Serum proteomic profiling reveals fragments of MYOM3 as potential biomarkers for monitoring the outcome of therapeutic interventions in muscular dystrophies

ORIGINAL ARTICLE Se um p o eomic p ofiling e eals agmen s o MYOM3 as po en ial bioma ke s o moni o ing he ou come o he apeu ic in e en ions in muscula dys ophies Jé émy Rouillon1,†, Jé ôme Poupio 1,†, Aleksanda Zoce ic1,†, Fa ima Amo 1, Thibau Lége 2, Camille Ga cia2, Jean-Michel Camad o2, B enda Wong3, Robin Pinilla1, Jé émie Cose e1, Anna M.L. Coenen-S ass4, G aham Mcclo ey4, Thomas C. Robe s4,5, Ma hew J.A. Wood4, Lau en Se ais6, Bja ne Udd7, Thomas Voi 8,9, Isabelle Richa d10 and Fedo S ina chouk1,* 1 Géné hon, E y, F ance, 2 Mass spec ome y Labo a o y, Ins i u Jacques Monod, UMR 7592, Uni e si y Pa is Dide o , CNRS, So bonne Pa is Ci é, F-75205 Pa is, F ance, 3 Di ision o Pedia ic Neu ology, Cincinna i Child en’s Hospi al Medical Cen e , Cincinna i, OH, USA, 4 Depa men o Physiology, Ana omy and Gene ics Ox o d, Ox o d, OX1 3QX, UK, 5 Depa men o Molecula and Expe imen al Medicine, The Sc ipps Resea ch Ins i u e, La Jolla, CA, USA, 6 Se ice o Clinical T ials and Da abases, Ins i u de Myologie, Pa is, F ance, 7 Folkhälsan Ins i u e o Gene ics and Depa men o Medical Gene ics, Haa man Ins i u e, Uni e si y o Helsinki, Helsinki, Finland, 8 UPMC Inse m, UMRS 974, CNRS FRE 3617, Pa is, F ance, 9 Uni e si é Pie e e Ma ie Cu ie- Pa is 6, Ins i u de Myologie, GH Pi ié-Salpê iè e, Pa is, F ance and 10 Gene hon, CNRS UMR 8587, E y, F ance *To whom co espondence should be add essed a : Géné hon, 1 bis ue de l′In e na ionale, 91002, E y, F ance. Tel: +33 169472535; Fax: +33 169472838; Email: [email p o ec ed] Abs ac The apy- esponsi e bioma ke s a e an impo an and unme need in he muscula dys ophy field whe e new ea men s a e cu en ly in clinical ials. By using a comp ehensi e high- esolu ion mass spec ome y app oach and wes e n blo alida ion, we ound ha wo agmen s o he myofib illa s uc u al p o ein myomesin-3 (MYOM3) a e abno mally p esen in se a o Duchenne muscula dys ophy (DMD) pa ien s, limb-gi dlemuscula dys ophy ype 2D (LGMD2D) and hei espec i e animal models. Le els o MYOM3 agmen s we e assayed in he apeu ic model sys ems: (1) es o a ion o dys ophin exp ession by an isense oligonucleo ide-media ed exon-skipping in mdx mice and (2) s able es o a ion o α-sa coglycan exp ession in KO-SGCA mice by sys emicinjec iono a i al ec o .Followingadminis a iono he he apeu icagen sMYOM3was es o ed owa dwild- ypele els. In heLGMDmodel,whe edi e en doseso ec o we eused,MYOM3 es o a ionwasdose-dependen .MYOM3 agmen sshowed lowe in e -indi idual a iabili y compa ed wi h he commonly used c ea ine kinase assay, and co ela ed be e wi h he es o a ion o he dys ophin-associa ed p o ein complex and muscle o ce. These da a sugges ha he MYOM3 agmen s hold p omise o minimally in asi e assessmen o expe imen al he apies o DMD and o he neu omuscula diso de s. † J. Rouillon, J. Poupio and A. Zoce ic con ibu ed equally o his wo k. Recei ed: Ap il 24, 2015. Re ised and Accep ed: June 4, 2015 © The Au ho 2015. Published by Ox o d Uni e si y P ess. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Human Molecula Gene ics, 2015, Vol. 24, No. 17 4916–4932 doi: 10.1093/hmg/dd 214 Ad ance Access Publica ion Da e: 9 June 2015 O iginal A icle 4916 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om In oduc ion The dys ophin-associa ed p o ein complex (DAPC) consis s o se e al ansmemb ane and in acellula sca olding elemen s implica ed in main aining he s uc u e and mo phology o e eb a e muscle fib es. Loss-o - unc ion mu a ions in genes encoding hese p o eins gi e ise o di e en o ms o muscula dys ophy. The absence o unc ional dys ophin o sa cogly- cans in he DAPC is accompanied by a s ong des abiliza ion o he complex a he sa colemma (1). As a consequence, muscle fib es become mo e sensi i e o mechanical damage leading o muscle degene a ion, ch onic inflamma ion and an inc ease in fib osis—hallma ks o he dys ophic pheno ype (2). The mos p e alen and se e e disease is Duchenne muscula dys ophy (DMD), an X-linked diso de caused by mu a ions in he dys- ophin gene, wi h a wo ld-wide incidence o 1/5000 male new- bo ns. DMD pa ien s usually lose he abili y o walk a ound he ageo 12anddiein hei hi do ou hdecadedue oca dio- espi a o y complica ions (3). Deficiencies in he sa coglycan genes a e usually less se e e bu can also be accompanied by ca diac p oblems (4,5). Recen ly, subs an ial p og ess in he de elopmen o he a- peu ic app oaches o he ea men o muscula dys ophies has been accomplished. The apies o DMD based on he deli - e y o minidys ophin (6) o an isense oligonucleo ide-media ed exon-skipping (7–10) a e in p e-clinical e alua ion o in phase I–III clinical ials. The small-molecule compound A alu en (11,12) has ecen ly ob ained condi ional ma ke ing au ho iza- ion o he ea men o DMD. Fu he mo e, a long- e m, sus- ained es o a ion o α-sa coglycan (Sgca) and γ-sa coglycan (Sgcg) exp ession was obse ed ollowing in amuscula gene ans e o muscles o pa ien s wi h limb-gi dle muscula dys- ophy ypes 2D (LGMD2D) (13)and2C(14) espec i ely.Wi h ecen p og ess in pha maco- o gene- he apy o muscula dys- ophies he e is a g owing need o minimally in asi e bio- ma ke s ha can be used o assess and moni o he e ficacy o he apy. Indeed, in o de o e alua e he e ficiency o a ea - men du ing animal s udies, esea che s ha e unlimi ed access o di e en ypes o biopsies o nec opsies. In con as , ials in humans impose e hical es ic ions equi ing minimally in a- si e me hods o assess and moni o he e ficacy o he apy. Cu - en me hods include unc ional e alua ion scales o measu e pa ien s’s a us (15–17), measu emen o he le el o a y infil- a ion by magne ic esonance imaging (MRI) (18)andquan ifi- ca ion o se um mic oRNAs (19–21) o u ina y p o eins (22). The bioma ke mos commonly used o DMD is se um c ea ine kinase (CK), which leaks in o he blood s eam upon muscle damage. Howe e , CK demons a es a ia ions due o physical ac- i i y, muscle inju y, c amping, oxic agen s o age (23,24). Thus, al hough se um CK measu emen is a use ul diagnos ic bioma k- e (25), i is no app op ia e o p edic he cou se o disease, se e - i y o pa hology o o moni o he e ficacy o ea men . Va ia ions in he composi ion o se um p o eome a e con- side ed a p omising sou ce o bioma ke s (26). In he p esen s udy, se um samples om DMD pa ien s and heal hy con ols we e compa ed using a comp ehensi e high- esolu ion mass spec ome y app oach and se e al ens o p o eins wi h al e ed le els we e e ealed by label- ee p o ein quan ifica ion ana- lysis. Among hese p o eins, he myofib illa s uc u al p o ein myomesin-3 (MYOM3), which was mo e abundan in DMD pa- ien se a han in heal hy con ols, was chosen o de ailed ana- lysis. MYOM3 was p esen in se a as wo in e nal agmen s o 100 and 130 kDa a he han as an in ac p o ein. Impo an ly, hese agmen s demons a ed lowe in e -indi idual a ia- ions compa ed o CK. High le els o hese MYOM3 agmen s we e also de ec ed in se a om LGMD2D pa ien s, as well as in animal models o DMD and se e al limb-gi dle muscula dys ophies. In he dys ophin-deficien mdx mouse, hese agmen s we e mo e eliable o he ea ly de ec ion o he disease and less sensi i e o physical exe cise when compa ed o CK. MYOM3 agmen s we e also supe io when compa ed o CK o he mon- i o ing he es o a ion o he DAPC and co ela ed o he escue o physical o ce a e gene he apy ea men o LGMD2D mouse model. Taken oge he , ou da a sugges ha MYOM3 agmen s a e bioma ke s o he de ec ion, e alua ion and ea men moni o ing o DMD, LGMD2D and po en ially o o he o ms o muscula dys ophy associa ed wi h inc eased u no e o sa come ic p o eins. Resul s De ec ion o se um p o eins wi h al e ed le els in DMD pa ien s by mass spec ome y Se um samples om 39 DMD pa ien s and 38 con ol subjec s col- lec ed in USA as pa o he Ad anced Diagnos ics o New The a- peu ic App oaches (ADNA) p ojec (h p://www.ins i u -me ieux. com/p oje ssan e_adna.php) we e analysed using a mass spec- ome y app oach. To educe he numbe o LC-MS/MS ana- lyses, he samples we e o ganized in o ou g oups (G1: young DMD om3 o10yea sold;G2:olde DMD om12 o20yea s old; G3: young con ols om 3 o 10 yea s old and G4: olde con- ols om 12 o 20 yea s old) subdi ided in a o al o 12 pools ac- co ding o he pa ien ’sage(Table1). Each pool included se a om a leas ou indi iduals whe e se um o each indi idual was equally ep esen ed. In o de o ensu e deep p o eome co e age, he pools we e immunodeple ed o he 12 majo se um p o eins. Mass spec ome y analysis o se um samples o all 12 sub- g oups enabled he iden ifica ion a o al o 3329 unique pep ides ma ching 378 p o eins (wi h a alse disco e y a e less han 0.01). Among hose, 69% o p o ein iden ifica ion calls (260 p o eins) we e based on spec a om wo o mo e pep ides. To e eal Table 1. Schema o samples assembling in o g oups and subg oups. DMD G1-1 o G1-4: se um om young DMD pa ien s o 3 o 10 yea s old; DMD G2-1 o G2-2: se um om DMD pa ien s o 12 o 20 yea s old. Heal hy con ols G3-1 o G3-4 and G4-1 o G4-2: age-ma ched heal hy con ols o he young and olde DMD pa ien s espec i ely. Numbe s below each pool indica e he in e al o age and he numbe o pa ien s (in b acke s) DMD G1–1G1–2G1–3G1–4G2–1G2–2 Age (numbe ) 3–4 (7) 4–6 (11) 6–7 (4) 7–10 (4) 12–16 (6) 16–20 (7) Heal hy con ols G3–1G3–2G3–3G3–4G4–1G4–2 Age (numbe ) 3–4 (5) 4–6 (6) 6–7 (5) 7–10 (5) 12–16 (10) 16–20 (7) Human Molecula Gene ics, 2015, Vol. 24, No. 17 |4917 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om p o eins di e en ially p esen in se a om DMD and heal hy indi iduals, he da a we e analysed by a label- ee quan ifica ion app oach using he ollowing pa ame e s: numbe o pep ides ≥2; Masco p o ein sco e (h p://www.ma ixscience.com/) ≥50 and old change ≥2. The analysis o young DMD pa ien s wi h hei age-ma ched con ols (G1 e sus G3 g oups) e ealed 24 p o eins mo e abundan in DMD and 13 in heal hy subjec s (Table 2). The op 10 p o eins wi h he lowes P- alue we e mo e abundan in DMD pa ien s and ei he in ol ed in muscle ene gy me abolism (py u a e kinase PKM, L-lac a e dehyd ogenase B chain, CK-M, alanine amino ans e ase 1, β-enolase, ca bonic anhyd ase 3, uc ose-bisphospha e aldolase A), in sa come e o ganiza ion (myomesin-3, myosin-7) o cos ame e o ganiza ion ( inculin). Compa ison o olde DMD pa ien s wi h hei age-ma ched con ols (G2 e sus G4) using he same pa ame e s esul ed in only nine al e ed p o eins: fi e p o eins mo e abundan in DMD (CK-M, adiponec in, uc ose-bisphospha e aldolase A, L-lac a e dehyd ogenase B chain and haemoglobin β) and ou in heal hy subjec s (gelsolin, phospha idylcholine-s e ol acyl ans e ase, cadhe in-13 and ca ilage acidic p o ein 1) (Table 3). Only ou o hese p o eins (CK-M, uc ose-bisphospha e aldolase A, L-lac- a e dehyd ogenase B chain and haemoglobin β) we e di e en- ially abundan in bo h DMD age g oups acco ding o he mass spec ome y analysis. Impo an ly, he exp ession a ios o hese ou p o eins in DMD e sus heal hy con ols we e subs an- ially lowe in olde DMD pa ien s as compa ed o he young DMD g oup (19.5; 3.3; 2.2 and 2.4 olds in olde DMD e sus 39.8; 14.2; 5.4 and 3.6 imes in young, espec i ely). The dec ease in he numbe o p o eins wi h al e ed le els and in magni ude o hei old changes is mos p obably due o he d as ic dec ease o muscle mass in olde DMD pa ien s and ela i e immobili y o hese pa- ien s. In e es ingly, label- ee analysis o young and olde DMD pa ien s (G1 e sus G2) e ealed eigh sec e ed p o eins ha in- c eased in abundance wi h pa ien age (dopamine β-hyd oxylase: 3- old, adiponec in: 3- old, se um amyloid P-componen : 3- old, insulin-like g ow h ac o -binding p o ein complex acid labile Table 2. Lis o p o eins wi h al e ed le els be ween G1 and G3 g oups (young DMD and age-ma ched heal hy con ols) classified by he dec ease in he a io DMD/heal hy ( old change) No. accession Desc ip ion Localiza ion Pep ides Sco e ANOVA (P- alue) Fold change MYG_HUMAN Myoglobin Cy oplasm 4 195 2.7e-03 234.8 MYOM2_HUMAN MYOM2 Myofib il 10 390 9.8e-05 100.1 MYOM3_HUMAN MYOM3 Myofib il 11 491 1.5e-05 49.7 TPIS_HUMAN T iosephospha e isome ase Cy oplasm 3 128 2.3e-03 48.4 AATC_HUMAN Aspa a e amino ans e ase Cy oplasm 3 75 4.7e-04 45.7 KCRM_HUMAN CK-M Cy oplasm 15 849 2.9e-05 39.8 MYH7_HUMAN Myosin-7 Myofib il 11 520 2.2e-05 38.3 ENOB_HUMAN β-enolase Cy oplasm 4 178 7.4e-05 34.8 G6PI_HUMAN Glucose-6-phospha e isome ase Cy oplasm/Sec e ed 4 130 1.6e-03 29.5 CAH3_HUMAN Ca bonic anhyd ase 3 Cy oplasm 5 182 8.6e-05 23.9 FLNC_HUMAN Filamin-C Myofib il 4 145 4.3e-04 19.4 ALAT1_HUMAN Alanine amino ans e ase 1 Cy oplasm 4 127 3.0e-05 15.6 ALDOA_HUMAN F uc ose-bisphospha e aldolase A Cy oplasm 15 729 9.3e-05 14.2 KPYM_HUMAN Py u a e kinase PKM Cy oplasm 16 845 1.1e-05 12.8 TITIN_HUMAN Ti in Myofib il 14 495 1.9e-03 10.8 VINC_HUMAN Vinculin Cy oplasm/Memb ane 2 74 7.2e-05 10.3 PYGM_HUMAN Glycogen phospho ylase, muscle o m Cy oplasm 8 257 6.1e-04 9.9 LDHA_HUMAN L-lac a e dehyd ogenase A chain Cy oplasm 8 378 9.1e-04 9.5 HPT_HUMAN Hap oglobin Sec e ed 29 1867 1.5e-04 7.6 HBD_HUMAN Haemoglobin subuni δCy oplasm 3 100 5.1e-03 6.2 LDHB_HUMAN L-lac a e dehyd ogenase B Cy oplasm 10 598 2.4e-05 5.4 HBB_HUMAN Haemoglobin subuni βCy oplasm 7 552 8.0e-03 3.6 HBA_HUMAN Haemoglobin subuni αCy oplasm 7 407 5.3e-03 3.4 TPM2_HUMAN T opomyosin βchain Myofib il 5 170 2.0e-02 2.6 VASN_HUMAN Vaso in Memb ane 4 135 4.0e-02 0.5 ALS_HUMAN Insulin-like g ow h ac o -binding p o ein complex Sec e ed 22 1096 1.0e-02 0.5 PHLD_HUMAN Phospha idylinosi ol-glycan-specific phospholipase D Sec e ed 9 533 4.7e-03 0.5 CHL1_HUMAN Neu al cell adhesion molecule L1-like p o ein Memb ane/Sec e ed 2 66 3.0e-02 0.5 COL11_HUMAN Collec in-11 Sec e ed 2 72 2.6e-03 0.4 CADH5_HUMAN Cadhe in-5 Memb ane 6 220 2.0e-03 0.4 CD109_HUMAN CD109 an igen Memb ane 2 59 3.0e-02 0.4 LBP_HUMAN Lipopolysaccha ide-binding p o ein Sec e ed 7 386 5.0e-03 0.4 CRAC1_HUMAN Ca ilage acidic p o ein 1 Sec e ed 6 223 2.0e-02 0.4 C4BPB_HUMAN C4b-binding p o ein Sec e ed 4 207 2.0e-02 0.4 CNDP1_HUMAN β-Ala-His dipep idase Sec e ed 8 294 4.2e-03 0.3 DPP4_HUMAN Dipep idyl pep idase 4 Memb ane/Sec e ed 5 162 5.5e-03 0.3 CETP_HUMAN Choles e yl es e ans e p o ein Sec e ed 7 296 8.2e-04 0.2 Top 10 p o eins wi h he lowes P- alue a e in bold. All shown p o eins passed h esholds o pep ide numbe s ≥2, a sco e≥50, a old change ≥2 and a P- alue ≤0.05. Pep ides: numbe o pep ides iden ified o a gi en p o ein. Sco e: Masco p o ein sco e. 4918 |Human Molecula Gene ics, 2015, Vol. 24, No. 17 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om subuni : 3- old, β-Ala-His dipep idase: 5- old, insulin-like g ow h ac o I: 5- old). Le els o MYOM3 demons a e less in e -indi idual a ia ions compa ed o CK in DMD pa ien s Ele a ed le els o cy osolic p o eins such as CK in he blood a e now widely used as he fi s s age o DMD diagnosis (24,27). The e o e, i was appealing o compa e se um le els o CK wi h one o he myofib illa s uc u al p o eins ound in he p esen s udy. Se um bioma ke s a e o en p esen ed by unca ed ag- men o he ull-leng h p o eins, hus making di ficul finding o comme cial an ibodies o he specific agmen (28). In ou hands, o he ele en an ibodies es ed agains he h ee myofi- b illa s uc u al p o eins (MYOM2, MYOM3 and Myosin 7) wi h he highes high old change and lowes P- alue be ween DMD and heal hy con ols (Table 2)onlyan ibodyagains MYOM3 was e ficien inimmunoblo analysis.MYOM3(UniP o KB# Q5VTT5), a p o ein o 1437 amino acids (162.2 kDa), is a membe o a amily o closely ela ed s uc u al p o eins de ec ed a he M-band o he sa come e in s ia ed skele al muscles: MYOM1, MYOM2 (o M p o ein) and MYOM3. These p o eins a e in ol ed in sa come e s abili y and esis ance du ing in ense o sus ained s e ching (29). Wes e n blo analysis o se um om DMD pa ien s wi h an an i-MYOM3 an ibody a ge ing amino acids 887–1178 o he p o ein e ealed he p esence o wo bands o 100 and 130 kDa espec i ely (Fig. 1). To iden i y he posi ion o hese agmen s wi hin he p o ein, hey we e pu ified by immunop ecipi a ion, sepa a ed by SDS-PAGE and analysed by mass spec ome y sepa a ely (Table 4). The ob ained pep ide co e ages o he ag- men s ( o al 55 pep ides co e ing amino acid 254–1331 o he uppe agmen and 38 pep ides co e ing he sequence om amino acid 476–1331 o he lowe agmen ) sugges ha bo h agmen s ha e simila C- e minal end bu di e en N- e minus. Minimum molecula weigh o he agmen s based on he posi ions o he mos N- e minal and C- e minal iden ified pep ides is equal o 121 kDa o he uppe agmen (1077 aa) and 96 kDa (855 aa) o he lowe agmen , which fi well wi h he size o he agmen s es ima ed by SDS-PAGE (130 and 100 kDa espec i ely). Impo an ly, agmen s o he same size we e ba ely de ec able in se a om heal hy subjec s by Wes e n blo analysis (Fig. 1), hus alida ing he mass spec ome y da a. We nex compa ed he le els o he MYOM3 agmen s and CK in all 103 subjec s om he US coho . The se um exp ession le- els o bo h MYOM3 agmen s we e de e mined by Wes e n blo analysis and CK assessed by measu ing i s enzyma ic ac i i y (Fig. 2). In acco dance wi h he mass spec ome y da a, esul s showed ha exp ession le els o bo h, CK and he MYOM3 ag- men s, we e much highe in young DMD pa ien s compa ed o he espec i e heal hy con ols ( a io DMD/Con ol: MYOM3 = 284 and CK = 193) (Fig. 2A and B). In olde DMD pa ien s, he exp es- sion le els o CK and MYOM3 agmen s we e espec i ely 14 Table 3. Lis o p o eins wi h al e ed le els in se um samples be ween G2 and G4 g oups (olde DMD and age-ma ched heal hy con ols) classified by he dec ease in he a io DMD/heal hy ( old change) No. accession Desc ip ion Localiza ion pep ides Sco e ANOVA (P- alue) Fold change KCRM_HUMAN CK-M Cy oplasm 3 96 1.0e-02 19.5 ADIPO_HUMAN Adiponec in Sec e ed 3 213 3.0e-02 4.4 ALDOA_HUMAN F uc ose-bisphospha e aldolase A Cy oplasm 2 84 3.0e-02 3.3 HBB_HUMAN Haemoglobin subuni βCy oplasm 9 632 8.4e-03 2.4 LDHB_HUMAN L-lac a e dehyd ogenase B chain Cy oplasm 6 223 4.0e-02 2.2 GELS_HUMAN Gelsolin Cy oplasm 32 2287 1.0e-02 0.5 LCAT_HUMAN Phospha idylcholine-s e ol acyl ans e ase Sec e ed 6 295 1.0e-02 0.4 CAD13_HUMAN Cadhe in-13 Memb ane 2 86 5.8e-03 0.4 CRAC1_HUMAN Ca ilage acidic p o ein 1 Sec e ed 3 105 1.0e-02 0.2 All shown p o eins passed h esholds o pep ide numbe s ≥2, a sco e ≥50, a old change ≥2andaP- alue ≤0.05. Pep ides: numbe o pep ides iden ified o a gi en p o ein. Sco e: Masco p o ein sco e. Figu e 1. Wes e n blo analysis o MYOM3 in pools o se a om subg oups o young (G1) and olde (G2) DMD pa ien s as well as young (G3) and olde (G4) heal hy subjec s. (A) no mal exposu e; (B) boos ed exposu e. Fo explana ion o g oups and subg oups see Table 1. Fi y mic og ams o se um p o eins we e loaded in each well. Human Molecula Gene ics, 2015, Vol. 24, No. 17 |4919 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om Table 4. Lis o MYOM3 pep ides iden ified by mass spec ome y in uppe (∼130 kDa) and lowe (∼100 kDa) bands om DMD pa ien se um # Posi ion Sequence Ion sco e (Uppe band) Ion sco e (Lowe band) 1 254–263 DAGFDSEIFK 24 2 265–283 STFGPSVEFTSVLKPVFAR 44 3 320–326 KILYTDR 19 4 321–326 ILYTDR 12 5 338–346 EDEGLYMVR 57 6 347–354 VPSPFGPR 44 7 355–363 EQSTYVLVR 48 8 364–380 DAEAENPGAPGSPLNVR 54 9 401–411 GNPITAYTIER 40 10 461–474 ASELVVMGDHDAAR 54 11 476–486 KTEIPFDLGNK 64 53 12 477–486 TEIPFDLGNK 19 43 13 487–513 ITISTDAFEDTVTIPSPPTNVHASEIR 92 50 14 514–527 EAYVVLAWEEPSPR 65 55 15 530–538 APLTYSLEK 66 37 16 539–556 SVIGSGTWEAISSESPVR 86 57 17 560–567 FAVLDLEK 40 37 18 560–568 FAVLDLEKK 33 35 19 569–574 KSYVFR 32 20 577–596 AMNQYGLSDPSEPSEPIALR 69 58 21 597–612 GPPATLPPPAQVQAFR 79 42 22 613–638 DTQTSVSLTWDPVKDPELLGYYIYSR 33 18 23 639–657 KVGTSEWQTVNNKPIQGTR 27 23 24 640–657 VGTSEWQTVNNKPIQGTR 67 79 25 658–664 FTVPGLR 25 21 26 675–692 SVSEAGVGESSAATEPIR 96 63 27 714–724 NEMVIGWKPPK 38 26 28 731–757 ILGYFLDQHDSEELDWHAVNQQPIPTR 12 29 761–773 VSDLHEGHFYEFR 29 30 796–809 EWTMPQPGPPYDVR 43 26 31 863–870 VSDLQPGK 42 15 32 935–944 DYKGPLDPQR 55 23 33 956–993 VILKEPGLEDLGTYSVIVTDADEDISASHTLTEEELEK 25 34 960–993 EPGLEDLGTYSVIVTDADEDISASHTLTEEELEK 17 35 1008–1019 LISGWNIDILER 64 45 36 1024–1030 LWLEVEK 14 20 37 1031–1044 LSPAAELHLIFNNK 52 55 38 1045–1052 EIFSSPNR 25 20 39 1045–1053 EIFSSPNRK 26 28 40 1053–1058 KINFDR 26 22 41 1061–1075 GLVEVIIQNLSEEDK 52 42 1061–1086 GLVEVIIQNLSEEDKGSYTAQLQDGK 33 21 43 1089–1102 NQITLTLVDDDFDK 72 96 44 1089–1105 NQITLTLVDDDFDKLLR 60 23 45 1118–1129 QGPYFERPLQWK 16 14 46 1151–1157 FQWFFQR 22 23 47 1189–1218 AMVSDDRGEDDTILDLTGDALDAIFTELGR 88 32 48 1219–1228 IGALSATPLK 62 57 49 1229–1237 IQGTEEGIR 46 56 50 1244–1251 YYNVEYMK 40 30 51 1252–1257 TTWFHK 16 52 1269–1284 TGTTLDEIWLHILDPK 19 21 53 1291–1299 YTLEIAAGK 37 35 54 1303–1322 QLSTDLSGQAFEDAMAEHQR 40 38 55 1325–1331 TLAIIEK 17 24 Ion sco es (Masco MS/MS ion sco es) a e shown o each pep ide iden ified in each band. The en i e leng h o MYOM3 is 1437 aa. The ob ained pep ide co e age o he MYOM3 sugges s ha bo h agmen s ha e simila C- e minal end bu di e en N- e minus. Minimum molecula weigh s o he agmen s based on he posi ions o he mos N- e minal and C- e minal iden ified pep ides a e equal o 121 kDa o he uppe agmen (1077 aa) and 96 kDa (855 aa) o he lowe . Thus es ima ed MW o he agmen s fi well wi h he posi ions o he agmen s on he SDS-PAGE (130 and 100 kDa espec i ely). 4920 |Human Molecula Gene ics, 2015, Vol. 24, No. 17 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om and 5 imes lowe han in young DMD pa ien s. O no e, in wo ou lie pa ien s o 16 and 20 yea s old he le els o he MYOM3 agmen s and CK we e lowe han he maximal alues in he e- spec i e heal hy con ols. The dec ease o hese p o eins wi h pa ien ’s age can be explained by he se e e loss o o al muscle mass due o he ad anced s age o he disease. Impo an ly, e en i bo h p o eins we e able o disc imina e DMD pa ien s and heal hy con ols, he e we e less in e -indi id- ual a ia ions in MYOM3 agmen le els compa ed o CK le els. While he CK le els in he young pa ien s a ied om 9000 o 60 000 IU/L (mean 27 130 IU/L ± 13 130), he alues o MYOM3 ag- men s emained be ween 11 a.u. and 24 a.u. (mean 19 a.u. ± 3). The low co ela ion obse ed be ween he le els o se um CK and MYOM3 agmen s in he g oup o young pa ien s (R 2 = 0.28) indica es ha di e en physiological mechanisms may accoun o he sec e ion/s abili y o hese p o eins a his age (Fig. 2C). Con e sely, hese wo bioma ke s we e well co ela ed in olde pa ien s (Fig. 2D). MYOM3 agmen s a e specifically p esen in se a om animal models o DMD The le els o MYOM3 agmen s we e quan ified in wo animal models o DMD: golden Re ie e muscula dys ophy (GRMD) which has a se e e pheno ype simila o DMD pa ien s (30)and dys ophin-deficien mdx mice. Wes e n blo analysis o GRMD and mdx se a e ealed he p esence o wo bands mig a ing a he same posi ions as human MYOM3 agmen s (Fig. 3A and B). Impo an ly, he abundance o hese agmen s was 100 imes highe han in he heal hy con ol dogs. Whe eas he le el o he MYOM3 agmen s in human DMD samples dec eased wi h age, exp ession o hese agmen s was e y simila in he se um o 2 and 18 mon hs old ambulan GRMD. We hypo hesize ha he high MYOM3 le els in elde GRMD can be due o he ambulan s a e o he dogs. Age-independen exp ession o he MYOM3 ag- men s could be an ad an age o u iliza ion o his bioma ke in gene he apy s udies conduc ed in dogs. MYOM3 agmen s a e ele a ed in se a o LGMD2D pa ien s and mouse models o LGMDs The p esence o he MYOM3 agmen s was also analysed in se um samples o h ee pa ien s wi h α-sa coglycanopa hy (LGMD2D). F agmen s o he same leng h (100 and 130 kDa) we e de ec ed a ele a ed le els in all hese pa ien s. O e all, he le el o hese agmen s in LGMD2D pa ien s was lowe compa ed o hei in ensi y in young DMD pa ien s (Fig. 4, uppe panel). The ollowing mouse models o limb-gi dle muscula dys o- phies we e included in his s udy: KO-Calpain 3 (models o LGMD2A) (31), KO-Dys e lin (models o LGMD2B) (32), KO-Sgcg (models o LGMD2C) (33) and KO-Sgca (models o LGMD2D) (34). These mouse models a e congenic s ains on he gene ic backg ound o he C57BL/6J mouse, which was included in he s udy as hei wild- ype (WT) con ol. Taking in o conside a ion he muscle impai men and ime o disease onse , hese mouse models can be classified in e ms o dec easing o de o se e i y: Figu e 2. Exp ession le els o se um MYOM3 agmen s (A) and CK (B) in se a om he en i e US coho including 39 young and 17 olde DMD pa ien s as well as 29 young and 18 olde heal hy con ols. To measu e le els o he MYOM3 agmen s, 50 μg o se um p o eins we e analysed by Wes e n blo , hen band in ensi ies we e quan ified and exp essed in a bi a y uni s (a.u). The CK enzyme ac i i y in se um is exp essed in in e na ional uni s pe li e (IU/L). (C) Linea eg ession analysis be ween se um le els o he MYOM3 agmen s and CK o young pa ien s. (D) Linea eg ession analysis be ween se um le els o he MYOM3 agmen s and CK o olde DMD pa ien s. The linea i y o he esponse by Wes e n blo o MYOM3 is demons a ed in Supplemen a y ma e ial, Figu e S2. Human Molecula Gene ics, 2015, Vol. 24, No. 17 |4921 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om KO-Sgca, KO-Sgcg, KO-dys and KO-Capn3. Se um om hese mouse models was collec ed a 1 and 6 mon hs o age, co e- sponding o he ea ly and ad anced s ages o he dys ophies, and he le els o he MYOM3 agmen s we e compa ed by Wes - e n blo . The highes le els o se um MYOM3 agmen s we e ob- se ed in he h ee mouse models wi h pe u ba ions in he DAPC (Fig. 4, lowe panel). In KO-Dys mice, hese agmen s we e ba ely de ec able a 1 mon h o age and hen inc eased a 6mon hs, eflec ing he agg a a ion o he disease a his age. MYOM3 agmen s we e ha dly de ec able in KO-Capn3 mice a any age. In mdx mice, he MYOM3 agmen s a e exp essed ea ly, demons a e less in e -indi idual a iabili y and a e less sensi i e o physical exe cise compa ed o CK In o de o iden i y he ea lies ime poin when he se um MYOM3 agmen s a e de ec able, we in es iga ed se a om mdx mice o di e en ages ( om bi h o 1-yea -old). The MYOM3 agmen s we e de ec ed in mdx mice a bi h, wi h a small dec ease in hei le els a 1 week o age and ollowed by a ise in abundance a 3 weeks (Fig. 5A). Impo an ly, he le els o hese agmen s in he age-ma ched con ol mice was lowe a all ages es ed (Fig. 5B). The kine ics o he MYOM3 agmen abun- dance in he se um o mdx mice co ela es wi h he iming o an acu e phase o muscle nec osis gene ally occu ing a 3–4 weeks o age, ollowed by an appa en s abiliza ion o he muscle pheno ype (35). The kine ics o se um CK le els in mdx mice we e di e en om ha o he MYOM3 agmen s du ing he fi s weeks o age. Consis en wi h p e ious s udies (36,37) se um CK was ele a ed in newbo n mice, bu hen became un- de ec able du ing he 1s and 2nd week o age (excep o 1 mouse), ising again a 3 and 12 weeks ollowed by a s abiliza ion (Fig. 5C). In heal hy mice, se um CK was also sligh ly ele a ed in newbo ns and 12- and 24-week-old animals (Fig. 5D). Impo an ly, less a ia ion was obse ed in he le els o MYOM3 agmen s in mice o he same age compa ed o he CK (maximum old change wo o MYOM3 e sus 110 o CK). The di e ence in age-dependen exp ession pa e ns be ween se um CK and MYOM3 agmen s in mdx mice is p obably ela ed o di e en mechanisms o biop ocessing o hese p o eins, especially du ing he ea ly phases o disease. To assess he impac o physical exe cise on he se um le els o he MYOM3 agmen s and CK, WT and mdx mice we e sub- jec ed o downhill unning o 30 min. This exe cise egimen is o en used o inc ease muscle inju y and wo sen he mdx pheno- ype (38,39). Se a we e collec ed 7 days be o e and 3, 24 and 48 h a e exe cise. Impo an ly, while in mdx mice CK concen a ion peaked a 3 h pos -exe cise (up o 10- old inc ease) ollowed by a subs an ial dec ease (Fig. 6C), physical exe cise had ela i ely li le impac on he se um le els o he MYOM3 agmen s (less han 2- old inc ease 48 h pos -exe cise) (Fig. 6A). In e es ingly, in heal hy mice, he e was a sligh inc ease in he le els o he MYOM3 agmen s 24 and 48 h a e exe cise, e en hough he maximum le el o he agmen s in heal hy mice was 50- old less han in mdx mice (Fig. 6B). Se um CK le els we e a iable in Figu e 3. MYOM3 agmen s a e specifically p esen in se a om animal models o DMD. (A) Wes e n Blo analysis o se um om GRMD and heal hy dogs. GRMD # 1–4: wo mon hs old; # 5–6: 18 mon hs old. Heal hy # 1–4: wo mon hs old; # 5–6: 18 mon hs old dogs. DMD: con ol se um om DMD pa ien . (B) Wes e n Blo analysis o se um om 6 mon hs old mdx and WT mice. WT: C57/BL10 s ain. Figu e 4. Uppe panel: Wes e n blo analysis o he MYOM3 agmen s in se um om 3 LGMD2D pa ien s (#1 is 35, #2 is 23 and #3 is 24 yea s old). Se um om wo DMD pa ien s (g oup G1) and h ee heal hy indi iduals (g oup G4) we e used as con ols. Lowe panel: Wes e n blo analysis o he MYOM3 agmen s in se um om mouse models o di e en muscula dys ophies a 1 and 6 mon hs o age. WT: C57BL/6J mouse; mdx (model o DMD); KO-Sgcg: model o LGMD2C; KO-Sgca: model o LGMD2D; KO-Dys : model o LGMD2B; KO-Capn3: model o LGMD2A. 4922 |Human Molecula Gene ics, 2015, Vol. 24, No. 17 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om heal hy mice wi hou no iceable co ela ion wi h physical exe - cise (Fig. 6D). Gi en ha MYOM3 is p edominan ly exp essed in slow and in e media e speed ( ype I and IIa) skele al fib es (29) which a e less a ec ed in DMD ela i e o as myofib es (40), i is possible ha he di e ence in he kine ics o hese bioma ke s is pa ially due o he di e en ial sensi i i y o hese muscle fib e ypes o exe cise-induced damage. MYOM3 agmen s enable moni o ing o pha maco- and gene- he apy ea men e ficacy The p esence o he MYOM3 agmen s in se um o DMD and LGMD2D pa ien s and hei espec i e mouse models p omp ed us o e alua e he u ili y o hese bioma ke s o moni o ing he esponse o expe imen al he apies in mdx and KO-Sgca mice. Res o a ion o dys ophin exp ession in mdx mouse muscles was achie ed by a single adminis a ion o an a ginine- ich cell- pene a ing pep ide (CPP) conjuga ed o a phospho odiamida e mo pholino oligonucleo ide (PMO) ha e ficien ly induces skip- ping o exon 23 and es o es dys ophin p o ein exp ession and muscle unc ion (41,42). In o de o e alua e he impac o he es o a ion o dys ophin exp ession on he se um le els o MYOM3 agmen s and CK, quad iceps emo is muscles and blood samples om ea ed mdx we e collec ed 2, 4 and 8 weeks pos -injec ion. Quad iceps and blood samples om non- ea ed mdx andWTcon ol12-week-oldmicewe e akenas con ols. In acco dance wi h he p e iously published da a (41–43), he es o a ion o he dys ophin exp ession and pe - cen age o exon skipping in quad iceps we e be ween 10 and 45% 2 weeks a e injec ions, ollowed by a dec ease a la e ime poin s (Fig. 7A and B). In a good ag eemen wi h he es o - a ion o dys ophin le els, wo weeks a e injec ion he le els o he MYOM3 agmen s in ea ed mdx mice subs an ially de- c eased (wi hou eaching he le el in he con ol mice) and hen g adually inc eased o e ime (Fig. 7C). In con as o he MYOM3 agmen s, CK le els did no eflec es o a ion o dys- ophin exp ession. Thus, 2 weeks a e injec ion he le el o se um CK was lowe in ea ed mdx mice compa ed o WT con ol mice (Fig. 7D), while dys ophin exp ession did no exceed 50% a ha ime. Mo eo e , 8 weeks a e he ea men , when he es ima ed le el o dys ophin-posi i e fib es was a ound 10%, CK le els we e highe in ea ed han in non- ea ed mdx mice. Di e en beha iou o he MYOM3 agmen s and CK a e pa - ial es o a ion o dys ophin exp ession may eflec he capaci y o hese bioma ke s o di e en ially e eal in acellula p ocess such as mic opa icle u no e (44) o inc eased myofib illa p o ein ca abolism (45,46). To es o e α-sa coglycan exp ession in KO-Sgca mice, we used ecombinan adeno-associa ed i us AAV2/8 ec o . Con ol C57BL/6J mice ecei ed an in a enous injec ion o PBS and ou g oups o KO-Sgca mice ecei ed in a enous injec ions o ei he PBS o low (1e11 g), medium (5e11 g) o high (1e12 g) Figu e 5. Le els o he MYOM3 agmen s (A,B) and CK-M (C,D) in se um om heal hy (B, D) and mdx (A, C) mice a di e en ages as es ima ed by Wes e n blo analysis. In ensi y o he bands (in a bi a y uni s, a.u.) on di e en gels was no malized by he espec i e bands o he posi i e con ol (50 μg o se um p o eins om he same mdx mouse p esen on each gel). Fi y mic og ams o se um p o eins we e used o he analysis. Age 0 co esponds o newbo n mice. Es ima ion o he CK-M le el by Wes e n blo analysis co ela ed well wi h he CK ac i i y (Supplemen a y ma e ial, Fig. S3). Human Molecula Gene ics, 2015, Vol. 24, No. 17 |4923 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om doses o AAV2/8 coding o hSGCA. Mice we e moni o ed o 3 mon hs a e he ea men . The ollowing assays we e com- pa ed in o de o define he mos app op ia e o he ollow-up o he ea men : his ological analysis o muscle biopsies (HPS s aining and es o a ion o he sa coglycan complex); o al phys- ical o ce 3 mon hs a e he ea men (1 week be o e animal sac ifice); biweekly analysis o se um CK and MYOM3 agmen s le els. His ological analysis o he gas ocnemius muscles demon- s a ed es o a ion o he complex in 5–30% (mean 15.6 ± 8.4), 60– 100% (mean 79.2 ± 16.7) and 84–100% (mean 94.6 ± 8.8) o fib es a e low, medium and high AAV dose ea men s, espec i ely (Fig. 8A and B). Impo an ly, by assessing he exp ession le el o α-sa coglycan (de e mined by immunos aining) he KO-Sgca, low, medium and WT mice could be clea ly dis inguished. Howe e , no s a is ically significan di e ence was ound be ween medium and high AAV doses by his me hod. Impo an ly, his analysis is high- ly labo ious, and he size o he biopsies makes i unsui able o he ollow-up o he he apeu ic e ec in small animals. Simila o his ological analysis, he con en ional whole body ension (WBT) me hod is an end-poin assay because mice be- come accus omed o he p o ocol (47). The WBT me hod was only able o disc imina e wo clus e s o animals: (1) KO-Sgca mice injec ed wi h PBS o low dose o AAV and (2) con ol C57BL/6J mice and KO-Sgca mice injec ed wi h medium o high doses o AAV (Fig. 8C). A h eshold 3000 IU/L o CK clea ly sepa a es KO-Sgca mice injec ed wi h PBS om all o he expe imen al g oups (Fig. 8D). Ne e heless, when applying he S uden ’s es (P- alue h eshold < 0.01), di e ences only be ween ew ime poin s/ injec ion doses appea ed as s a is ically significan (Fig. 8F). Changing o he P- alue h eshold o <0.05 pe mi s o dis inguish mo e expe imen al g oups o mice (Fig. 8F). Lowe CK le els in all g oups o mice a day 90 (one week a e he o al o ce measu e- men s) (Fig. 8D) could be explained by he ac ha an inc ease o CK le els a e physical exe ion is ollowed by a subs an ial dec ease pe sis ing o 2 weeks (48). In e -indi idual a ia ions o he MYOM3 agmen le els we e lowe compa ed o se um CK in he case o all expe imen alg oups (Fig. 8D and E). In acco dance wi h a p e ious s udy showing p o- g essi e de elopmen o muscula dys ophy in KO-Sgca mice (34) he le els o he MYOM3 agmen s in he con ol mice injec ed wi h PBS inc eased g adually wi h age (Fig. 8E). E en he lowes dose o AAV (1e11 g) s abilized he MYOM3 agmen le els, while medium and high doses educed MYOM3 agmen le els 5- old and 8- old, espec i ely. Due o he low in e -indi idual a iabili y, measu emen o he MYOM3 agmen s enabled nea ly all g oups o mice o be dis inguished wi h ei he o he h esholds (P< 0.01 o 0.05) a he majo i y o ime poin s (Fig. 8E and F). Fu he mo e, MYOM3 agmen abundance was be e co ela ed (R 2 = 0.71) wi h muscle o ce as measu ed by he escape es com- pa ed wi h CK (R 2 = 0.59) (Supplemen a y ma e ial, Fig. S1). Figu e 6. Le els o he MYOM3 agmen s (A,B) and CK-M (C,D) in se um om heal hy (B, D) and mdx (A, C) mice a di e en ime a e physical exe cise es ima ed by Wes e n blo analysis. Band in ensi y on di e en gels was no malized by he espec i e bands o he posi i e con ol (50 μg o se um p o eins om he same mdx mouse p esen on each gel). Fi y mic og ams o mouse se um we e used o he analysis. 4924 |Human Molecula Gene ics, 2015, Vol. 24, No. 17 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om 37. Wooddell, C.I., Zhang, G., G i fin, J.B., Hegge, J.O., Huss, T. and Wol , J.A. (2010) Use o E ans blue dye o compa e limb mus- cles in exe cised young and old mdx mice. Muscle Ne e,41, 487–499. 38. B ussee, V., Ta di , F. and T emblay, J.P. (1997) Muscle fibe s o mdx mice a e mo e ulne able o exe cise han hose o no - mal mice. Neu omuscul. Diso d.,7, 487–492. 39. Vilquin, J.T., B ussee, V., Asselin, I., Kinoshi a, I., Ging as, M. and T emblay, J.P. (1998) E idence o mdx mouse skele al muscle agili y in i o by eccen ic unning exe cise. Muscle Ne e,21, 567–576. 40. Webs e , C., Silbe s ein, L., Hays, A.P. and Blau, H.M. (1988) Fas muscle fibe s a e p e e en ially a ec ed in Duchenne muscula dys ophy. Cell,52, 503–513. 41. Yin, H., Saleh, A.F., Be s, C., Camelli i, P., Seow, Y., Ash a , S., A zumano , A., Hammond, S., Me i , T., Gai , M.J. e al. (2011) Pip5 ansduc ion pep ides di ec high e ficiency oligonucleo- ide-media ed dys ophin exon skipping in hea and pheno- ypic co ec ion in mdx mice. Mol. The .,19, 1295–1303. 42. Be s, C., Saleh, A.F., A zumano , A.A., Hammond, S.M., God ey, C., Cou sindel, T., Gai , M.J. and Wood, M.J. (2012) Pip6-PMO, A New Gene a ion o Pep ide-oligonucleo ide Conjuga es Wi h Imp o ed Ca diac Exon Skipping Ac i i y o DMD T ea men . Mol. The . Nucleic Acids,1, e38. 43. Jea awi iyapaisa n, N., Moul on, H.M., Sazani, P., Kole, R. and Willis, M.S. (2010) Long- e m imp o emen in mdx ca diomy- opa hy a e he apy wi h pep ide-conjuga ed mo pholino oligome s. Ca dio asc. Res.,85, 444–453. 44. Duguez, S., Duddy, W., Johns on, H., Laine, J., Le Bihan, M.C., B own, K.J., Bigo , A., Ha hou , Y., Bu le -B owne, G. and Pa - idge, T. (2013) Dys ophin deficiency leads o dis u bance o LAMP1- esicle-associa ed p o ein sec e ion. Cell. Mol. Li e Sci., 70, 2159–2174. 45. Mussini, E., Co nelio, F., Colombo, L., De Pon e, G., Giudici, G., Co ellessa, L. and Ma cucci, F. (1984) Inc eased myofib illa p o ein ca abolism in duchenne muscula dys ophy mea- su ed by 3-me hylhis idine exc e ion in he u ine. Muscle Ne e,7, 388–391. 46. McKe an, R.O., Halliday, D. and Pu kiss, P. (1977) Inc eased myofib illa p o ein ca abolism in Duchenne muscula dys- ophy measu ed by 3-me hylhis idine exc e ion in he u ine. J. Neu ol. Neu osu g. Psychia y,40, 979–981. 47. Ca lson, C.G., Ru e , J., Bledsoe, C., Singh, R., Ho , H., B uem- me , K., Ses i, J., Ga i, F., Be ge, J. and McCa hy, L. (2010) A simple p o ocol o assessing in e - ial and in e -examine eliabili y o wo nonin asi e measu es o limb muscle s eng h. J. Neu osci. Me hods,186, 226–230. 48. Kobayashi, Y.M., Rade , E.P., C aw o d, R.W. and Campbell, K. P. (2012) Endpoin measu es in he mdx mouse ele an o muscula dys ophy p e-clinical s udies. Neu omuscul. Diso d., 22,34–42. 49. Ha hou , Y., Ma a hi, R.L., Raya a apu, S., Zhang,A., B own, K.J., Seol, H., Go dish-D essman, H., Ci ak, S., Bello, L., Naga aju, K. e al. (2014) Disco e y o se um p o ein bioma ke s in he mdx mouse model and c oss-species compa ison o Duchenne muscula dys ophy pa ien s. Hum. Mol. Gene .,23, 6458–6469. 50. Mendell, J.R. and Lloyd-Pu yea , M. (2013) Repo o MDA muscle disease symposium on newbo n sc eening o Du- chenne muscula dys ophy. Muscle Ne e,48,21–26. 51. Tu ne , D.C., Wallimann, T. and Eppenbe ge , H.M. (1973) A p o ein ha binds specifically o he M-line o skele al muscle is iden ified as he muscle o m o c ea ine kinase. P oc. Na l. Acad. Sci. U. S. A.,70, 702–705. 52. Wallimann, T., Mose , H. and Eppenbe ge , H.M. (1983) Isoen- zyme-specific localiza ion o M-line bound c ea ine kinase in myogenic cells. J. Muscle Res. Cell Mo il.,4, 429–441. 53. S olz, M. and Wallimann, T. (1998) Myofib illa in e ac ion o cy osolic c ea ine kinase (CK) isoenzymes: alloca ion o N- e minal binding epi ope in MM-CK and BB-CK. J. Cell Sci., 111(P 9), 1207–1216. 54. He, B., Tang, R.H., Weislede , N., Xiao, B., Yuan, Z., Cai, C., Zhu, H., Lin, P., Qiao, C., Li, J. e al. (2012) Enhancing muscle mem- b ane epai by gene deli e y o MG53 amelio a es muscula dys ophy and hea ailu e in del a-Sa coglycan-deficien hams e s. Mol. The .,20, 727–735. 55. Mas e s, C.J., Reid, S. and Don, M. (1987) Glycolysis–new con- cep s in an old pa hway. Mol. Cell. Biochem.,76,3–14. 56. Gizak, A., Rakus, D. and Dzugaj, A. (2003) Immunohis ochem- ical localiza ion o human uc ose-1,6-bisphospha ase in subcellula s uc u es o myocy es. His ol. His opa hol.,18, 135–142. 57. Wang, Y., Kinzie, E., Be ge , F.G., Lim, S.K. and Baumann, H. (2001) Hap oglobin, an inflamma ion-inducible plasma p o- ein. Redox Rep.,6, 379–385. 58. Le Bo gne, F., Guyo , S., Loge o , M., Beney, L., Ge ais, P. and Dema quoy, J. (2012) Explo a ion o lipid me abolism in ela ion wi h plasma memb ane p ope ies o Duchenne muscula dys ophy cells: influence o L-ca ni ine. PloS one, 7, e49346. 59. S aple on, D.I., Lau, X., Flo es, M., T ieu, J., Geh ig, S.M., Chee, A., Naim, T., Lynch, G.S. and Koopman, R. (2014) Dys unc ion- al muscle and li e glycogen me abolism in mdx dys ophic mice. PloS one,9, e91514. 60. Aga ko a, I. and Pe ia d, J.C. (2005) The M-band: an elas ic web ha c osslinks hick filamen s in he cen e o he sa co- me e. T ends Cell Biol.,15, 477–485. 61. Lange, S., Himmel, M., Aue bach, D., Aga ko a, I., Hayess, K., Fu s , D.O., Pe ia d, J.C. and Ehle , E. (2005) Dime isa ion o myomesin: implica ions o he s uc u e o he sa come ic M-band. J. Mol. Biol.,345, 289–298. 62. Schoenaue , R., Be oncini, P., Machaidze, G., Aebi, U., Pe - ia d, J.C., Hegne , M. and Aga ko a, I. (2005) Myomesin is a molecula sp ing wi h adap able elas ici y. J. Mol. Biol.,349, 367–379. 63. Alde on, J.M. and S einha d , R.A. (2000) How calcium influx h ough calcium leak channels is esponsible o he ele a ed le els o calcium-dependen p o eolysis in dys ophic myo ubes. T ends Ca dio asc. Med.,10, 268–272. 64. Cai, C., Masumiya, H., Weislede , N., Ma suda, N., Nishi, M., Hwang,M.,Ko,J.K.,Lin,P.,Tho n on,A.,Zhao,X.e al. (2009) MG53 nuclea es assembly o cell memb ane epai ma- chine y. Na . Cell Biol.,11,56–64. 65. Ca pen e , S. and Ka pa i, G. (1979) Duchenne muscula dys- ophy: plasma memb ane loss ini ia es muscle cell nec osis unless i is epai ed. B ain,102, 147–161. 66. Lange, S., Xiang, F., Yako enko, A., Vihola, A., Hackman, P., Ros ko a, E., K is ensen, J., B andmeie , B., F anzen, G., Hedbe g, B. e al. (2005) The kinase domain o i in con ols muscle gene exp ession and p o ein u no e . Science,308, 1599–1603. 67. Robe s, T.C., Blombe g, K.E., McClo ey, G., El Andaloussi, S., God ey, C., Be s, C., Cou sindel, T., Gai , M.J., Smi h, C.I. and Wood, M.J. (2012) Exp ession analysis in mul iple muscle g oups and se um e eals complexi y in he mic oRNA an- sc ip ome o he mdx mouse wi h implica ions o he apy. Mol. The . Nucleic Acids,1, e39. Human Molecula Gene ics, 2015, Vol. 24, No. 17 |4931 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om 68. Ayoglu, B., Chaouch, A., Lochmulle , H., Poli ano, L., Be ini, E., Spi ali, P., Hille , M., Niks, E.H., Gualandi, F., Pon en, F. e al. (2014) A fini y p o eomics wi hin a e diseases: a BIO- NMD s udy o blood bioma ke s o muscula dys ophies. EMBO Mol. Med.,6, 918–936. 69. Chapman, V.M., Mille , D.R., A ms ong, D. and Caskey, C.T. (1989) Reco e y o induced mu a ions o X ch omosome- linked muscula dys ophy in mice. P oc. Na l. Acad. Sci. U. S. A., 86, 1292–1296. 70. Lau e, L., Suel, L., Roudau , C., Bou g, N., Ouali, A., Ba oli, M., Richa d, I. and Daniele, N. (2009) Ca diac anky in epea p o- ein is a ma ke o skele al muscle pa hological emodelling. FEBS J.,276, 669–684. 71. Boisge aul , F., G oss, D.A., Fe and, M., Poupio , J., Da ocha, S., Richa d, I. and Galy, A. (2013) P olonged gene exp ession in muscle is achie ed wi hou ac i e immune ole ance using mic o RNA 142.3p- egula ed AAV gene ans e . Hum. Gene The .,24, 393–405. 72. Smi h, R.H., Le y, J.R. and Ko in, R.M. (2009) A simplified baculo i us-AAV exp ession ec o sys em coupled wi h one-s ep a fini y pu ifica ion yields high- i e AAV s ocks om insec cells. Mol. The .,17, 1888–1896. 73. Ca lson, C.G. and Makiejus, R.V. (1990) A nonin asi e p oced- u e o de ec muscle weakness in he mdx mouse. Muscle Ne e,13, 480–484. 74. Fouge ousse, F., Ba oli, M., Poupio , J., A andel, L., Du and, M., Gue che , N., Gicquel, E., Danos, O. and Richa d, I. (2007) Pheno ypic co ec ion o alpha-sa coglycan deficiency by in a-a e ial injec ion o a muscle-specific se o ype 1 AAV ec o . Mol. The .,15,53–61. 4932 |Human Molecula Gene ics, 2015, Vol. 24, No. 17 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Sep embe 27, 2016h p://hmg.ox o djou nals.o g/Downloaded om