudnicki MA. Wnt7a activates theplanar cell polarity pathway to drive the symmetric expansion of satellite•  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submit

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