Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy3K/dy3K mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange. org/dataset/PXD000978).131130013013Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; 2014-10-6; CAPES; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; KAW2007.0118; Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Allamand, V., Guicheney, P., Merosin-deficient muscular dystrophy, autosomal recessive (mdc1a, mim#156225, lama2 gene coding for -2 chain of laminin) (2002) Eur. J. Hum. Genet., 10, pp. 91-94Helbling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tomé, F.M.S., Guicheney, P., Mutations in the laminin -2 chain gene (lama2) cause merosin-deficient muscular dystrophy (1995) Nat. Genet., 11, pp. 216-218Voit, T., Tomé, F.S., The congenital muscular dystrophies (2004) Myology, 2, pp. 1203-1238. , (Angel, A., and Franzini-Armstrong, C., eds McGraw-Hill, New YorkIbraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J., Slaughter, C.A., Sernett, S.W., Campbell, K.P., Primary structure of dystrophin-Associated glycoproteins linking dystrophin to the extracellular matrix (1992) Nature, 355, pp. 696-702Talts, J.F., Andac, Z., Gohring, W., Brancaccio, A., Timpl, R., Binding of the g domains of laminin -1 and -2 chains and perlecan to heparin, sulfatides, -dystroglycan and several extracellular matrix proteins (1999) EMBO J., 18, pp. 863-870Von Der Mark, H., Williams, I., Wendler, O., Sorokin, L., Von Der Mark, K., Pöschl, E., Alternative splice variants of -7-1 integrin selectively recognize different laminin isoforms (2002) J. Biol. Chem., 277, pp. 6012-6016Gawlik, K.I., Durbeej, M., Skeletal muscle laminin and mdc1a: Pathogenesis and treatment strategies (2011) Skelet. Muscle, 1, p. 9Han, R., Kanagawa, M., Yoshida-Moriguchi, T., Rader, E.P., Ng, R.A., Michele, D.E., Muirhead, D.E., Campbell, K.P., Basal lamina strengthens cell membrane integrity via the laminin g domain-binding motif of -dystroglycan (2009) Proc. Natl. Acad. Sci. U.S.A., 106, pp. 12573-12579Vachon, P.H., Xu, H., Liu, L., Loechel, F., Hayashi, Y., Arahata, K., Reed, J.C., Engvall, E., Integrins (-7-1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy (1997) J. Clin. Invest., 10, pp. 1870-1881Mayer, U., Integrins: Redundant or important players in skeletal muscle and beyond (2003) J. Biol. Chem., 279, pp. 14587-14590Gawlik, K.I., Mayer, U., Blomberg, K., Sonnenberg, A., Ekblom, P., Durbeej, M., Laminin -1 chain mediated reduction of laminin -2 chain deficient muscular dystrophy involves integrin -7-1 and dystroglycan (2006) FEBS Lett., 580, pp. 1759-1765Moll, J., Barzaghi, P., Lin, S., Bezakova, G., Lochmuller, H., Engvall, E., Muller, U., Ruegg, M.A., An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy (2001) Nature, 413, pp. 302-307Doran, P., Martin, G., Dowling, P., Jockusch, H., Ohlendieck, K., Proteome analysis of the dystrophin-deficient mdx diaphragm reveals a drastic increase in the heat shock protein cvhsp (2006) Proteomics, 6, pp. 4610-4621Gardan-Salmon, D., Dixon, J.M., Lonergan, S.M., Selsby, J.T., Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice (2011) Eur. J. Appl. Physiol., 111, pp. 2763-2773Carberry, S., Zweyer, M., Swandulla, D., Ohlendieck, K., Proteomics reveals drastic increase of extracellular matrix proteins collagen and dermatopontin in the aged mdx diaphragm model of duchenne muscular dystrophy (2012) Int. J. Mol. Med., 30, pp. 229-234Ge, Y., Molloy, M.P., Chamberlain, J.S., Andrews, P.C., Proteomic analysis of mdx skeletal muscle: Great reduction of adenylate kinase 1 expression and enzymatic activity (2003) Proteomics, 3, pp. 1895-1903Guevel, L., Lavoie, J.R., Perez-Iratxeta, C., Rouger, K., Dubreil, L., Feron, M., Talon, S., Megeney, L.A., Quantitative proteomic analysis of dystrophic dog muscle (2010) J. Proteome Res., 10, pp. 2465-2449Matsumura, C.Y., Menezes De Oliveira, B., Durbeej, M., Marques, M.J., Isobaric tagging-based quantification for proteomic analysis: A comparative study of spared and affected muscles from mdx mice at the early phase of dystrophy (2013) PLoS One, 8, p. e65831Rayavarapu, S., Coley, W., Cakir, E., Jahnke, V., Takeda, S., Aoki, Y., Grodish-Dressman, H., Nagaraju, K., Identification of disease specific pathways using in vivo silac proteomics in dystrophin deficient mdx mouse (2013) Mol. Cell. Proteomics, 12, pp. 1061-1073Lewis, C., Carverry, S., Ohlendieck, K., Proteomic profiling of x-linked muscular dystrophy (2009) J. Muscle Res. Cell. Motil., 30, pp. 267-279De La Torre, C., Illa, I., Faulkner, G., Soria, L., Robles-Cedeno, R., Pereles-Dominguez-Perles, R., De Luna, N., Gallardo, E., Proteomics identification of differentially expressed proteins in the muscle of dysferlin myopathy patients (2009) Proteomics Clin. Appl., 3, pp. 486-497De Morrée, A., Hensbergen, P.J., Van Haagen, H.H., Dragan, I., Deelder, A.M., T'Hoen, P.A., Frants, R., Van Den Maarel, S.M., Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity (2010) PLoS One, 5, p. e13854Washburn, M., Wolters, D., Yates, J., III, Large scale analysis of the yeast proteome by multidimensional protein identification technology (2001) Nat. Biotechnol., 19, pp. 242-247Thompson, A., Schäfer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., Neumann, T., Hamon, C., Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by ms/ms (2003) Anal. Chem., 75, pp. 1895-1904Carmignac, V., Quere, R., Durbeej, M., Proteasome inhibition improves the muscle of laminin -2 chain-deficient mice (2011) Hum. Mol. Genet., 20, pp. 541-552Vizcaíno, J.A., Deutsch, E.W., Wang, R., Csordas, A., Reisinger, F., Ríos, D., Dianes, J.A., Hermjakob, H., Proteome-xchange provides globally co-ordinated proteomics data submission and dissemination (2014) Nat. Biotechnol., 32, pp. 223-226Karp, N.A., McCormick, P.S., Russell, M.R., Lilley, K.S., Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in-gel electrophoresis (2007) Mol. Cell. Proteomics, 6, pp. 1354-1364Storey, J.D., Tibshirani, R., Statistical significance for genome wide studies (2003) Proc. Natl. Acad. Sci. U.S.A., 100, pp. 9440-9445Levin, Y., The role of statistical power analysis in quantitative proteomics (2011) Proteomics, 11, pp. 2565-2567Gawlik, K., Miyagoe-Suzuki, Y., Ekblom, P., Takeda, S., Durbeej, M., Laminin -1 chain reduces muscular dystrophy in laminin -2 chain deficient mice (2004) Hum. Mol. Genet., 13, pp. 1775-1784Kline, K.G., Wu, C.C., Mudpit analysis: Application to human heart tissue (2009) Methods Mol. Biol., 528, pp. 281-293Martins De Souza, D., Oliveira, B.M., Castro-Dias, E., Winck, F.V., Horiuchi, R.S., Baldasso, P.A., Caetano, H.T., Novello, J.C., The untiring search for the most complete proteome representation: Reviewing the methods (2008) Brief Funct. Genomic. Proteomic., 7, pp. 312-321Miyagoe, Y., Hanaoka, K., Nonaka, I., Hayasaka, M., Nabeshima, Y., Arahata, K., Nabeshima, Y., Takeda, S., Laminin -2 chain-null mutant mice by targeted disruption of the lama2 gene: A new model of merosin (laminin 2)-deficient congenital muscular dystrophy (1997) FEBS Lett., 415, pp. 33-39Häger, M., Bigotti, M.G., Meszaros, R., Carmignac, V., Holmberg, J., Allamand, V., Åkerlund, M., Durbeej, M., Cib2 binds integrin -7b-1d and is reduced in laminin -2 chain-deficient muscular dystrophy (2008) J. Biol. Chem., 283, pp. 24760-247695Deconinck, N., Dan, B., Pathophysiology of duchenne muscular dystrophy: Current hypotheses (2007) Pediatr. Neurol., 26, pp. 1-7Vandebrouck, C., Martin, D., Colson-Van Schoor, M., Debaix, H., Gailly, P., Involvement of trpc in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers (2002) J. Cell Biol., 158, pp. 1089-1096Millay, D.P., Sargent, M.A., Osinska, H., Baines, C.P., Barton, E.R., Vuagniaux, G., Sweeney, H.L., Molkentin, J.D., Genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy (2008) Nat. Med., 14, pp. 442-447Millay, D.P., Goonasekera, S.A., Sargent, M.A., Maillet, M., Aronow, B.J., Molkentin, J.D., Calcium influx is sufficient to induce muscular dystrophy through a trpc-dependent mechanism (2009) Proc. Natl. Acad. Sci. U.S.A., 106, pp. 19023-19028Culligan, K.G., Ohlendieck, K., Abnormal calcium handling in muscular dystrophy (2002) Basic Appl. Myol., 12, pp. 147-157Straub, V., Rafael, J.A., Chamberlain, J.S., Campbell, K.P., Animal models for muscular dystrophy show different patterns of sarcolemmal disruption (1997) J. Cell Biol., 139, pp. 375-385Gawlik, K.I., Åkerlund, M., Carmignac, V., Elamaa, H., Durbeej, M., Distinct roles for laminin globular domains in laminin -1 chain mediated rescue of murine laminin -2 chain deficiency (2010) PLoS One, 5, p. e11549Rescher, U., Gereke, V., Annexins-unique membrane binding proteins with diverse functions (2004) J. Cell Sci., 117, pp. 2631-2639Probst-Cousinm, S., Berghoff, C., Neundörfer, B., Heuss, D., Annexin expression in inflammatory myopathies (2004) Muscle Nerve, 30, pp. 102-110Jeudi, S., Wardrop, K.E., Alessi, A., Dominov, J.A., Bcl-2 inhibits the innate immune response during early pathogenesis of murine congenital muscular dystrophy (2011) PLoS One, 6, p. e22369Taniguchi, M., Kurahashi, H., Noguchi, S., Sese, J., Okinaga, T., Tsukahara, T., Guicheney, P., Toda, T., Expression profiling from fukuyama-type congenital muscular dystrophy and laminin-2 deficient congenital muscular dystrophyIs congenital muscular dystrophy a primary fibrotic disease? (2006) Biochem. Biophys. Res. Commun., 342, pp. 489-502Kuang, W., Xu, H., Vachon, P.H., Engvall, E., Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models (1998) J. Clin. Invest., 102, pp. 844-852Meinen, S., Barzaghi, P., Lin, S., Lochmuller, H., Rüegg, M.A., Linker molecules between laminins and dystroglycan ameliorate laminin-2-deficient muscular dystrophy at all disease stages (2007) J. Cell Biol., 176, pp. 979-993Doe, J.A., Wuebbles, R.D., Allred, E.T., Rooney, J.E., Elorza, M., Burkin, D.J., Transgenic overexpression of the -7 integrin reduces muscle pathology and improves viability in the dy(w) mouse model of merosin-deficient congenital muscular dystrophy type 1a (2011) J. Cell Sci., 124, pp. 2287-2297Girgenrath, M., Dominov, J.A., Kostek, C.A., Miller, J.B., Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy (2004) J. Clin. Invest., 114, pp. 1635-1639Erb, M., Meinen, S., Barzaghi, P., Sumanovski, L.T., Courdier-Fruh, I., Rüegg, M.A., Meier, T., Omigapil ameliorates the pathology of muscle dystrophy caused by laminin-2 deficiency (2009) J. Pharmacol. Exp. Ther., 331, pp. 787-795Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M., Musclespecific expression of insulin-like growth factor 1 improves outcome in lama2dy-w mice, a model for congenital muscular dystrophy type 1a (2011) Hum. Mol. Genet., 20, pp. 2333-2343Carmignac, V., Svensson, M., Körner, Z., Elowsson, L., Matsumura, C., Gawlik, K., Allamand, V., Durbeej, M., Autophagy is increased in laminin -2 chain-deficient muscle and inhibition improves muscle morphology in a mouse model of mdc1a (2011) Hum. Mol. Genet., 20, pp. 4891-4902Rooney, J.E., Knapp, J.R., Hodges, B.L., Wuebbles, R.D., Burkin, D.J., Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy (2011) Am. J. Pathol., 180, pp. 1593-1602Elbaz, M., Yanay, N., Aga-Mizrachi, S., Brunschwig, Z., Kassis, I., Ettinger, K., Barak, V., Nevo, Y., Losartan, a therapeutic candidate in congenital muscular dystrophy: Studies in the dy2j/dy2j mouse (2012) Ann. Neurol., 71, pp. 699-708Meinen, S., Lin, S., Rüegg, M.A., Angiotensin ii type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-2-deficient congenital muscular dystrophy (mdc1a) (2012) Skelet. Muscle, 2, p. 18Turk, R., Sterrenburg, E., Van Der Wees, C.G., De Meijer, E.J., De Menezes, R.X., Groh, S., Campbell, K.P., Hoen, T.P.A., Common pathological mechanisms in mouse models for muscular dystrophies (2006) FASEB J., 20, pp. 127-129Chen, Y.W., Zhao, P., Borup, R., Hoffman, E.P., Expression profiling in the muscular dystrophies: Identification of novel aspects of molecular pathophysiology (2000) J. Cell Biol., 151, pp. 1321-1336Chinet, A.E., Even, P.C., Decrouy, A., Dystrophin-dependent efficiency of metabolic pathways in mouse skeletal muscles (1994) Experientia, 59, pp. 602-605Even, P.C., Decrouy, A., Chinet, A., Defective regulation of energy metabolism in the mdx-mouse skeletal muscles (1994) Biochem. J., 304, pp. 649-654Groh, S., Zong, H., Goddeeris, M.M., Lebakken, C.S., Venzke, D., Pessin, J.E., Campbell, K.P., Sarcoglycan complex: Implications for metabolic defects in muscular dystrophies (2009) J. Biol. Chem., 284, pp. 19178-19182Onopiuk, M., Brutkowski, W., Wierzbicka, K., Wojciechowska, S., Szczepanowska, J., Fronk, J., Lochmuller, H., Zablocki, K., (2009) Biochem. Biophys. Res. Commun., 386, pp. 463-466Le Borgne, F., Guyot, S., Logerot, M., Beney, L., Gervais, P., Demarquoy, J., Exploration of lipid metabolism in relation with plasma membrane properties of duchenne muscular dystrophy cells: Influence of l-carnitine (2012) PLoS One, 7, p. e49346Matsumura, C.Y., Pertille, A., Albuquerge, T.C., Santo Neto, H., Marques, M.J., Diltiazem and verapamil protect dystrophin-deficient muscle fibers of mdx mice from degeneration: A potential role in calcium buffering and sarcolemmal stability (2009) Muscle Nerve, 39, pp. 167-176Jørgensen, L.H., Blain, A., Greally, E., Lavala, S.H., Blamire, A.M., Davison, B.J., Brinkmeier, H., Lochmüller, H., Long-term blocking of calcium channels in mdx mice results in differential effects on heart and skeletal muscle (2011) Am. J. Pathol., 178, pp. 273-283Goonasekera, S.A., Lam, C.K., Millay, D.P., Sargent, M.A., Hajjar, R.J., Kranias, E.G., Molkentin, J.D., Mitigation of muscular dystrophy in mice by serca overexpression in skeletal muscle (2011) J. Clin. Invest., 121, pp. 1044-1052Horiuchi, K., Amizuka, N., Takeshita, S., Takamatsu, H., Katsuura, M., Ozawa, H., Toyama, Y., Kudo, A., Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor (1999) J. Bone Miner. Res., 14, pp. 1239-1249Lorts, A., Schwanekamp, J.A., Baudino, T.A., McNally, E.M., Molkentin, J.D., Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor- pathway (2012) Proc. Natl. Acad. Sci. U.S.A., 109, pp. 10978-10983Ting, L., Rad, R., Gygi, S.P., Haas, W., Ms3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics (2011) Nat. Methods, 8, pp. 937-940Christoforou, A., Lilley, K.S., Taming the isobaric tagging elephant in the rooms in quantitative proteomics (2011) Nat. Methods, 8, pp. 911-913