Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, and Increased Expression of Extracellular Matrix Proteins in Laminin α2 Chain–deficient Muscle

Abstract

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). 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). Congenital muscular dystrophy with laminin α2 chain deficiency, also known as MDC1A, 1The abbreviations used are:MDC1Amerosin congenital muscular dystrophy type 1ATMTtandem mass tags. 1The abbreviations used are:MDC1Amerosin congenital muscular dystrophy type 1ATMTtandem mass tags. is a severe muscle wasting disease for which there is no cure. MDC1A is caused by mutations in the LAMA2 gene that lead to complete or partial deficiency of laminin α2 chain (1Allamand V. Guicheney P. Merosin-deficient muscular dystrophy, autosomal recessive (MDC1A, MIM#156225, LAMA2 gene coding for α2 chain of laminin).Eur. J. Hum. Genet. 2002; 10: 91-94Crossref PubMed Scopus (112) Google Scholar, 2Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tomé F.M.S. Schwartz K. Fardeau M. Tryggvason K. Guicheney P. Mutations in the laminin α2 chain gene (LAMA2) cause merosin-deficient muscular dystrophy.Nat. Genet. 1995; 11: 216-218Crossref PubMed Scopus (566) Google Scholar, 3Voit T. Tomé F.S. The congenital muscular dystrophies.in: Angel A. Franzini-Armstrong C. Myology. McGraw-Hill, New York2004: 1203-1238Google Scholar). Although the primary defect in MDC1A is known, the secondary molecular mechanisms eventually leading to muscle degeneration are not fully understood. In normal muscle, laminin α2 chain binds to the cell surface receptors dystroglycan and integrin α7β1, which both indirectly bind the cytoskeleton (4Ibraghimov-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.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1195) Google Scholar, 5Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. Binding of the G domains of laminin α1 and α2 chains and perlecan to heparin, sulfatides, α-dystroglycan and several extracellular matrix proteins.EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar, 6von der Mark H. Williams I. Wendler O. Sorokin L. von der Mark K. Pöschl E. Alternative splice variants of α7β1 integrin selectively recognize different laminin isoforms.J. Biol. Chem. 2002; 277: 6012-6016Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 7Gawlik K.I. Durbeej M. Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies.Skelet. Muscle. 2011; 1: 9Crossref PubMed Scopus (86) Google Scholar). Both of these adhesion complexes are important for normal skeletal muscle function, and laminin α2 chain binding to dystroglycan contributes to the maintenance of sarcolemmal integrity and protects muscles from damage (8Han R. Kanagawa M. Yoshida-Moriguchi T. Rader E.P. Ng R.A. Michele D.E. Muirhead D.E. Kunz S. Moore S.A. Iannaccone S.T. Miyake K. McNeil P.L. Mayer U. Oldstone M.B.A. Faulkner J.A. Campbell K.P. Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of α-dystroglycan.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12573-12579Crossref PubMed Scopus (117) Google Scholar), whereas laminin α2 chain binding to integrin α7β1 promotes myofiber survival (9Vachon P.H. Xu H. Liu L. Loechel F. Hayashi Y. Arahata K. Reed J.C. Wewer U.M. Engvall E. Integrins (α7β1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.J. Clin. Invest. 1997; 10: 1870-1881Crossref Scopus (183) Google Scholar, 10Mayer U. Integrins: redundant or important players in skeletal muscle and beyond.J. Biol. Chem. 2003; 279: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In MDC1A, laminin α2 chain is absent or severely reduced, and the expression of dystroglycan and α7β1 is also dysregulated in MDC1A (9Vachon P.H. Xu H. Liu L. Loechel F. Hayashi Y. Arahata K. Reed J.C. Wewer U.M. Engvall E. Integrins (α7β1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.J. Clin. Invest. 1997; 10: 1870-1881Crossref Scopus (183) Google Scholar, 11Gawlik K.I. Mayer U. Blomberg K. Sonnenberg A. Ekblom P. Durbeej M. Laminin α1 chain mediated reduction of laminin α2 chain deficient muscular dystrophy involves integrin α7β1 and dystroglycan.FEBS Lett. 2006; 580: 1759-1765Crossref PubMed Scopus (44) Google Scholar, 12Moll 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.Nature. 2001; 413: 302-307Crossref PubMed Scopus (198) Google Scholar). Thus, the structural link is broken, and the yet to be determined downstream intracellular signaling pathways are also interrupted. Consequently, laminin α2 chain–deficient muscle fibers undergo degeneration–regeneration cycles, but rather quickly regeneration fails and muscle fibers die by apoptosis/necrosis followed by a major replacement of muscle tissue with connective tissue (3Voit T. Tomé F.S. The congenital muscular dystrophies.in: Angel A. Franzini-Armstrong C. Myology. McGraw-Hill, New York2004: 1203-1238Google Scholar, 7Gawlik K.I. Durbeej M. Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies.Skelet. Muscle. 2011; 1: 9Crossref PubMed Scopus (86) Google Scholar). In order to unravel novel secondary molecular mechanisms, which could indicate new therapeutic targets, we decided to evaluate the protein expression profile in laminin α2 chain–deficient dy3K/dy3K muscle. Several proteomic profiling studies of dystrophin-deficient muscles (Duchenne muscular dystrophy) have been performed (13Doran 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.Proteomics. 2006; 6: 4610-4621Crossref PubMed Scopus (113) Google Scholar, 14Gardan-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. 2011; PubMed Scopus Google Scholar, S. M. D. Ohlendieck K. reveals drastic increase of extracellular matrix proteins and in the mdx diaphragm model of muscular J. PubMed Scopus (44) Google Scholar, Y. Proteomic analysis of mdx skeletal reduction of expression and 2003; PubMed Scopus Google Scholar, L. C. K. L. M. S. M. proteomic analysis of dystrophic Proteome 10: Scholar, Durbeej M. for proteomic a comparative of and affected muscles from mdx the phase of PubMed Scopus Google Scholar, S. W. E. V. S. Y. H. E.P. Y. K. of disease pathways using in in dystrophin deficient mdx Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. Ohlendieck K. Proteomic profiling of muscular dystrophy.J. 2009; PubMed Scopus Google Scholar), as well as with muscles muscular dystrophy type C. I. Faulkner G. L. R. R. E. identification of differentially expressed proteins in the muscle of Clin. Appl. 2009; PubMed Scopus Google Scholar, A. I. der S.M. Proteomic analysis of the protein for sarcolemmal maintenance and PubMed Scopus Google Scholar). a of proteins that were differentially expressed in different dystrophic muscles and different (13Doran 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.Proteomics. 2006; 6: 4610-4621Crossref PubMed Scopus (113) Google Scholar, 14Gardan-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. 2011; PubMed Scopus Google Scholar, S. M. D. Ohlendieck K. reveals drastic increase of extracellular matrix proteins and in the mdx diaphragm model of muscular J. PubMed Scopus (44) Google Scholar, Y. Proteomic analysis of mdx skeletal reduction of expression and 2003; PubMed Scopus Google Scholar, L. C. K. L. M. S. M. proteomic analysis of dystrophic Proteome 10: Scholar, Durbeej M. for proteomic a comparative of and affected muscles from mdx the phase of PubMed Scopus Google Scholar, S. W. E. V. S. Y. H. E.P. Y. K. of disease pathways using in in dystrophin deficient mdx Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. Ohlendieck K. 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Durbeej M. the muscle of laminin α2 Genet. 2011; PubMed Scopus Google Scholar). were in the of to the All mouse by the for were by of and the diaphragm and gastrocnemius muscles were in and using a and we used the as Durbeej M. for proteomic a comparative of and affected muscles from mdx the phase of PubMed Scopus Google Scholar). different for and were of muscles from in and via were for and The protein determined using a were to the for the mass and of proteins were in of were to the and the to a of with of were and for of and were for from were with of protein were in of and were and of were used to of to be a of of the used to the protein for Our design a in order to to The for and were with of for and were by using of of and and as The were and in of for gastrocnemius and diaphragm muscles and and with and The a of in a new were the the and were into and with were with to The were a and a phase Both were with The the by a to and the as to to were as for MS mass The in a with one MS of followed by and of the most and in MS The for the were as for mass of with a of and for with a of of a for used for one most of the to be for All were a as and also a using of a the identification the in the MS data from one and MS were for and identification using Proteome The performed with the using the in protein from MS of of one with and The protein in the to a using the and identified proteins were by to the of in from were used to via the to the for a protein were for to or proteins of the with a of and the of were a to or the of protein and the of used for protein the were into for data The mass data have been deposited to the ProteomeXchange via the J.A. R. A. F. D. J.A. Z. T. I. M. Mayer G. L. A. S. R. L. H. ProteomeXchange data and PubMed Scopus Google with the identifier the for and for the which proteins were differentially proteins in for the were were and performed to compared proteins should a of and a protein or In order to were and to in studies using 6: Full Text Full Text PDF PubMed Scopus Google Scholar, R. for Natl. Acad. Sci. U.S.A. 2003; PubMed Scopus Google Scholar). Also, to the of the using the to Y. The of analysis in 2011; 11: PubMed Scopus Google for were as were and proteins were as but from a different for of protein were performed to using in a followed by were in in with were with primary mouse mouse and for and for in with were in for and with secondary with with were in to and the to the in protein expression for Skeletal muscle and of were for K. Y. Ekblom P. S. Durbeej M. Laminin α1 chain muscular dystrophy in laminin α2 chain deficient Genet. PubMed Scopus Google with and were using a were using with the been used as the proteomic to dystrophic muscle (13Doran 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.Proteomics. 2006; 6: 4610-4621Crossref PubMed Scopus (113) Google Scholar, 14Gardan-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. 2011; PubMed Scopus Google Scholar, S. M. D. Ohlendieck K. reveals drastic increase of extracellular matrix proteins and in the mdx diaphragm model of muscular J. PubMed Scopus (44) Google Scholar, Y. Proteomic analysis of mdx skeletal reduction of expression and 2003; PubMed Scopus Google Scholar, L. C. K. L. M. S. M. proteomic analysis of dystrophic Proteome 10: Scholar). several with we decided to a proteomic and the multidimensional protein identification technology In of are with a to identification and of proteins in the In we used and were with for A. J. K. S. J. G. T. R. C. mass a novel for comparative analysis of protein by Chem. 2003; PubMed Scopus Google Scholar). were using with and phase to and protein were and and the identification and of in using a mass were identified and by of from to Biol. 2009; PubMed Scopus Google Scholar, D. E. S. J.C. The for the most complete the PubMed Scopus Google Scholar). gastrocnemius and diaphragm muscles from laminin α2 chain–deficient dy3K/dy3K and dy3K/dy3K muscles as or fibers indicate in muscle of and the of or and fibrotic tissue V. R. Durbeej M. the muscle of laminin α2 Genet. 2011; PubMed Scopus Google Scholar, Y. K. I. M. Y. Arahata K. Y. S. Laminin α2 by of the a new model of congenital muscular Lett. 1997; PubMed Scopus Google Scholar). the multidimensional protein identification technology we identified 700 proteins in gastrocnemius and diaphragm muscles 113 and 101 proteins, respectively, were to be differentially in by a of in dy3K/dy3K gastrocnemius and diaphragm muscle to muscles. In laminin α2 chain–deficient gastrocnemius muscle, there were and proteins, and in diaphragm muscle, proteins were and were of the proteins and of the proteins in the muscles were a of the differentially expressed proteins were intracellular proteins, but also several extracellular and membrane proteins were used the to the proteins with expression into different the proteins were proteins involved in metabolic processes, of and processes, processes, processes, and the proteins were proteins involved in processes, metabolic processes, cell processes, and proteins, and proteins were the most proteins in dy3K/dy3K whereas proteins, proteins, and extracellular matrix proteins were the most proteins A of the proteins to the of metabolic of and as metabolic processes, and metabolic processes, and chain of these pathways is the proteins were in laminin α2 chain–deficient and and in and metabolic and of and metabolic and and of and of in and Also, in a gene expression profiling we that several were to metabolic M. R. V. J. V. M. S. Brancaccio A. Mayer U. Durbeej M. binds integrin and is in laminin α2 muscular dystrophy.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). performed analysis to the proteomic and in laminin α2 chain–deficient muscle An increase in calcium been to the of muscular dystrophy of muscular Full Text Full Text PDF Scopus Google Scholar), but is not yet calcium is in In muscular muscular the muscle cell membrane is and could lead to of calcium in the calcium a of The calcium to of the muscle mechanisms C. Martin D. M. H. P. of in the calcium in dystrophic mouse skeletal muscle Biol. 2002; PubMed Scopus Google Scholar, M.A. H. G. J. and of muscular dystrophy.Nat. PubMed Scopus Google Scholar, S.A. M.A. M. is to muscular dystrophy a Natl. Acad. Sci. U.S.A. 2009; 106: PubMed Scopus Google Scholar). Also, the expression of proteins involved in the of intracellular calcium could be in muscular dystrophy Ohlendieck K. calcium in muscular Appl. 2002; Scholar). 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V. membrane binding proteins with Sci. PubMed Scopus Google Scholar). Several of the and are differentially expressed in normal muscle to muscular dystrophy, and the are expressed in and in dystrophin-deficient muscle Durbeej M. for proteomic a comparative of and affected muscles from mdx the phase of PubMed Scopus Google Scholar, S. C. D. expression in PubMed Scopus Google Scholar). The could have a in the in muscular dystrophy, and MDC1A is characterized by S. A. J.A. the pathogenesis of congenital muscular 2011; 6: PubMed Scopus Google Scholar). several of the proteins are extracellular matrix gene expression profiling have that of extracellular matrix is in congenital muscular in muscular dystrophy M. H. S. J. T. T. Guicheney P. K. I. S. 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