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• W3090995810 abstract "The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC–MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging. The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC–MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging. Duchenne muscular dystrophy (DMD) is a severe, X-linked, pediatric neuromuscular disorder characterized by progressive muscle wasting, loss of ambulation around age 10, and cardiorespiratory failure that is ultimately fatal (1Moriuchi T. Kagawa N. Mukoyama M. Hizawa K. Autopsy analyses of the muscular dystrophies.Tokushima J. Exp. Med. 1993; 40: 83-93PubMed Google Scholar, 2Chiang D.Y. Allen H.D. Kim J.J. Valdes S.O. Wang Y. Pignatelli R.H. Lotze T.E. Miyake C.Y. Relation of cardiac dysfunction to rhythm abnormalities in patients with Duchenne or Becker muscular dystrophies.Am. J. Cardiol. 2016; 117: 1349-1354Abstract Full Text Full Text PDF PubMed Google Scholar, 3Ishikawa Y.Y. Miura T. Ishikawa Y.Y. Aoyagi T. Ogata H. Hamada S. Minami R. Duchenne muscular dystrophy: Survival by cardio-respiratory interventions.Neuromuscul. Disord. 2011; 21: 47-51Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 4Ricotti V. Ridout D.A. Scott E. Quinlivan R. Robb S.A. Manzur A.Y. 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Van Ommen G.J.B. Den Dunnen J.T. Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule.Muscle Nerve. 2006; 34: 135-144Crossref PubMed Scopus (402) Google Scholar, 6Bladen C.L. Salgado D. Monges S. Foncuberta M.E. Kekou K. Kosma K. Dawkins H. Lamont L. Roy A.J. Chamova T. Guergueltcheva V. Chan S. Korngut L. Campbell C. Dai Y. Wang J. Barišić N. Brabec P. Lahdetie J. Walter M.C. Schreiber-Katz O. Karcagi V. Garami M. Viswanathan V. Bayat F. Buccella F. Kimura E. Koeks Z. van den Bergen J.C. Rodrigues M. Roxburgh R. Lusakowska A. Kostera-Pruszczyk A. Zimowski J. Santos R. Neagu E. Artemieva S. Rasic V.M. Vojinovic D. Posada M. Bloetzer C. Jeannet P.-Y. Joncourt F. Díaz-Manera J. Gallardo E. Karaduman A.A. Topaloğlu H. El Sherif R. Stringer A. Shatillo A.V. Martin A.S. Peay H.L. Bellgard M.I. Kirschner J. Flanigan K.M. Straub V. Bushby K. Verschuuren J. Aartsma-Rus A. Béroud C. Lochmüller H. The TREAT-NMD DMD global database: Analysis of more than 7,000 duchenne muscular dystrophy mutations.Hum. Mutat. 2015; 36: 395-402Crossref PubMed Scopus (272) Google Scholar). Dystrophin is important for mechanical force transduction and is also involved in signaling functions (7Hoffman E.P. Brown R.H. Kunkel L.M. Dystrophin: the protein product of the Duchene muscular dystrophy locus. 1987.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 8Levine B.A. Moir A.J.G. Patchell V.B. Perry S.V. The interaction of actin with dystrophin.FEBS Lett. 1990; 263: 159-162Crossref PubMed Scopus (79) Google Scholar, 9Samitt C.E. Bonilla E. Immunocytochemical study of dystrophin at the myotendinous junction.Muscle Nerve. 1990; 13: 493-500Crossref PubMed Scopus (74) Google Scholar, 10Muntoni F. Torelli S. Ferlini A. Dystrophin and mutations: One gene, several proteins, multiple phenotypes.Lancet. Neurol. 2003; 2: 731-740Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 11van Westering T.L.E. Betts C.A. Wood M.J.A. Current understanding of molecular pathology and treatment of cardiomyopathy in duchenne muscular dystrophy.Molecules. 2015; 20: 8823-8855Crossref PubMed Scopus (51) Google Scholar), in part because of its role as an organizing center for the dystrophin-associated protein complex (DAPC). Absence of dystrophin leads to myofiber fragility, sensitivity to contractile damage, chronic cycles of myonecrosis and regeneration, persistent inflammation, and progressive fibro/fatty muscle degeneration (12Pastoret C. Sebille A. mdx mice show progressive weakness and muscle deterioration with age.J. Neurol. Sci. 1995; 129: 97-105Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 13Messina S. Bitto A. Aguennouz M. Minutoli L. Monici M.C. Altavilla D. Squadrito F. Vita G. Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice.Exp. Neurol. 2006; 198: 234-241Crossref PubMed Scopus (117) Google Scholar, 14Yokota T. Lu Q.-L. Morgan J.E. Davies K.E. Fisher R. Takeda S. Partridge T.A. Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration.J. Cell Sci. 2006; 119: 2679-2687Crossref PubMed Scopus (65) Google Scholar). The DMD gene consists of 79 exons and contains at least seven internal promoters (15Doorenweerd N. Mahfouz A. van Putten M. Kaliyaperumal R. T' Hoen P.A.C. Hendriksen J.G.M. Aartsma-Rus A.M. Verschuuren J.J.G.M. Niks E.H. Reinders M.J.T. Kan H.E. Lelieveldt B.P.F. Timing and localization of human dystrophin isoform expression provide insights into the cognitive phenotype of Duchenne muscular dystrophy.Sci. Rep. 2017; 712575 Crossref PubMed Scopus (40) Google Scholar) that give rise to the various dystrophin isoforms (e.g. Dp427, Dp260, Dp140, Dp116, Dp71, and Dp40) (supplemental Fig. S1). Some isoforms are ubiquitously expressed (e.g. Dp71) (16Austin R.C. Howard P.L. D'Souza V.N. Klamut H.J. Ray P.N. Cloning and characterization of alternatively spliced isoforms of Dp71.Hum. Mol. Genet. 1995; 4: 1475-1483Crossref PubMed Google Scholar), whereas others are expressed in a more tissue-restricted pattern (such as Dp260, which is the retinal isoform of dystrophin) (17D'Souza V.N. Nguyen T.M. Morris G.E. Karges W. Pillers D.A. Ray P.N. A novel dystrophin isoform is required for normal retinal electrophysiology.Hum. Mol. Genet. 1995; 4: 837-842Crossref PubMed Google Scholar). As a result, the genomic locations of DMD-causing mutations may differentially affect the expression of these various isoforms, and by extension disease manifestation. For example, loss of the Dp71 isoform has been associated with cognitive impairment (18Daoud F. Angeard N. Demerre B. Martie I. Benyaou R. Leturcq F. Cossée M. Deburgrave N. Saillour Y. Tuffery S. Urtizberea A. Toutain A. Echenne B. Frischman M. Mayer M. Desguerre I. Estournet B. Réveillère C.P.-B. Cuisset J.M. Kaplan J.C. Héron D. Rivier F. Chelly J. Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression.Hum. Mol. Genet. 2009; 18: 3779-3794Crossref PubMed Scopus (88) Google Scholar, 19Moizard M.P. Toutain A. Fournier D. Berret F. Raynaud M. Billard C. Andres C. Moraine C. Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening.Eur. J. Hum. Genet. EJHG. 2000; 8: 552-556Crossref PubMed Scopus (0) Google Scholar). Several dystrophic mouse strains have been developed to investigate DMD pathophysiology, and test novel therapeutics in vivo (20McGreevy J.W. Hakim C.H. McIntosh M.A. Duan D. Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy.Dis. Model. Mech. 2015; 8: 195-213Crossref PubMed Scopus (205) Google Scholar). The most used model is the mdx mouse, which carries a nonsense mutation in exon 23 leading to loss of the major muscle dystrophin isoform Dp427 (supplemental Fig. S1) (21Bulfield G. Siller W.G. Wight P.A. Moore K.J. X chromosome-linked muscular dystrophy (mdx) in the mouse.Proc. Natl. Acad. Sci. U S A. 1984; 81: 1189-1192Crossref PubMed Google Scholar, 22Sicinski P. Geng Y. Ryder-Cook A.S. Barnard E.A. Darlison M.G. Barnard P.J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.Science. 1989; 244: 1578-1580Crossref PubMed Google Scholar). Although the mdx mouse recapitulates some aspects of DMD pathology (23Duddy W. Duguez S. Johnston H. Cohen T.V. Phadke A. Gordish-Dressman H. Nagaraju K. Gnocchi V. Low S. Partridge T. Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia.Skelet. Muscle. 2015; 5: 16Crossref PubMed Scopus (56) Google Scholar), it is generally considered to exhibit mild muscular dystrophy with only a small reduction in life-span (24Chamberlain J.S. Metzger J. Reyes M. Townsend D. Faulkner J.A. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.FASEB J. Off. 2007; 21: 2195-2204Crossref PubMed Scopus (0) Google Scholar). mdx mice undergo a brief period of degeneration and regeneration between 2 and 12 weeks of age (25Dangain J. Vrbova G. Muscle development in mdx mutant mice.Muscle Nerve. 1984; 7: 700-704Crossref PubMed Google Scholar, 26Turk R. Sterrenburg E. de Meijer E.J. van Ommen G.-J.B. den Dunnen J.T. 't Hoen P.A.C. Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling.BMC Genomics. 2005; 6: 98Crossref PubMed Scopus (69) Google Scholar, 27Roig M. Roma J. Fargas A. Munell F. Longitudinal pathologic study of the gastrocnemius muscle group in mdx mice.Acta Neuropathol.). 2004; 107: 27-34Crossref PubMed Scopus (0) Google Scholar), with more pronounced muscle pathology and cardiomyopathy manifesting much later in life (28Lefaucheur J.P. Pastoret C. Sebille A. Phenotype of dystrophinopathy in old mdx mice.Anat. Rec. 1995; 242: 70-76Crossref PubMed Google Scholar, 29Stuckey D.J. Carr C.A. Camelliti P. Tyler D.J. Davies K.E. Clarke K. In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy.PLoS ONE. 2012; 7e28569 Crossref PubMed Scopus (48) Google Scholar). Importantly, the exon 23 mutation observed in the mdx mouse does not typically occur in boys with DMD (30The UMD-TREAT-NMD DMD Locus Specific Databases The UMD TREAT-NMD DMD mutations database.Google Scholar), which has motivated the development of more patient-relevant dystrophic mouse models. To this end, Araki et al. generated the mdx52 mouse model in which Dmd exon 52 is deleted, leading to the absence of the Dp260 and Dp140 isoforms in addition to Dp427 (supplemental Fig. S1) (31Araki E. Nakamurab K. Nakaob K. Kameyac S. Kobayashid O. Nonakad I. Kobayashia T. Katsuki M. Araki E. Nakamura K. Nakao K. Kameya S. Kobayashi O. Nonaka I. Kobayashi T. Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy.Biochem. Biophys. Res. Commun. 1997; 238: 492-497Crossref PubMed Scopus (81) Google Scholar). Deletions in the so-called “hot-spot” region (DMD exons 45-55) are some of the most observed mutations in boys with DMD, thereby making this model more patient-relevant than the more widely used mdx mouse (6Bladen C.L. Salgado D. Monges S. Foncuberta M.E. Kekou K. Kosma K. Dawkins H. Lamont L. Roy A.J. Chamova T. Guergueltcheva V. Chan S. Korngut L. Campbell C. Dai Y. Wang J. Barišić N. Brabec P. Lahdetie J. Walter M.C. Schreiber-Katz O. Karcagi V. Garami M. Viswanathan V. Bayat F. Buccella F. Kimura E. Koeks Z. van den Bergen J.C. Rodrigues M. Roxburgh R. Lusakowska A. Kostera-Pruszczyk A. Zimowski J. Santos R. Neagu E. Artemieva S. Rasic V.M. Vojinovic D. Posada M. Bloetzer C. Jeannet P.-Y. Joncourt F. Díaz-Manera J. Gallardo E. Karaduman A.A. Topaloğlu H. El Sherif R. Stringer A. Shatillo A.V. Martin A.S. Peay H.L. Bellgard M.I. Kirschner J. Flanigan K.M. Straub V. Bushby K. Verschuuren J. Aartsma-Rus A. Béroud C. Lochmüller H. The TREAT-NMD DMD global database: Analysis of more than 7,000 duchenne muscular dystrophy mutations.Hum. Mutat. 2015; 36: 395-402Crossref PubMed Scopus (272) Google Scholar). We recently reported differences in the number of dystrophin-positive revertant fibers and regenerating fibers between mdx and mdx52 mice (32Echigoya Y. Lee J. Rodrigues M. Nagata T. Tanihata J. Nozohourmehrabad A. Panesar D. Miskew B. Aoki Y. Yokota T. Mutation types and aging differently affect revertant fiber expansion in dystrophic mdx and mdx52 mice.PLoS ONE. 2013; 8e69194 Crossref PubMed Scopus (16) Google Scholar). Specifically, mdx mice contained higher numbers of revertant fibers at all ages tested (2–18 months of age), whereas mdx52 mice contained elevated numbers of centrally-nucleated fibers at 2 months of age only (32Echigoya Y. Lee J. Rodrigues M. Nagata T. Tanihata J. Nozohourmehrabad A. Panesar D. Miskew B. Aoki Y. Yokota T. Mutation types and aging differently affect revertant fiber expansion in dystrophic mdx and mdx52 mice.PLoS ONE. 2013; 8e69194 Crossref PubMed Scopus (16) Google Scholar). Importantly, the mdx52 model also allows for the testing of patient mutation-relevant exon skipping strategies in vivo (e.g. targeting exon 51 or exon 53) (33Echigoya Y. Aoki Y. Miskew B. Panesar D. Touznik A. Nagata T. Tanihata J. Nakamura A. Nagaraju K. Yokota T. Long-term efficacy of systemic multiexon skipping targeting Dystrophin exons 45-55 with a cocktail of vivo-morpholinos in Mdx52 mice.Mol. Ther. - Nucleic Acids. 2015; 4: e225Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 34Echigoya Y. Lim K.R.Q. Trieu N. Bao B. Miskew Nichols B. Vila M.C. Novak J.S. Hara Y. Lee J. Touznik A. Mamchaoui K. Aoki Y. Takeda S. Nagaraju K. Mouly V. Maruyama R. Duddy W. Yokota T. Quantitative antisense screening and optimization for exon 51 skipping in Duchenne muscular dystrophy.Mol. Ther. 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Although the global quantification of nucleic acid transcripts is relatively simple using digital gene expression analysis (i.e. RNA-sequencing) or hybridization methods (e.g. DNA microarrays), proteomics analysis is substantially more challenging. Early proteomics studies in dystrophic muscle used 2D-electrophoresis to identify differentially abundant proteins, but results were limited to only a handful of differential expression calls (44Gardan-Salmon D. Dixon J.M. Lonergan S.M. Selsby J.T. Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice.Eur. J. Appl. Physiol. 2011; 111: 2763-2773Crossref PubMed Scopus (43) Google Scholar, 45Carberry S. Zweyer M. Swandulla D. Ohlendieck K. Profiling of age-related changes in the tibialis anterior muscle proteome of the mdx mouse model of dystrophinopathy.J. Biomed. Biotechnol. 2012; 2012: 1-11Crossref PubMed Scopus (22) Google Scholar). The use of other methodologies such as iCAT, in vivo SILAC, and label-free approaches resulted in the identification of further differentially expressed proteins in dystrophic muscle, although these studies were still limited by their low proteomic coverage (∼1000 proteins quantified, or less) (46Guevel L. Lavoie J.R. Perez-Iratxeta C. Rouger K. Dubreil L. Feron M. Talon S. Brand M. Megeney L.A. Quantitative proteomic analysis of dystrophic dog muscle.J. Proteome Res. 2011; 10: 2465-2478Crossref PubMed Scopus (59) Google Scholar, 47Rayavarapu S. Coley W. Cakir E. Jahnke V. Takeda S. Aoki Y. Grodish-Dressman H. Jaiswal J.K. Hoffman E.P. Brown K.J. Hathout Y. Nagaraju K. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.Mol. Cell. Proteomics. 2013; 12: 1061-1073Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 48Capitanio D. Moriggi M. Torretta E. Barbacini P. De Palma S. Viganò A. Lochmüller H. Muntoni F. Ferlini A. Mora M. Gelfi C. Comparative proteomic analyses of Duchenne muscular dystrophy and Becker muscular dystrophy muscles: changes contributing to preserve muscle function in Becker muscular dystrophy patients.J. Cachexia. Sarcopenia Muscle. 2020; 11: 547-563Crossref PubMed Scopus (13) Google Scholar). Global proteome profiling in fibrous tissues such as skeletal muscle is complicated by the presence of very high concentrations of a few structural proteins, such as actins and myosins (49Ohlendieck K. Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques.Skelet. Muscle. 2011; 1: 6Crossref PubMed Scopus (80) Google Scholar). Peptides derived from these proteins mask signals from lowly abundant proteins and thereby limit the depth of proteome coverage that can be achieved. To increase analytical depth, we recently applied high-resolution sample pre-fractionation based on narrow-range isoelectric focusing of peptides to quantify expression of over 3272 proteins in mouse muscle (39Roberts T.C. Johansson H.J. McClorey G. Godfrey C. Blomberg K.E.M. Coursindel T. Gait M.J. Smith C.I.E. Lehtiö J. EL Andaloussi S. Wood M.J.A. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.Hum. Mol. Genet. 2015; 24: 6756-6758Crossref PubMed Scopus (27) Google Scholar). To date, there have been relatively few high-resolution proteomics studies in dystrophic muscle, and fewer still that have measured global changes in protein expression in muscle throughout the progression of pathology over time (39Roberts T.C. Johansson H.J. McClorey G. Godfrey C. Blomberg K.E.M. Coursindel T. Gait M.J. Smith C.I.E. Lehtiö J. EL Andaloussi S. Wood M.J.A. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.Hum. Mol. Genet. 2015; 24: 6756-6758Crossref PubMed Scopus (27) Google Scholar, 47Rayavarapu S. Coley W. Cakir E. Jahnke V. Takeda S. Aoki Y. Grodish-Dressman H. Jaiswal J.K. Hoffman E.P. Brown K.J. Hathout Y. Nagaraju K. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.Mol. Cell. Proteomics. 2013; 12: 1061-1073Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 50Murphy S. Zweyer M. Raucamp M. Henry M. Meleady P. Swandulla D. Ohlendieck K. Proteomic profiling of the mouse diaphragm and refined mass spectrometric analysis of the dystrophic phenotype.J. Muscle Res. Cell Motil. 2019; 40: 9-28Crossref PubMed Scopus (11) Google Scholar). Here, we have performed MS-based proteomic profiling in both the mdx and mdx52 DMD mouse models compared with WT controls (WT) at three ages representing different stages of dystrophic pathology. We show that this state-of-the-art proteomic strategy has uncovered previously unidentified pathological pathways in dystrophic mouse models, which are potential therapeutic targets for DMD. Mice were housed under 12:12 h light-dark conditions with food and water ad libitum. All experimental protocols in this study were approved by the Experimental Animal Care and Use Committee of the National Institute of Neuroscience, NCNP, Japan. mdx52 mice were generated at our facility at the NCNP (31Araki E. Nakamurab K. Nakaob K. Kameyac S. Kobayashid O. Nonakad I. Kobayashia T. Katsuki M. Araki E. Nakamura K. Nakao K. Kameya S. Kobayashi O. Nonaka I. Kobayashi T. Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy.Biochem. Biophys. Res. Commun. 1997; 238: 492-497Crossref PubMed Scopus (81) Google Scholar) and have been back-crossed with C57BL/6 mice for more than 10 generations. mdx mice on a C57BL/6 background were kindly provided by Dr T. Sasaoka (Brain Research Institute, Niigata University, Niigata, Japan). C57BL/6 mice were used as controls to match the background of the dystrophic strains (i.e. mdx52 and mdx). Serum and tissues from each strain were collected at 4, 8, 16, 24, 48, and 80 weeks of age (n = 3–5 per group). Tibialis anterior (TA) muscle of all strains at 8, 16, and 80 weeks (n = 3) was subsequently cryosectioned, collecting 50 sections of 10 μm for each sample. C2C12 myoblasts were cultured at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% antibiotics/antimycotics (growth medium: GM) (all Invitrogen, Carlsbad, CA). DMEM supplemented with 2% horse serum (HS) and 1% antibiotics/antimycotics (differentiation medium: DM) was used to differentiate C2C12 myoblasts for 3–6 days to form multinucleated myotubes. C2C12 myoblasts were seeded in 24-well and 6-well plates at 100,000 cells and 400,000 cells per well respectively. For transfections, cells were incubated in GM before addition of 50 nm siRNA complexes (either targeting Mvp or a control siRNA, ON-TARGETplus siRNA: Dharmacon, Cambridge, UK). Complex formation was performed in the absence of serum, and cells collected after 3 days in DM. Serum (50 µl) from each sample (n = 2–4) was mixed with TRIzol LS (ThermoFisher Scientific), supplemented with 3 µl of a spike-in of 5 nm cel-miR-39 (5ʹ-UCACCGGGUGUAAAUCAGCUUG-3ʹ) per sample as an exogenous reference control. RNA was then extracted according to manufacturer's instructions, with modifications previously described in (51Roberts T.C. Coenen-Stass A.M.L. Betts C.A. Wood M.J.A. Detection and quantification of extracellular microRNAs in murine biofluids.Biol. Proced. Online. 2014; 16: 5Crossref PubMed Scopus (23) Google Scholar). Samples were stored at −80 °C. TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA) was used for cDNA synthesis of miRNAs. Primers for miR-1a-3p (5ʹ-UGGAAUGUAAAGAAGUAUGUAU-3ʹ), miR-133a-3p (5ʹ-UUUGGUCCCCUUCAACCAGCUG-3ʹ), miR-206-3p (5ʹ-UGGAAUGUAAGGAAGUGUGUGG-3ʹ), miR-223-3p (5ʹ-UGUCAGUUUGUCAAAUACCCCA-3ʹ) and cel-miR-39 were used, following manufacturer's instructions. TaqMan Gene Expression Master Mix (Applied Biosystems) was used for qPCR, following manufacturer's instructions. Primers for miR-1a-3p, miR-133a-3p, miR-206-3p, miR-223-3p and cel-miR-39 were obtained from ThermoFisher and data were normalized as previously described (52Roberts T.C. Coenen-Stass A.M.L. Wood M.J.A. Assessment of RT-qPCR normalization strategies for accurate quantification of extracellular microRNAs in murine Serum.PLoS ONE. 2014; 9e89237 Crossref" @default.
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• W3090995810 title "Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy" @default.
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