Mighty is a novel promyogenic factor in skeletal myogenesis

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Research Article

Mighty is a novel promyogenic factor in skeletal myogenesis

Amy Marshalla,b,1, Mônica Senna Salernoa, Mark Thomasa, Todd Daviesa, Carole Berrya,Kelly Dyera, Jeremy Bracegirdlea, Trevor Watsona, Marie Dziadekb, Ravi Kambadura,d,Rob Bowerc, Mridula Sharmaa,⁎aFunctional Muscle Genomics, AgResearch, Hamilton, New ZealandbSchool of Biological Sciences, University of Auckland, Auckland, New ZealandcOrico Limited, Dunedin, New ZealanddSchool of Biological Sciences, Nanyang Technological University, Singapore

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Department of BiochDrive, Singapore 117597, Singapore. Fax: +65

E-mail address: [email protected] (M.1 née Bishop.

0014-4827/$ – see front matter © 2008 Elsevidoi:10.1016/j.yexcr.2008.01.004

A B S T R A C T

Article Chronology:Received 16 August 2007Revised version received27 December 2007Accepted 6 January 2008Available online 14 January 2008

Genetic analysis has revealed an important function inmyogenesis forMyostatin, amemberof the TGF-β superfamily. However, the cascade of genes that responds to Myostatinsignalling to regulate myogenesis is not well understood. Thus, a suppressive subtractionhybridization to identify such genes was undertaken and here we report the cloning andcharacterization of a novel gene, Mighty. Mighty is expressed in a variety of different tissuesbut appears to be specifically regulated by Myostatin in skeletal muscle. Overexpression ofMighty in C2C12 cells results in early withdrawal of myoblasts from the cell cycle, enhancedand accelerated differentiation and hypertrophy of myotubes. Most importantly, Mightyoverexpression leads to increased and earlier expression of MyoD and increased secretion ofanother known differentiation inducing factor, IGF-II. Furthermore, viral expression ofMighty in mdx mice resulted in an increase in the number of larger healthy muscle fibers.Given its role in myogenesis, we propose that Mighty is a critical promyogenic factor whichplays a key role in the signalling pathway downstream of Myostatin.

© 2008 Elsevier Inc. All rights reserved.

Keywords:MyoblastsDifferentiationIGF-IIMyostatinMyogenesisSkeletal muscle

Introduction

The process of skeletal muscle myogenesis involves initialdetermination of mesodermal cells towards a myogenic line-age. These myogenic precursor cells proliferate, then irrever-sibly withdraw from the cell cycle and differentiate intomultinucleated myotubes. The process of myogenic differen-tiation is regulated by a variety of positive and negative growthfactors. One negative factor, Myostatin (GDF-8) is a member ofthe transforming growth factor β (TGF-β) superfamily. Myosta-tin is predominantly expressed in skeletal muscle and Myos-

emistry, Yong Loo Lin Sch6779 1453.Sharma).

er Inc. All rights reserved

tatin-null mice show a dramatic increase in skeletal musclemass [1]. Loss of functional Myostatin also results in increasedskeletal muscle mass referred to as “double muscling” in avariety of different cattle breeds [2–5]. This increase in skeletalmuscle in both cases is due to an increase in fiber number(hyperplasia) and an increase in fiber size (hypertrophy) [5–8].

It has been proposed thatMyostatin inhibits skeletalmusclehyperplasia by controlling the proliferation rate of myoblasts.Myostatin also inhibits myogenic differentiation via inhibitionofMyoD expression and activity.Myostatin signals to theMyoDpromoter via activation of Smad3 signalling [9]. Interaction of

ool of Medicine, National University of Singapore, MD7, 8 Medical

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http://dx.doi.org/10.1016/j.yexcr.2008.01.004

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MyoD proteinwith activated Smad3 inhibits MyoD transactiva-tional activity [10] and in turn inhibitsmyogenic differentiation[9]. In addition to effects on embryonic myogenesis, Myostatinalso appears to regulate postnatal myogenesis.

Recent studies have shown that Myostatin is involved insatellite cell quiescence and that lack of Myostatin leads toincreased activation of satellite cells and improved musclehealing [11]. AlthoughMyostatin's role inmyogenesis is clearlydocumented through genetic analysis, few genes that respondto Myostatin signalling to control myogenesis have been char-acterized. Here, we showa functional relationship between lackof Myostatin and enhanced differentiation of myoblasts whichismediated throughapreviouslyuncharacterized gene,Mighty.To delineate the role ofMighty inmyogenesiswe examined theeffect of Mighty overexpression in myoblasts and found thatMighty controls myoblast differentiation by regulating thewithdrawal of myoblasts from the cell cycle. Overexpressionof Mighty also induces hypertrophy of C2C12 myotubes in vitroand in vivo in the muscle of mdx mice characterized by anincrease in the cross-sectional area of the myofibers.

Materials and methods

Animals

Myostatin-null mice (C57BL/10 background) and mdx micewere obtained from S.-J. Lee (The Johns Hopkins University,Baltimore, MD) and Animal Resources Centre, Western Aus-tralia respectively. Wild-type mice (C57BL/10) were bred at theRuakura Small Animal Colony (Hamilton). Animal manipula-tions described in this paper were approved by the RuakuraAnimal Ethics Committee (AgResearch, Hamilton, NZ).

Suppressive subtraction hybridization (SSH)

A SSH, comparing mRNA expression between Myostatin-nullmouse muscle (‘tester’) and wild-type mouse muscle (‘driver’)was performed exactly as described by Siriett et al. [12].

Sequence analysis

Blast searches were performed on the NCBI-Blast website(http://www.ncbi.nlm.nih.gov/BLAST). Sequence alignmentswere performed using EBI clustalW alignment software(http://www.ebi.ac.uk/clustalw).

Semiquantitative RT-PCR

Total RNAwas derived fromwild-type orMyostatin-nullmouseskeletal muscles and tissues using TRIzol (Invitrogen, Carlsbad,CA) according to the manufacturer's instructions. RT-PCRreactions for Mighty gene expression were carried out using1 μg total RNA and Superscript™ First-strand Synthesis System(Invitrogen, Carlsbad, CA) according to the manufacturer'sprotocol. The primers used for the PCR were, 5′TGAAGCGGCC-CATGGAGTTC-3′ and 5′TTGGCCTTGTCCCGTATCGC-3′ or 5′-GGTGGGCTGGTCCTTCTTCA-3′. The reaction included 1× Qsolution (Qiagen Scientific, Germantown, MD) and the anneal-ing temperature was 62 °C. The number of PCR cycles (28 or 30)

was standardised for the linear amplification of Mighty cDNA.The PCR products were analysed on an agarose gel and nor-malised to tubulin.

Recombinant Myostatin and Mstn-ant1

Production and purification of Myostatin (processed, C-term-inal 266–375 amino acids) has been described previously [13].For the recombinantMstn-ant1,Myostatin cDNAwas truncatedat the amino acid 350, producing a truncated portion of theprocessed region (266–350 amino acids) and cloned into vectorpET 16-B (Novagen, Madison, WI), as previously described [14].The protein was purified utilizing a Ni-NTA agarose affinitycolumn (Qiagen Scientific, Germantown, MD) [14].

Western blotting

To determine levels of Mighty, MyoD,Myogenin, p21, MHC andGAPDH protein during differentiation, C2C12 myoblasts wereplated at 25,000 cells/cm2 and switched to differentiationmedia (DMEM 2% HS). All cells were grown at 37 °C, 5% CO2.The cells were harvested at 0, 12, 24, 48, 72 and 96 h timepoints, the proteins were extracted and Western blotting wasperformed according to Thomas et al. [15].

For Western blot analysis, the following primary antibodyconcentrationswere used: p21, 1:400 dilution of purifiedmousemonoclonal anti-p21 antibody (SX118; PharMingen, San Diego,CA); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (SC-304; Santa Cruz Biotechnology, Santa Cruz,CA); Myogenin, 1:200 dilution of purified rabbit polyclonal anti-Myogenin antibody (SC-576; Santa Cruz Biotechnology, SantaCruz, CA);Myosinheavy chain (MHC), 1:2000 dilutionof purifiedmousemonoclonal anti-MHCantibody (MF-20; gift fromDonaldFischman); Mighty, 1:2000 dilution of affinity purified rabbitpolyclonal anti-Mighty peptide antibody (QED Biosciences,USA); α-tubulin, 1:4000 dilution of purified mouse monoclonalanti-α-tubulin antibody (DM 1A; Sigma, St. Louis, MO) and V5,1:5000 dilution of the mouse monoclonal anti-V5 antibody(Invitrogen, Carlsbad, CA).

Northern blot analyses

To findout ifMightymRNAexpression is regulatedbyMyostatin,C2C12cellswereplatedat 25,000 cells/cm2and incubated for 24hin DMEM 10% FBS. The cells were differentiated in DMEM 2%HSwith orwithoutMyostatin for 72 h. Total RNAwas extracted andNorthern blotting was performed essentially as described bySambrook et al. [16]. TheMighty cDNAprobe containing the ORFwas amplified from a ∼650 bp BamHI and KpnI fragment of theIMAGE: clone 3498569 (Resgene, Invitrogen, Carlsbad, CA) usingthe forward primer 5′-CACCATGGCGTGCGGGGCGACACTG-3′and the reverse primer 5′-GGATACATAGCTTGTTGGCCT-3′.The PCR reaction was performed with PWO DNA polymerase(Roche Diagnostics GmbH, Roche Applied Sciences, Mannheim,Germany) and 1× Q solution.

For Northern blotting of IGF-II mRNA, mouse IGF-II cDNAfragment for the probe was amplified by PCR using the forwardprimer 5′-TGTTGGTGCTTCTCATCTCT-3′ and the reverse pri-mer 5′-TCACTGATGGTTGCTGGACA-3′which amplifies a cDNAfragment from nucleotides 111–628 of the mouse IGF-II mRNA

http://www.ncbi.nlm.nih.gov/BLAST

http://www.ebi.ac.uk/clustalw

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sequence (Genbank accession number BC058615). An α-tubulinprobewasamplified ina 35 cycle Taq (RocheDiagnosticsGmbH,Roche Applied Sciences, Mannheim, Germany) PCR using theforward primer 5′-GCTTCTTGGTTTTCCACAGC-3′ and thereverse primer 5′-CATGGTAGGCTTTCTCAGCA-3′.

Isolation of the Mighty promoter region

TheMighty 2.1 kb promoter (−1963 through +129)was amplifiedfrom mouse genomic DNA using Expand long PCR kit (RocheDiagnostics GmbH, Roche Applied Sciences, Mannheim, Ger-many) and 1× Q solution using the forward primer with a NheIrestriction site 5′-GCTAGCCCACATTCACTGTGCAAG-3′ and thereverse primer with a BglII restriction site 5′-AGATCTGATC-CAACTCTTCAGCTAG-3′. PCRswere performedwith 35 cycles of95 °C for 15 s, 52 °C for 30 s and 68 °C for 3 min, with a finalextension at 68 °C for 7 min. The resulting PCR product waspurified and cloned into the pGEMT-Easy vector (Promega,Madison, WI) and sequenced. The 1.1 kb promoter (−960through +129) was derived from the 2.1 kb promoter sequenceby ScaI and BglII restriction digestion, cloned into the SmaI andBglII sites of pGL3b (Promega, Madison, WI) to drive luciferaseexpression. The 0.287 kb (−158 through +129) promoterfragment was amplified using the primers 5′-GCTAGCTCCGG-CAGAGAGCGTGAAG-3′ and 5′-AGATCTGATCCAACTCTTCAGC-TAG-3′ and the PCR reaction was identical to that describedabove. The promoter fragmentwas subcloned into theNheI andBglII restriction sites of pGL3b to drive luciferase expression.

Inhibition of the Mighty promoter activity by Myostatin

Mighty promoter truncation 0.287 kb (2 μg) was cotransfectedwith control β-gal expression vector pCH110 (AmershamPharmacia, Piscataway, NJ) (1 μg) using Lipofectamine 2000(Invitrogen, Carlsbad, CA) into C2C12 cells (15,000 cells/cm2).After 24 h the cells were treated with 0, 2, 4, 6 and 8 μg/ml ofMyostatin as described previously [17]. After the treatment,cell lysates were made, luciferase assays were performed andthe results normalised to β-gal values [17].

For the rescue of Myostatin inhibition on Mighty promoterC2C12myoblasts were seeded into 24-well plates (Nalge Nunc,Rochester, NY) and transfected with 0.5 μg 0.287 kb Mightypromoter construct and 0.125 μg pCH110. After 24 h DMEM 5%FBS media containing either 3 μg/ml Myostatin or 3 μg/mlMyostatin with 40 μg/ml of Mstn-ant1 [14] was added to thecultures. Myostatin and Mstn-ant1 were incubated for 1 h onice prior to their addition to the cultures. At the end of thetreatment the cell lysates were made and assayed forluciferase and β-gal activities. The luciferase values werenormalised to β-gal values. Each transfectionwas performed aminimum of three times in triplicate.

Analysis of Myostatin signalling pathways

Dominant negative (dn) Activin receptor type II B (dnActRIIB),Activin receptor like kinase (dnALK5), Smad2 and Smad3constructs have been described previously [9,17,18]. Transfec-tions of C2C12 cells (15,000 cells/cm2) were performed using2 μg of the 1.1 kb Mighty promoter (−960 through +129), 1 μg ofpCH110 and 2 μg of either pcDNA3, dnActRIIB, dnALK5,

dnSmad2, dnSmad3 constructs according to Forbes et al. [17].Twenty four hours after transfection the cells were rinsedonce with DMEM without serum and then incubated withDMEM without serum for a further 24 h with or withoutMyostatin. At the end of the treatment cell lysates were made,luciferase assays were performed and luciferase activity wasnormalised to β-gal activity.

For MAPK and PI3K inhibitor use, C2C12 cells were firsttransfected with 1.1 kb Mighty promoter construct asdescribed above. After 24 h, the cells were pretreated with10 μM SB20350, 50 μM PD98059, 10 μM LY294002 or vehicle(DMSO) alone for 1 h before Myostatin treatment. Cells werethen treated with inhibitors with or without Myostatin for12 h. Cell lysates weremade, luciferase assayswere performedand values normalised to β-gal values. Each transfection wasperformed at least three times in triplicate.

Stable overexpression of Mighty in C2C12 myoblasts

The Mighty ORF was amplified from the IMAGE: clone 3498569(Resgene, Invitrogen, Carlsbad, CA) encoding the entire mouseMighty mRNA. The fragment was amplified using the forwardprimer 5′-CACCATGGCGTGCGGGGCGACACTG-3′ and thereverse primer 5′-GGATACATAGCTTGTTGGCCT-3′ and cloneddirectly into the pcDNA3.1D/V5-His-TOPO vector (Invitrogen,Carlsbad, CA). The PCRwas carried out using PWO polymerasewith 1× Q solution and an annealing temperature of 62 °C. Inorder to produce stable cell lines overexpressingMighty V5 Histagged protein or LacZ as a control, C2C12 cells wereelectroporated with the Mighty pcDNA3.1D/V5-His-TOPOplasmid or LacZ pcDNA3.1D/V5-His-TOPO plasmid essentiallyby the method of Grieshammer et al. [19] and seeded onto10 cm dishes (Nalge Nunc, Rochester, NY) at clonal density.Geneticin (Invitrogen, Carlsbad, CA) at 0.6 mg/ml was used toselect for resistant clones. Clones were subsequently isolatedand expanded. To confirm the overexpression phenotype, cellextracts were harvested and Western blotting was performedwith a 1:5000 dilution of the monoclonal mouse anti-V5antibody (Invitrogen, Carlsbad, CA).

BrdU incorporation

Mightyoverexpressingclones7and11orcontrol cell lineLacZorC2C12 were seeded at 15,000 cells/cm2 for actively growingconditions, or 25,000 cells/cm2 for differentiation conditions intriplicateonThermanoxcoverslips (NalgeNunc, Rochester,NY).Cellswere then treatedwith differentiationmedia for 0, 6 or 12hand then pulsed with 10 μM BrdU for 15 min. ICCs were thenperformed using 1:1000 dilution of mouse monoclonal anti-BrdU antibody (B2531, Sigma). BrdU positive nuclei werecounted as a percentage of total nuclei. At least 1000 nucleiwere counted per sample; averages were derived from triplicatewells.

Primary myoblast culture

Primary myoblasts were generated from day 90 semitendino-sus muscle tissue of foetuses from normal-muscled (NM) anddouble-muscled (DM) cattle according to Thomas et al. [15].The cells were seeded on 10 cm plates and grown in DMEM


10% FBS until approximately 70% confluent. The media waschanged to differentiation media (DMEM 2% HS) and the cellsharvested at 0, 6, 12, 24, and 48 h after the addition ofdifferentiation media for Western blotting.

Transfection of Mighty-siRNA in C2C12 myoblasts

Mighty-siRNA duplexes were procured from Qiagen (QiagenScientific, Germantown, MD). The target sequence for thesiRNA was CTG CAA ATA CGT GGT GAG AAA. C2C12 cells insix-well plates were transfected with 5 nM of siRNA or non-target siRNA using HiPerfect (Qiagen Scientific, Germantown,MD) transfecting agent according to the manufacturer's proto-col. The cells were harvested 48 h after the transfection. Theprotein lysatesweremadeandanalysedbyWesternblotting forMighty, MyoD, Myogenin, p21 and GAPDH expressions.

Radioimmunoassay for IGF-II

Radioimmunoassays (RIAs) for IGF-II were performed accord-ing to Hua et al. [20] to determine the amount of IGF-II secretedinto the media. Mighty overexpressing clones 7 and 11 andLacZ expressing control cells were plated at 25,000 cells/cm2

and left to attach for 24 h in DMEM 10% FBS at 37 °C, 5% CO2.Cells were then switched to differentiation media for 0, 24, 48,72 or 96 h. Conditioned media was then collected from cellsand the amount of IGF-II secreted from cells was measured byRIA.

Treatment of Mighty overexpressing clone with IGF-IIantibody

Mighty overexpressing clone 11 cells were seeded either ontoThermanox coverslips (Nalge Nunc, Rochester, NY), or directlyinto 24-well plates at a density of 25,000 cells/cm2. After a 24 hattachment period media was changed to differentiationmedia containing either 100 μg/ml of monoclonal antihumanIGF-II antibody (clone 75015, R & D Systems, Minneapolis, MN)or 100 μg/ml of monoclonal IgG1 isotype control antibody(clone 11711, R & D Systems, Minneapolis, MN). After 48 h ofincubation, cultures on the coverslips were fixed and subse-quently processed for MHC immunocytochemistry [15]. Thenumber of nuclei in MHC positive myotubes was counted.Three replicates were run for each treatment. Cultures grownin 24-well plates were lysed in protein lysis buffer andprocessed for MHC expression by Western blotting.

Real time PCR

RNA (1 μg) from wild-type and Myostatin-null muscle tissuewas reverse transcribed using Superscript II (Invitrogen,Carlsbad, CA). Real time PCRs for IGF-II and 28S were carriedout using a LightCycler 2.0 (Roche Diagnostics GmbH, RocheApplied Sciences, Mannheim, Germany). PCR amplificationwas performed with LightCycler Fast start SYBR Green DNAMastermix (Roche Diagnostics GmbH, Roche Applied Sciences,Mannheim, Germany) in triplicate. The sequences of theprimers and the annealing temperatures for IGF-II and 28Swere according to the methods of Armand et al. [21] andSimpson et al. [22] respectively.

Transfections of IGF-II promoter

The pIZB (Mouse 5.6 kb BamHI and KpnI fragment containingIGF-II P2andP3promoters and6kbH19enhancer linked toβ-galexpression) construct was a kind gift from Hiroyuki Sasaki [23].Two micrograms of pIZB and 0.25 μg pRL (Renilla luciferase,Promega, Madison, WI) per well were transfected into Mightyoverexpressing clone 11 andC2C12myoblasts (15,000 cells/cm2)in serum free medium (3 wells per transfection). After 24 h themedium was changed to DMEM 2% HS medium and the cellswere incubated for further 24 h. The cell lysates weremade andβ-gal activity was determined and normalised to renillaluciferase activity. The transfections were performed at leastthree times in triplicate.

Measurement of cell length, width and area

Differentiated myotubes (60–72 h in differentiation medium)were stained with Gill's haematoxylin and 1% eosin, and thecoverslips were permanently mounted in DPX mountant(BDH, Poole, Dorset, UK). Micrographs were taken using anOlympus BX50F microscope (Olympus Optical, PA) and a SpotRT™-KE slider camera (Diagnostics Instruments, StirlingHeights, MI). Myotubes were selected that contained 3 or 4myonuclei. The length, width and area of these myotubeswere determined by quantitative image analysis using theWindows version 4.0.1 SPOT Basic and Advanced software(Diagnostic Instruments, Stirling Heights, MI).

Measurement of the fusion index of differentiating cultures

Mighty overexpressing clones 7 and 11, LacZ and C2C12myoblast control cells were differentiated for 72 and 96 h inDMEM 2% HS. Cells were fixed and then stained with Gill'shaematoxylin. Coverslipswere thenmountedandmicrographswere taken as above. The percentage ofmyonuclei contained inmyotubes out of the total number of nuclei (Fusion Index) wascalculated. This was performed on triplicate experiments, withthree microscope fields counted per coverslip.

Isolation of reserve cell populations and RT-PCR for CD34

Reserve cellswere isolated from cultures of both normal C2C12cells and from stably-transfected cell lines expressing Mighty(clones 7 and 11) according to the method of Kitzmann et al.[24]. For semiquantitative RT-PCR for CD34, RNA was isolatedfrom the reserve cells and cDNA synthesised according to themanufacturer's protocol (Invitrogen, Carlsbad, CA). The num-ber of PCR cycles was standardised for the linear amplificationof truncated CD34. Truncated CD34 was PCR amplified using24 cycles with the primers (forward: 5′-AGCACAGAACTTCC-CAGCAA-3′ and reverse: 5′CCTCCACCATTCTCCGTGTA-3′) at94 °C for 20 s, 58 °C for 45 s, and 72 °C for 45 s. The finalelongation was for 5 min at 72 °C. The PCR products wereanalysed on an agarose gel and normalised to tubulin.

Cloning of Mighty cDNA into retroviral vector

The Mighty ORF was amplified from the IMAGE clone 3498569(Resgene, Invitrogen, Carlsbad, CA) encoding the entire mouse


Mighty mRNA. The PCR reaction was performed with PWODNA polymerase and 1× Q solution. Mighty cDNA was clonedinto the BglII and StuI sites of pLNCX2 viral vector (Clontech,Mountain View, CA).

Generation of Mighty overexpressing viral supernatant

RetroPack PT67 cells (Clontech, Mountain View, CA) at 80%confluence were electroporated with 10 μg of Mighty over-expressing viral vector Mighty-pLNCX2 or pLNCX2 emptyvector, at 0.24 kV, 960 μF using Gene Pulser (BioRad, USA).Individual clones were isolated and the supernatant collected,aliquoted and frozen at −80 °C for use in transduction assays.The viral titer was determined by infecting NIH 3T3 cells withserially diluted viral supernatants.

Transduction of cells with viral supernatant

C2C12 cells were plated at 15,000 cells/well in 4-well Permanoxculture slides (Invitrogen, Carlsbad, CA). The cells weretransduced with Mighty overexpressing clone supernatant orcontrol clone supernatant at 1:5 dilutions in DMEM containing8 μg/ml polybrene (Sigma). Twenty four hours after transduc-tion the medium was changed to differentiation medium andthe cells allowed to differentiate for 12 or 24 h. The cells werethen fixed and ICC for myosin heavy chain (MHC) wasperformed as described by Thomas et al. [15].

Injection of mdx mice with viral supernatant

Five- to seven-week old mdx mice were injected in the left m.tibialis anterior (TA) with 50 μl of Mighty overexpressingretroviral clone supernatant and in the right TA with control

Fig. 1 –Mighty mRNA expression is increased in the skeletalmuscle of Myostatin-null mice. (A) Reverse Northernanalysis. Dot blots of the Mighty cDNA sequence derivedfrom the SSH were probed with Myostatin-null (KO) orwild-type (WT) 32P radiolabelledm. biceps femoris (BF) cDNA.RT-PCR analysis of Mighty mRNA expression in BF ofwild-type mice and Myostatin-null mice shows an increasein Mighty mRNA levels in KO muscle. Levels of Mightytranscripts were normalised to α-tubulin mRNA. (B) RT-PCRof Mighty mRNA expression comparing wild-type (WT) andMyostatin-null (KO) tissues: liver, kidney and m. masseter(Mas).α-Tubulin RT-PCR is provided as a control. (C)Westernblots and corresponding graphs showing expression ofMighty,MyoD,Myogenin, p21, andMHCduringdifferentiationof C2C12 myoblasts. Maximum expression was termed 1.0and relative expression at various time points was plotted.Each point represents the relative mean±SE of at least threeseparate experiments. GAPDH proteinWestern blot isprovided to demonstrate even loadings. (D) Mighty mRNAexpression is negatively regulated by Myostatin indifferentiating C2C12 myoblast cultures. Northern blotanalysis of Mighty mRNA expression in 72 h differentiated(Diff) C2C12 myoblasts treated with or without Myostatin isshown and 28S rRNA level is shown as loading control.


clone supernatant (containing 8 μg/ml polybrene) on day 0 andday 7. The mice were sacrificed on day 14, the muscles werecollected and mounted in Tissue Tek O.C.T. and frozen inisopentane for histology. The muscle sections were stainedwith haematoxylin and eosin and the fiber size was deter-mined. To visualize the fibrosis in the muscle, the sectionswere stained with Van Geisen stain. The immunocytochem-istry for laminin was performed using rabbit anti-lamininantibody (DAKO) at 1:100 dilution according to the protocoldescribed in McCroskery et al. [11].

Statistical analysis

The results were analysed by ANOVA and/or student's t-testand a P value of ≤0.05 was considered significant.

Results

Mighty gene

The Mighty gene (accession number BC003291, cDNA cloneMGC:7743 IMAGE:3498569) was isolated as a novel downstreamtarget gene of Myostatin in a SSH. The mouse Mighty genecontains five exons and maps to chromosome 4D2.D. ThemouseMighty gene sequence encodes a 2271 bpmRNAwith anopen reading frame of 576 nucleotides. Reverse Northernblotting revealed that Mighty mRNA expression was ∼60%upregulated in muscle of Myostatin-null mice, indicating thatthe Mighty gene is negatively regulated by Myostatin (Fig. 1A).The Mighty protein sequence does not share a large degree of


homologywithanyprotein sequenceof known function.Resultsfrom the BLOCKS database showed that Mighty protein maycontaina forkheaddomain spanned fromaminoacids49–89andshowed homology to the following sequence: qmQTPPASLqQ-pAPPGSERRlpTPEQIfQnIKQEYnrYQRw where the bold capitalsindicate homologous amino acids.

Mighty mRNA is upregulated in Myostatin-null skeletalmuscle

Northern and RT-PCR analyses were performed to determinetheMightymRNAexpression indifferentmuscles ofMyostatin-null and wild-type RNA samples. Mighty expression appears tobe specifically regulated in different skeletal muscles by thepotent inhibitorofmuscle growth,Myostatin. The expressionofthe Mighty gene is increased in Myostatin-null m. bicepsfemoris (BF), m. masseter (Mas) (Figs. 1A and B), m. quadricepsfemoris, m. tibialis anterior (TA), m. gastrocnemius, and m.diaphragm over wild-type mice. Mighty expression was com-paratively low in skeletal muscle compared to other tissues;brain, testes, lung, kidney, intestine and liver (data not shown).However, an increase in Mighty gene expression in Myostatin-null skeletal muscle indicates that Myostatin specificallyregulates Mighty expression in skeletal muscle. No significantincrease in Mighty gene expression was seen in liver, kidney(Fig. 1B), testesandbrainbetweenwild-type andMyostatin-nulltissues. Thus theupregulationofMighty inMyostatin-nullmiceis restricted to skeletal muscle tissue.

Mighty expression precedes MyoD in the myogenic program

In order to assess Mighty gene expression with respect toknown myogenic genes the levels of Mighty protein in C2C12cells undergoing differentiation were quantified by Westernblotting. Fig. 1C shows that the peak expression of Mighty wasdetected at 12 h of differentiation. Mighty expression appears

Fig. 2 – Myostatin signalling to Mighty promoter. (A) Mighty promyoblasts were transiently transfected with the 0.287 kb (−158 ttreated with increasing concentrations of recombinant Myostatinnormalised to β-gal activity. Bars are mean±SE (n=6) and *** denMyostatin added). The experimentwas performed aminimumofrescues the inhibition of Mighty expression. C2C12 myoblasts wtreated with Myostatin in the absence or presence of Mstn-ant1.respect to the control (n=6). A minimum of three independent trfrom Myostatin alone-treated cells. (C) C2C12 myoblasts were trathrough +129) in the presence or absence of dnActRIIB or dnALK5The reporter luciferase activity was measured and normalised towas performed at least three times. *** denotes significance (Pbbbb0(D) C2C12 cells were transiently transfected with the 1.1 kb MighdnSmad3 following treatment with or without recombinant Myoβ-gal activity. Bars are mean±SE (n=6) and a minimum of threesignificance (Pbbbb0.05) from pcDNA3+Myostatin-treated cells. (E) MActivity of the 1.1 kb Mighty promoter construct in C2C12 cells inwithout recombinant Myostatin in differentiation medium was dindependent experiments were performed. *** denotes significancontrol+Myostatin-treated cells. (F) Mighty protein expression istagged Mighty protein expression detected by V5 antibody is enrclones 7 and 11. Sp1 protein levels show enrichment of nuclear

to peak much earlier than MyoD, Myogenin and p21 (Fig. 1C).After having confirmed the expression of Mighty in C2C12myoblasts, we wanted to determine the negative regulation ofMighty by Myostatin in these cells. Hence, C2C12 myoblastswere treated with recombinant Myostatin during both pro-liferation and differentiation and Mighty gene expression wasanalysed by Northern blotting. In both actively growing anddifferentiating (Fig. 1D) conditions Myostatin exposurereduced the expression of Mighty. Myostatin inhibited theexpression of the Mighty gene after 12 h in differentiationmedia (Fig. 1D) and this inhibition of expression was main-tained up to 72 h (data not shown). Myostatin inhibition ofMighty mRNA expression was not blocked in the presence ofcycloheximide (result not shown) indicating that de novoprotein synthesis is not required for Myostatin inhibition ofMighty mRNA expression.

Mighty expression is regulated at the transcriptional level byMyostatin

To determine if the Myostatin regulation of the Mighty geneoccurs at the transcriptional level the 0.287 kbMightypromoterfragment fused to the luciferase reporter gene was used intransfection assays. C2C12 cells were transfected with the0.287 kb Mighty promoter construct, and the following day,treated for 24 h with increasing concentrations of Myostatin.The results show that Myostatin induced downregulation ofthe Mighty promoter (Fig. 2A) confirming that Myostatinregulates Mighty gene activity, at least in part, by repressionof Mighty gene transcription. Furthermore, addition of aMyostatin antagonist Mstn-ant1 which is a truncated matureMyostatin [14] in the medium during Myostatin treatmentresulted in an increase in Mighty promoter activity (Fig. 2B).

Previously, Myostatin has been shown to signal via type IIreceptor ActRIIB, type I receptor ALK5, and Smad2/3 [9,25].dnActRIIB and dnALK5 receptors, and dnSmad2/3 signalling

moter activity is inhibited by Myostatin treatment. C2C12hrough +129) Mighty promoter construct. Cells were thenprotein. The reporter luciferase activity was determined andotes significance (Pbbbb0.001) when compared to control (0, nothree times. (B) TreatmentwithMyostatin antagonist partiallyere transfected with the 0.287 kb promoter construct andThe bars show the relative change in luciferase activity withansfections were performed, ** denotes significance (Pbbbb0.01)nsiently transfected with the 1.1 kb Mighty promoter (−960following treatment with or without recombinant Myostatin.β-gal activity. Bars are mean±SE (n=6) and each transfection.05) when compared to pcDNA3+Myostatin-treated cells.ty promoter in the presence or absence of dnSmad2 orstatin. Luciferase activity was measured and normalised toindependent transfections were carried out. *** denotesyostatin appears to signal via MEK to the Mighty promoter.the presence of SB20350, PD98059 and LY294002 with oretermined. Bars are mean±SE (n=6) and at least threece (Pbbbb0.001), ** denotes significance (Pbbbb0.01) fromenriched in nuclear extracts. A Western blot showing V5iched in nuclear extracts of C2C12 Mighty overexpressingextracts.


intermediates were used to investigate the mechanism ofMyostatin signalling to theMighty promoter. Cotransfection ofthe 1.1 kbMighty promoter constructwith either the dnActRIIB

Fig. 3 – Mighty overexpressing clones show an enhanced differedifferentiation markers. (A) Micrographs showing Mighty overex72 h of culture in differentiation media. Cells were immunostainhaematoxylin (20X, scale bar=25 μm). (B) Mighty overexpressioncells. C2C12 cells were transduced with Mighty expressing viraldifferentiate. MHC immunostaining was performed on the cellspercentage of MHC positive myotubes which were significantly hcontrol-treated (n=9, * Pbbbb0.01). A minimum of three independentlevels of p21, MyoD,Myogenin, andMHC protein inMighty overexcultured in differentiation media for 0, 3, 6, 12, 24, 48, 72, and 96 hby their respective antibodies. Tubulin protein levels, detected bloadings. (D) BrdU incorporation of Mighty overexpressing clonemyonuclei incorporating BrdU of total myonuclei, as detected bywere counterstainedwith Gill's haematoxylin to visualize non-Br** or $$ Pbbbb0.01, * or $ Pbbbb0.05 as compared to C2C12 (*) or LacZ ($)

or dnALK5 receptor or dnSmad2 or dnSmad3 rescued thelevels of promoter expression in the presence of exogenousMyostatin (Figs. 2C and D). The levels of Mighty promoter

ntiation phenotype and accelerated expression of myogenicpressing clones 7 and 11, and control C2C12 cells after 0 anded using anti-MHC antibody and counterstained with Gill'sby transduction leads to enhanced differentiation in C2C12

supernatant or control supernatant and allowed toafter 12 h in the differentiation media. The graph shows theigher in the Mighty-treated cells compared to theexperiments were performed. (C) Western blots showing thepressing clone 11 compared to C2C12 control cells. Cells were. Expression of p21, MyoD, Myogenin and MHCwas detectedy anti-tubulin antibodies, are included to demonstrate evens during myogenic differentiation. The percentage ofimmunostaining with anti-BrdU antibodies, is shown. CellsdU labelled nuclei. Bars aremean±SE (n=3). *** or $$$ Pbbbb0.001,values respectively.


expression in the absence of Myostatin treatment were alsoincreased in the presence of dominant negative signallingintermediates likely due to the inhibition of endogenousMyostatin signalling. In addition to the Smad pathway,Myostatin also signals through the ERK1/2 MAPK pathway[26]. Mighty 1.1 kb promoter construct was used to investigatethe effect of MEK MAPK inhibitors on Myostatin signalling tothe Mighty promoter (Fig. 2E). The results show that Mightypromoter activity is repressed by SB20350 and LY294002,inhibitors of p38 mitogen activated protein kinase and PI3Krespectively, in the presence or absence of exogenous Myos-tatin. In the presence of PD98059, but not other inhibitors, analmost 2-fold induction of Mighty promoter activity wasobserved in the presence of exogenous Myostatin. In theabsence of Myostatin a similar fold induction was seen,presumably due to the inhibition of endogenous Myostatin

Fig. 4 – Western blot showing Mighty (A), MyoD (B), Myogenin (Cand double-muscled (DM) bovine myoblasts during differentiatioexpression at various time points was plotted. Each point represprovided to demonstrate even loadings.

signalling (Fig. 2E). Furthermore, treatment of C2C12 cells withMyostatin resulted in the phosphorylation of MEK after 15minof treatment (data not shown).

Mighty overexpression enhances myogenic differentiation

In order to study the possible function of Mighty duringmyogenesis, two independent clones (clones 7 and 11) thatstablyoverexpressedMightywereselected (Fig. 2F). Clones7and11were induced todifferentiate and theextent ofdifferentiationwas determined by MHC staining which was visible at 48 h inboth Mighty overexpressing clones and C2C12 cell cultures. By72 h the degree of differentiation was far greater in clones 7 and11 than in control cells (Fig. 3A). Mighty overexpressing clonestypically form short, thick myotubes, which, as differentiationprogresses, can become rounded and contain a large number of

), and p21 (D) expressions in primary normal-muscled (NM)n. Maximum expression was termed 1.0 and relativeents the relative mean±SE (n=3). Tubulin protein levels are

Fig. 5 – Mighty (A), MyoD (B), Myogenin (C), and p21 (D) expressions in differentiating C2C12 myoblasts treated withMighty-siRNA. Western blots and corresponding graphs of specific protein expression in C2C12 myoblasts treated withMighty-siRNA compared to cells treated with non-target siRNA during differentiation. Control expression was termed 100 andrelative expression of Mighty-siRNA-treated cells was plotted. Each point represents the relative mean±SE of at least threeseparate experiments. ** Pbbbb0.01, * Pbbbb0.05 denote significance when compared to non-target siRNA. GAPDH protein levels areprovided to demonstrate even loadings.


nuclei. Similarly, Mighty overexpression in C2C12 myoblastsusing retroviral vector also resulted in an enhanced differentia-tion phenotype (Fig. 3B).

Fig. 6 – Effect of Mighty on IGF-II expression. (A) IGF-II mRNA exclones. Northern blot analysis comparing IGF-II expression inMig72, and 96 h of differentiation. The twomurine isoforms of IGF-IIprotein in conditioned media from Mighty overexpressing clonedifferentiation for 24, 48, 72, and 96 h in differentiation media. Basignificance when compared to the conditioned media from LacZexpression of MHC. i) Western blot showing MHC protein level itreatment. The bars show the densitometry analysis of the relativwith IGF-II antibody or control antibody in the differentiation meperformed and the number of nuclei in MHC immunostained celPCR analyses of IGF-II demonstrate an increase in IGF-II mRNA inMyostatin-null mice (n=6). *** denotes significance (Pbbbb0.001) fromtransiently cotransfected with the IGF-II promoter/enhancer andincubated in differentiation medium for 24 h, then β-gal activityactivity. The bars represent the mean±SE (n=9) and at least thresignificance (Pbbbb0.001) when compared to C2C12 cells.

To further characterize the enhanced differentiation phe-notype seen inMighty overexpressing clones, the expression ofmyogenic differentiation markers p21, MyoD, Myogenin and

pression is grossly upregulated in Mighty overexpressinghty overexpressing clones and control C2C12 cells at 0, 24, 48,are visible as a 3.6 kb and a 1.2 kb mRNA transcripts. (B) IGF-IIs 7 and 11 and control LacZ expressing cells, followingrs are mean±SE (n=3). *** Pbbbb0.001, ** Pbbbb0.01, * Pbbbb0.05 denoteexpressing cells. (C) IGF-II antibody treatment decreases the

n Mighty overexpressing cells (clone 11) after IGF-II antibodyeMHC protein levels in the cells. ii) Clone 11 cells were treateddia and allowed to differentiate for 48 h. The ICC for MHC wasls was counted and compared with the control. (D) Real timethe m. biceps femoris (BF) and m. masseter (Mas) muscle ofthe wild-typemuscle. (E) C2C12 and clone 11myoblasts wererenilla luciferase expression construct pRL. Cells werewas determined and values normalised to renilla luciferasee independent transfections were performed. *** denotes


MHC was investigated during the process of myogenic differ-entiation (Fig. 3C). Higher levels of p21 expression were seen inclone 7 and 11 immediately after switching to differentiation

medium (0h) and the elevated levelsweremaintained at 24h inthis medium. Similarly, MyoD expression is also elevated inclone 7 and clone 11 as compared to control cells throughout


differentiation. The expression of Myogenin was detectedconsiderably earlier at 24 h in differentiationmedium in clones7 and 11 compared to control cells, where the expression wasdetected at 48 h. Furthermore, increased levels of Myogeninwere observed at all the time points tested during differentia-tion. Consistent with earlier differentiation, higher levels ofMHC expression were observed in clones 7 and 11 noticeablyearlier than in control cells (Fig. 3C). Thus the gene expressionanalyses confirm that ectopic expression of Mighty leads toaccelerated differentiation of myoblasts.

In order to examine whether accelerated differentiation ofMighty overexpressing clones is due to early withdrawal fromthe cell cycle, BrdU labelling of cells was utilized. Underactively growing conditions about 40% of cells in all four celllines (clone 7, 11, LacZ, C2C12) incorporate BrdU (Fig. 3D). Thisindicates that all cells show similar cell cycle dynamics inproliferating conditions. Immediately after switching todifferentiation medium (0 h) a significantly lower number ofclone 7 and 11 cells incorporate BrdU as compared to controlcell lines. A similar decrease can be seen in clones 7 and 11 at12 h of differentiation indicating that overexpression of theMighty gene effectively predisposes cells to rapidly withdrawfrom the cell cycle.

Myoblasts derived from double-muscled cattle (DM)express non-functional Myostatin, and have been shown tohave increased levels ofMighty (Fig. 4A). To further confirm thepromyogenic activity of Mighty, we monitored the differentia-tion of these DMmyoblasts and compared to that ofmyoblastscultured from normal-muscled cattle (NM). The results indi-cate that there is accelerated differentiation in the cultures ofDM myoblasts as compared to the NM myoblasts. Consistentwith the enhanced differentiation, increased expression isseen for Mighty, MyoD, Myogenin, and p21 in the DM cellscompared to theNMcells (Fig. 4). The largest increases are seenwithMyoDandp21which also peak earlier in theDMcells thanthe NM cells. Both Mighty and Myogenin show the samegeneral pattern of expression between the NM and DM cells,butwithhigher levels seen in theDMcompared to theNMcells.

The above mentioned results clearly show that high levelsof Mighty expression lead to upregulation of MyoD (Fig. 4B). Tofurther confirm that this is indeed the case, we downregulatedMighty expression by siRNA. Fig. 5 shows the knockdown ofMighty protein expression byMighty-siRNA. The expression ofMighty in C2C12 myoblasts transfected with Mighty-siRNAwas reduced by approximately 67% compared to cells trans-fected with non-target siRNA (Fig. 5A). A reduction in MyoDprotein expression by approximately 30% (Fig. 5B) and anapproximate 38% reduction in Myogenin (Fig. 5C) expressionby Mighty-siRNA were observed. The expression of p21 wasalso reduced by approximately 32% (Fig. 5D) by Mighty-siRNA.These results further confirm that Mighty regulates expres-sion of MyoD and its downstream target genes.

IGF-II is upregulated in Mighty overexpressing differentiatingC2C12 Cells

IGF-II has been shown to enhance the differentiation ofmyoblasts [27–29]. Interestingly, Myostatin-null mice havehigher levels of IGF-II as compared to wild-type mice (Fig. 6D)thus IGF-II expression was analysed in Mighty overexpressing

clones. Analysis of the earliest time point (0 h) when the cellswere switched to the differentiation medium showed that the3.6 kb isoform of IGF-II mRNA was ∼10-fold upregulated inMighty overexpressing clones 7 and 11 compared to wild-typeC2C12 cells, while the levels of 1.2 kb isoform of IGF-II werebelow the limit of detection in all cell lines (Fig. 6A). Duringmyogenic differentiation IGF-II mRNA expression increased inboth control and Mighty overexpressing cells, however theexpression of 3.6 kb isoform and the 1.2 kb isoform of IGF-IImRNAwas consistently higher inMighty overexpressing clonescompared to control C2C12 cells (Fig. 6A). Densitometricanalysis of Northern blots showed that after 24 h in differentia-tion media, Mighty overexpressing clones have a ∼50-foldincrease in the expression of IGF-II compared to control cells,∼10-fold at 48 and 72 h, and ∼4-fold by 96 h. RIA was used todetermine the amount of IGF-II in the media of Mightyoverexpressing clones and control cells (Fig. 6B). IGF-II proteinis increased in Mighty overexpressing clones after 48 h indifferentiation medium by 2.9-fold and 3.5-fold in clone 7 andclone11 respectivelywhencompared toLacZexpressingcontrolcells. LacZ expressing control cells also show an increase in IGF-II proteinwhen compared toun-transfectedC2C12 cells. At 96h,significantly more IGF-II was still present in the medium fromMighty overexpressing clones compared to the medium fromcontrol cells. Addition of IGF-II antibody to the differentiationmedium of clone 11 resulted in a significant reduction in thelevels of MHC protein (Fig. 6C i), and in the number of nuclei indifferentiated myotubes at 48 h in the differentiation medium(Fig. 6C ii). To determine if an increase in IGF-II is due toincreased IGF-II mRNA stability, C2C12 control cells and Mightyoverexpressing clones 7 and 11 were treated with the RNAsynthesis inhibitor Actinomycin D. IGF-II mRNA expressionfrom all three cell lines remained stable for over 12 h (data notshown) indicating that upregulation of IGF-II mRNA in Mightyoverexpressing clones is not due to enhanced mRNA stability.Due to the fact that Mighty overexpressing clones showed nodifference in IGF-II mRNA stability compared to control cells,transcription regulation of IGF-II by Mighty was investigated.Transient transfection of the IGF-II promoter/enhancer β-galreporter construct in Mighty overexpressing clone 11 led to asignificant increase in the IGF-II promoter activity (Fig. 6E).

Mighty overexpression in C2C12 induces hypertrophy andfusion

Quantitative image analysis and flow cytometry using forwardangle light scatter of actively growing Mighty overexpressingclones revealed that cell size of clones 7 and 11was larger thancontrol cells (data not shown). Similarly, Mighty overexpres-sing clones after 72 h in differentiation media also appearhypertrophied as compared to control cells. Quantitativeimage analysis of myotubes containing 3 nuclei showed thatMighty overexpressing clones 7 and 11 demonstrated a ∼2-fold increase in area (Fig. 7A), width (1.7-fold) and length (1.5-fold) over control cells. After 72 and 96 h in differentiationmedia the fusion index (percentage of total nuclei that areincorporated in multinucleated myotubes) of Mighty over-expressing clones 7 and 11 was compared to that of controlcells. Mighty overexpressing clones 7 and 11 show a 2–3-foldincrease at 72 h (Fig. 7B) and a ∼4-fold increase at 96 h (data


not shown), respectively, in fusion index over LacZ controlcells. In addition to an increased fusion index Mighty over-expressing clones 7 and 11, show a 2–3-fold increase inmyonuclei number at 72 h and a 4-fold increase at 96 h overcontrol cells (Fig. 7C). During differentiation of primarycultures or C2C12 cells a small subpopulation of myoblastsremains quiescent but is capable of self renewal anddifferentiation. This subpopulation is known as reserve cellsand expresses CD34, a marker of satellite cells, differentially[24,30]. The full-length transcript of CD34 is expressed inactivated satellite cells while the truncated form is expressedin quiescent satellite cells. Reserve cells were isolated fromC2C12 and Mighty clones and analysed for CD34 expression.

Fig. 7 – Mighty overexpression leads to hypertrophy and increashypertrophied. Morphometric analysis of myotube area (only myoverexpressing clones by quantitative image analysis shows thatare mean±SE of three microscope fields per culture on three indC2C12 control cells. (B) Mighty overexpressing clones show an innuclei per myotube was determined by counting three microscorepresent themean±SE. *** denotes significance (Pbbbb0.001) from Lashow an increased fusion index. Mighty overexpressing clones 7differentiated for 72 and 96 h. Cells were stained with Gill's haemcontaining NNNN2 nuclei per myotube, divided by the total number oper culture on three independent cultures. The bars represent thexpressing control cells). (D) CD34 expression is downregulated ifor CD34 was performed on the RNA from the reserve cells. The gCD34 with respect to C2C12 reserve cells normalised to tubulin eindicates significance when compared to C2C12 reserve cells.

The overall levels of truncated CD34 were dramaticallyreduced in the reserve cells of the two Mighty overexpressionclones as compared to C2C12 reserve cells (Fig. 7D).

Mighty overexpression in vivo increases fiber size

To determine if Mighty overexpression in vivo has the samephenotype as seen in C2C12 myoblasts, mdx mice wereinjected with the viral supernatant of the Mighty expressingclone or empty vector and TAmuscles were collected after thetreatment. As shown in Fig. 8A, TA muscles injected withMighty expressing clones weighed significantly more thancontrol-treated contra-lateral TA muscles.

ed fusion. (A) Mighty overexpressing myotubes areotubes with 3 nuclei were measured) of Mightyclone 7 and 11 derivedmyotubes are larger than control. Dataependent cultures. *** denotes significance (Pbbbb0.001) fromcrease in number of myonuclei per myotube. The number ofpe fields per culture on three independent cultures. The barscZ expressing control cells. (C) Mighty overexpressing clonesand 11, and control cell lines LacZ and C2C12 wereatoxylin. Fusion index (number of nuclei within myotubes

f nuclei) was determined by counting three microscope fieldse mean±SE (*Pbbbb0.05, ** Pbbbb0.01, *** Pbbbb0.001 compared to LacZn the reserve cells of Mighty clones. Semiquantitative RT-PCRraph represents relative percent change in the expression ofxpression. The values are the mean±SE (n=4), *** Pbbbb0.001

Fig. 8 – Mighty overexpression increases muscle fiber size in mdx mice. (A) Muscle weights of mdx mice treated with Mighty.Treatment of mdxmicewithMighty expressing viral vector shows an increase inweights of the treated TAmuscle (n=10). Dataare expressed asmean±SE. * Pbbbb0.05 indicates level of significance when compared to control-treated TAmuscle. (B) Treatmentof mdx mice with Mighty expressing viral vector shows an increase in fiber size. Average mode (y axis) represents the mostfrequent occurring value of fiber size for each treatment in the TA muscles of mdx mice. The results are an average of sixanimals per treatment (mean±SE). * Pbbbb0.05 indicates level of significance from control-treated muscle. (C) Treatmentwith Mighty ameliorates the mdx phenotype. Van Geisen staining shows the fibrosis in the muscle of mdx muscle (control)(4X, scale bar=400 μm). The arrows indicate the damaged areas in the mdx muscle (control) and arrowhead indicates thesmall diameter fibers (n=6). Laminin ICC of TA muscle (10X, scale bar=500 μm) shows mdx muscle treated with Mighty hasmore well-defined muscle fibers and reduced muscle damage (n=6).


The muscle sections were stained with haematoxylin andeosin and the size of the muscle fibers determined. Fig. 8Bshows that expression of Mighty in the muscle results in anincrease in the number of larger fibers as compared to thecontrols. In general the fibers of Mighty-treatedmice appearedhealthier and better defined (Fig. 8C). Also, a decrease in thefibrosis, detected by Van Geisen staining, was observed in themuscles of the Mighty injected animals as compared to thecontrols (Fig. 8C) indicating a reduction in degeneration.

Discussion

During myogenesis, fusion competent myoblasts undergopostmitotic differentiation to form myotubes. In this commu-

nication, we report the identification and characterization of anovel gene Mighty and its involvement in key signallingpathways controlling the specification of postmitotic differen-tiation of myoblasts and subsequent fusion to formmyotubes.

Alignment of Mighty protein sequence with all knownproteins revealed that Mighty does not have a particularlyhigh homology with any well characterized protein domains.However, there is some homology between Mighty andforkhead domains listed in the NCBI protein domain data-bases (smart00339.10, FH; pfam00250.11, Fork_head; andcd00059.2, FH). There is a great deal of variation within theforkhead domains of known forkhead transcription factorsand given the small size of theMighty protein, it is possible forMighty protein to form a forkhead domain structure similar tothose present in DNA binding proteins, E2F4 and DP2 [31]. The


functional analysis presented here does indicate that Mightyhas an effect on the transcription of certain genes. Indeed thisnotion is further supported by the presence of a nuclearlocalisation sequence in the Mighty protein and its nuclearand cytoplasmic localisations (Fig. 2F). Whether Mighty is atranscription factor or a co-factor remains to be elucidated.

Expressionanalysis shows that theMightygene is expressedin many tissues. However, regulation of Mighty expression byMyostatin is seen only in skeletal muscle which may be due tothe predominant expression of Myostatin in skeletal muscleand the autocrine fashion in which Myostatin functions. Thisimplicates Mighty as playing a role in skeletal muscle down-stream of Myostatin in the signalling pathway. This is furthersupported by the observation that exposure to increased levelsof Myostatin downregulated Mighty in both proliferating anddifferentiating C2C12 cells. This downregulation is a directeffect of Myostatin on Mighty transcription as cycloheximidetreatmentdoesnot preventMyostatin inhibitionofMightygeneexpression and Myostatin treatment inhibits expression fromthe Mighty promoter. Furthermore, this inhibition is partiallyrescued by the antagonistic effect of Mstn-ant1 on Myostatinactivity (Fig. 2B). Myostatin appears to signal to theMighty geneby the previously characterized Smad signalling pathway[9,17,25]. Myostatin is able to inhibit luciferase activity of the1.1 kb Mighty promoter construct and the truncations thereofindicating that the elements responding toMyostatin signallingmay be present within 200 bp of Mighty upstream sequences(Fig. 2A, C andD). Sequence analysis ofMighty upstream regionrevealed the presence of one CAGA box at position −81. Smad3and Smad4 complexes have been shown to bind to CAGA boxesin the promoters of TGF-β and Activin downstream genes [32].Furthermore, Smad3 and Smad4 complexes can represstranscription depending on their associated factors [33]. Theexpression ofmyc is downregulated in response to TGF-βwhena complex containing the transcription factors E2F4/5, DP1 andthe corepressor p107 and Smad3 and 4 assemble on theadjacent Smad and E2F sites [33]. The binding of Smad3 and 4and the nature of the repression complex on CAGA box of theMighty promoter needs to be characterized.

Myostatin also inhibitsMighty expression throughMEK/ERKsignalling pathway (Fig. 2E). Recently, Myostatin has beenshown to stimulate ERK1/2 phosphorylation resulting in down-regulation ofMyogenin expression and thus differentiation in aMEK dependent manner [26]. Considering these results, wepropose that the effects of Myostatin levels on Mighty genetranscription may be mediated by more than one mechanism.Interestingly, upon inhibition of p38 MAPK or PI3K pathways arepression of Mighty promoter activity is observed indicatingthat both p38 MAPK and PI3K may have a positive effect onMighty transcription. The muscle specific bHLH transcriptionfactors, MyoD, Myogenin, MRF4 and Myf5 are considered to bethe master regulators of myogenesis [34,35]. These myogenicfactors further activate genes that are required for muscledetermination and/or differentiation. During the course ofmuscle differentiation MyoD is known to upregulate p21 [36]and Myogenin gene expression. p21 inhibits progressionthrough the cell cycle [37] and Myogenin expression leads themyoblasts into terminal differentiation [38]. Results presentedhere show that Mighty protein is expressed in proliferatingmyoblasts but upregulated during differentiation. During

myoblast differentiation the peak expression of Mighty isobserved considerably earlier thanMyoD in C2C12 and primarybovinemyoblasts, indicating thatMighty functionsupstreamofMyoD. Consistent with this prediction, the knockdown ofMighty gene by RNAi in C2C12 myoblasts also reduced theexpression of MyoD. Subsequent reductions were also seen inthe levels of Myogenin and p21 indicating that the knockdownof Mighty decreases myoblast differentiation. Furthermore,Mighty overexpressing clones withdraw from the cell cycleearlier than control C2C12 cells, showaccelerated expression ofdifferentiation markers p21, MyoD, Myogenin and MHC anddifferentiate faster than control cells. This results in Mightyoverexpressingclones formingmultinucleatedMHCexpressingmyotubes considerably earlier than control cells.

Similarly, higher levels of Mighty in DM myoblastscorrespond with increased levels of MyoD, Myogenin, andp21, indicating that Mighty overexpression in thesemyoblastsmimics the enhanced differentiation phenotype seen in DMmyoblasts. Given that higher levels of Mighty are present inG1/S in NMmyoblasts (data not shown) and overexpression ofMighty increases the withdrawal of myoblasts from the cellcycle, we propose that Mighty is involved in the recruitment ofmyoblasts into the differentiation pathway.

A similar enhanced differentiation phenotype to that ofMighty overexpressing clones is seen in differentiating culturestreatedwith IGF-II. The formation ofmyotubes is accelerated inthe presence of IGF-II during myogenic differentiation[28,39,40], accompanied by the expression of myogenic mar-kers, Myogenin and MHC [28,41]. Our results show that Mightyoverexpression in C2C12 myoblasts induces the expression ofIGF-II mRNA and secreted protein expression is upregulated inMighty overexpressing clones during the process of myogenicdifferentiation. An increase in the IGF-II protein was alsoobserved in the LacZ control cell line as compared to un-transfected C2C12 cells (Fig. 6B). The LacZ control cells are apool ofmixed clones and therefore, the stable integration of Lacgene could be at several loci of the C2C12 genome. Therefore,the observed increased levels of IGF-II in LacZ control cellscould be due to site of integrations of LacZ cassette. It isnoteworthy however, that two independent clonal cell linesthat ectopically express Mighty, have increased expression ofIGF-II which is not only higher as compared to un-transfectedC2C12 cells but also significantly higher than the stablyintegrated LacZ control cells. Both the major IGF-II transcriptsof 3.6 kb and 1.2 kb which arise from the use of alternate pro-moters [27] were upregulated in Mighty overexpressing clones(Fig. 6A). Corroborating these results, higher levels of IGF-IIwerealso seen in the muscles of Myostatin-null mice (Fig. 6D).Mighty enhanced IGF-II mRNA expression is most likely by theupregulation of IGF-II gene transcription since transienttransfection of the IGF-II-promoter–reporter with a Mightyover expressing plasmid resulted in upregulation of the IGF-IIpromoter activity in thepresenceof anHDAC inhibitor (datanotshown). A significant increase in IGF-II promoter activity wasalso seen when IGF-II promoter–reporter plasmid was trans-fected inMighty overexpressing clone 11 (stable) in the absenceof HDAC inhibitor (Fig. 6E). At present, the DNA binding prop-erties ofMighty are not known, thus characterization ofMightyasa factoror a co-factor that can regulategeneexpressionat thegenetic and/or epigenetic level remains to be elucidated.


In keeping with high levels of IGF-II, the downstreamsignalling molecule phosphorylated Akt was also elevated inMighty overexpressing clones (data not shown). An increase inthe expression of phospho-Akt Ser473 has been associatedwith an increase in IGF-II signalling, confirming that signallingwas occurring via the PI3K pathway [28,41]. Akt has beenfound to stimulate MyoD and MEF2 indirectly and subse-quently Myogenin promoter activity during muscle differen-tiation [42,43]. Consistent with these results, we observed anoverall increase in the mean number of nuclei per myotubeand size of Mighty overexpressing C2C12 myotubes in vitro.Furthermore, an increase in the myofiber size and a decreasein fibrosis in the muscle were noted in Mighty-treated mdxmice (Figs. 8B and C) confirming enhanced myogenic activityin Mighty-treated mice.

Induction of hypertrophy in vivo may be due to increasedactivation of quiescent satellite cells and increased fusion ofmyonuclei derived from the myoblasts. Indeed this mechan-ism is also supported in Mighty gain of function C2C12 cells.An increase in the fusion index of Mighty overexpressingmyoblasts indicates that a larger proportion of cells formmyotubes; this is confirmed by a decrease in the expression ofCD34 (quiescent form) in the reserve cell population of Mightyclones (Fig. 7D). The quiescent reserve cells are heterogeneousfor CD34 expression, thus exhibiting satellite cell character-istics and CD34− reserve cells are considered to be fusioncompetent [44]. By this analogy Mighty, by downregulatingCD34, could be rendering the satellite cell/reserve cell fusioncompetent. Previous studies have shown that IGF-I treatmentin vitro can also lead to hypertrophy as a consequence ofenhanced protein synthesis and increased fusion index ofhuman myotubes [45].

In conclusion, the results presented here indicate thatMighty occupies a key role in the signalling cascade betweenextra-cellular Myostatin, and the set of downstream intra-cellular transcription factors that govern skeletal myogenesis.Additionally, Mighty overexpression appears to improvemuscle regeneration in mdx mice.

Acknowledgments

We are grateful to Dr. Hiroyuki Sasaki for the IGF-II promoterconstructs and Dr. Serhiy Souchelnytskyi and Dr. Lisa Choy forthe dnSmad2 and Smad3-F constructs respectively. Thesupport of Orico Ltd. for funding is also acknowledged. Ourthanks to Craig McFarlane, An-chi Tsuei, Leanne Platt andother members of the Functional Muscle Genomics team also.

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Mighty is a novel promyogenic factor in skeletal myogenesis

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