- Email: [email protected]
EM.P.3.03 Differential expression of genes involved in muscular degeneration in four dystrophic mouse models
574
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
used to estimate mdx mouse respiratory impairment, systemic functional assessments compared with histopathological analysis have not been demonstrated. Here we report a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected during early insufficiency in the mdx mouse as well as an assessment of muscle tetanic force. In result, the dystrophic changes in the diaphragm lead to respiratory dysfunctions. Moreover, we propose gene therapy approaches targeting diaphragm with delivery of viral vectors. These total assessment system for rodent respiratory function, including whole-body plethysmography, may be useful to evaluate the therapeutic approaches for the neuro-muscular disease models. doi:10.1016/j.nmd.2009.06.097 INVESTIGATIONS OF THE EXTRACELLULAR MATRIX; POSTER PRESENTATIONS EM.P.3.01 The adult phenotype of congenital muscular dystrophy (MDC1A) due to mutation of LAMA2 N. Canki-Klain1, C. Béroud 2, N.F. Clarke 3, I. Kovac 4, S. Chambert 2, P. Guicheney 3 1 Zagreb University School of Medicine and University Hospital Centre Zagreb, Croatian Institute for Brain Research& Dept. Neurol., Zagreb, Croatia, 2 INSERM, U827, Université Montpellier 1, Montpellier, France, 3 Inserm UMR S956, CHU Pitié-Salpêtrière, Paris, France, 4 University Hospital Centre Zagreb,Institute for Rehabilitation and Orthopaedic Devices, Department of Physical Medicine and Rehabilitation, Zagreb, Croatia
Mutations in the laminin alpha 2-chain gene (LAMA2) were originally identified in a subset of patients with autosomal recessive merosin-deficient congenital muscular dystrophy linked to chromosome 6 (MDC1A). The clinical phenotype of these patients is characterized by severe muscle weakness at birth or in the first six months of life, delayed motor milestones, inability to walk, markedly raised creatine kinase, and abnormal white matter signal on brain MRI with normal intelligence. We report a 48-year-old man, born to unaffected parents who were second cousins. He had profound hypotonia at birth, started to walk at 6 years and was independently mobile until age 14 years. He could never fully elevate his arms and lost the ability to sit in vertical position from age 17 years. At his current age of 48 he has end-stage muscular atrophy, with his neck in fixed extreme extension, and a severe thoracolumbal scoliosis with torsion. His contractures made it impossible to perform brain MRI. The patient has normal intellect, progressive swallowing difficulties, signs of severe obstructive and restrictive pulmonary disease, and normal CK. Genome-wide SNP analysis of the proband, and close relatives using the Illumina 6000 SNP panel showed a large region of homozygosity around theLAMA2 gene. We confirmed homozygous linkage to LAMA2 by analysis of 8 flanking microsatellite markers and analysed LAMA2 for mutations from genomic DNA by sequencing the 65 exons and flanking intronic regions. We identified a novel homozygous mutation c.IVS15+3A>C (c.2208+3A>G) that is predicted to alter splicing of exon 15. The splicing defect induced by this mutation is unknown (no muscle available) but, as suggested by bioinformatics analysis and previous results in other genes, a reduced level of normal transcript is expected potentially resulting in partial laminin alpha 2 deficiency. This might explain why the patient achieved ambulation. doi:10.1016/j.nmd.2009.06.098
EM.P.3.02 Intracellular signaling pathway alterations in laminin a2 chain deficient skeletal muscle and brain V. Carmignac, M. Durbeej-Hjalt Biomedical Center B12, Division for Cell and Matrix Biology, University of Lund, Department of Experimental Medical Science, Lund, Sweden Laminin a2 chain, an extracellular matrix protein, is mainly expressed in the neuromuscular system. Homozygous mutations of the laminin a2 chain gene are associated with congenital muscular dystrophy 1A (MDC1A). Patients affected by MDC1A develop function abnormalities in skeletal muscle, peripheral nerve and central nervous system. To gain a greater insight into the molecular mechanisms by which loss of laminin a2 chain causes MDC1A, we have begun to characterize intracellular signaling cascades in muscle and brain. We found that expression of heat shock protein 70 and 90 is significantly reduced in skeletal muscle and brain from 4week-old dy3K/dy3K mice (a mouse model for MDC1A). Also, the global ubiquitination of proteins is also significantly raised in these tissues. Notably, the changes are detected before the dystrophic phenotype becomes evident (in 6-day-old mice). In skeletal muscle, the MAP kinase and AKT pathways remained intact, whereas the MAP kinase signaling pathway is upregulated in brain. Finally, all signaling abnormalities are restored in laminin a2 chain deficient mice overexpressing laminin a1 chain. In summary, we have started to assemble a map of the main components of the laminin a2 chain induced signal transduction pathways in skeletal muscle and brain in the hope that we can apply this knowledge to the treatment of MDC1A. doi:10.1016/j.nmd.2009.06.099
EM.P.3.03 Differential expression of genes involved in muscular degeneration in four dystrophic mouse models Onofre-Oliveira, P.C.M. Martins, V.L. Ferreira, P.C.G. D. Ayub-Guerrieri, M. Vainzof Human Genome Research Center – IB – USP, Genetics and Evolutive Biology, Sao Paulo, Brazil Muscle weakness in muscular dystrophies is the consequence of an imbalance between successive cycles of degeneration and regeneration, with further replacement of the degraded muscle fibers by adipose and connective tissues. One important marker for degeneration is the expression of TGFB1, which is an inflammatory cytokine with a possible role in the stimulation of fibrosis in the dystrophic muscle through the activation of genes related to the expression of collagen. Several mouse models for genetic muscular dystrophies myd are recognized. Among them, mdx, SJL/J, Lama2dy 2J/J and Large are models for Duchenne, LGMD2B, CMD1A and CMD1D muscular dystrophies, respectively, presenting variable histopathological and clinical findings. High expression of Tgfb1 and alpha 2 subunit of collagen 1 (Col1a2) genes has been found in the mdx mouse. Here we studied the expression of these two genes in three additional mouse models, carrying different defects in muscle proteins, to better understand the involved pathophysiological mechanisms, aiming future therapies. Using Real-Time PCR, we quantified expression of the dy Tgfb1 and Col1a2 genes in the mdx, SJL/J, Lama2 2J/J and Largemyd strains and normal controls. The expression data was compared to histopathological alterations in the respective muscles. Tgfb1 was found to be highly expressed in all four models. However, the Col1a2 gene was upregulated in mdx and SJL/J, both of which showed the most preserved muscle histology, but exhibited lower expression
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
in the more severely affected muscles from Lama2dy 2J/J and Largemyd strains. The results suggest that Tgfb1 gene is activated in the dystrophic process in all types/stages of degeneration while activation of Col1a2 gene possibly occurs in earliest stages of this process. Funding by FAPESP, CNPq-INCT, FINEP, ABDIM. doi:10.1016/j.nmd.2009.06.100
EM.P.3.04 Muscle-derived Duchenne muscular dystrophy fibroblasts show altered production of extra-cellular matrix components S. Zanotti, S. Gibertini, S. Saredi, F. Blasevich, R. Mantegazza, L. Morandi, M. Mora Fondazione IRCCS Istituto Neurologico C. Besta, Neuromuscular Diseases and Neuroimmunology Unit, Milano, Italy In order to increase understanding of pro-fibrotic mechanisms in dystrophic muscle, we isolated primary human fibroblasts from DMD and control muscle biopsies, and investigated their proliferating activity and soluble collagen production, as well as transcript and protein levels of decorin, myostatin, TGF-b1, MMP-1 (interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the MMP inhibitors TIMP-1, TIMP-2, TIMP-3 and TIMP-4. We found significantly greater proliferation rate and soluble collagen production in DMD fibroblasts than controls. Decorin, myostatin and MMP-7 transcripts and proteins were significantly upregulated, and MMP-1 and TIMP-3 were significantly downregulated in DMD fibroblasts compared to controls. The study has revealed several differences between fibroblasts from DMD muscle and those from normal muscle, particularly as regards factors likely to affect ECM turnover. Although the detailed significance of these differences is not clear, they show that DMD fibroblasts are characterized by a profibrotic phenotype. Dystrophin absence itself could exert a direct influence on ECM homeostasis by allowing leakage of cellular components to the extracellular space, or abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes. By such mechanisms dystrophin absence may influence muscle fibroblasts either directly by altering their adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. doi:10.1016/j.nmd.2009.06.101
EM.P.3.05 Expression of fibulins 1–5 during myogenesis in vitro and in skeletal muscle regenerating in vivo, and in dystrophic mdx muscles M.D. Grounds, T. Shavlakadze, J. Vukovic The University of Western Australia, School of Anatomy & human Biology, Perth, Australia Many myopathies are due to gene defects that result in absent or altered extracellular matrix (ECM) and numerous ECM molecules are in intimate contact with myofibres and with the quiescent muscle precursors (satellite cells). Complex interactions between ECM components and different cells play a crucial role in muscle function and all aspects of new muscle formation after damage. Fibulins (FBLN) are members of an adhesive glycoprotein family that are associated with fibrosis in some conditions. The role of FBLN 1–5 was investigated using immunostaining and qPCR in cultured myogenic C2C12 cells, in tissue sections of regenerating whole muscle grafts,
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in dystrophic (mdx) quadriceps and diaphragm, and in normal adult (2 month) and geriatric (23 month) mouse muscle. Cultured myoblasts expressed all fibulins and a subset was differentially regulated during myogenesis in myotubes. Immunostaining of regenerating myogenic cells (especially myotubes) in whole muscle grafts (at 6 days) generally confirmed this myogenic expression pattern. Dystrophic mdx quadriceps muscle (at 3 month) showed increased immunostaining for FBLN-1, -2 and -4 proteins around regenerating (small diameter) myofibres. While FBLN-3 and -5 were absent from dystrophic quadriceps aged up to 6 month, they were present in 3-month mdx diaphragm where they co-localised with fibronectin. These novel results indicate that different fibulins are differentially expressed and are involved in myogenesis and myofibre regeneration. A significant finding is that FBLN-3 and -5 are associated with the severe dystrophic pathology in the mdx diaphragm. Since fibrosis is a major complication resulting from progressive myofibre damage in dystrophic muscle, it is of especial interest to fully understand the roles of all ECM molecules, including fibulins, associated with this process. doi:10.1016/j.nmd.2009.06.102
EM.P.3.06 Transient upregulation of matrilin-2 gene expression suggests a role in early steps of skeletal muscle regeneration É. Korpos 1, L. Mátés 1, L. Mendler 2, M. Kiricsi 3, Á. Zvara 4, F. Deák 1, Z. Rottenberger 1, A. Keller-Pintér 2, L. Puskás 4, L. Dux2, I. Kiss 1 1
Biological Research Center of the Hungarian Academy of Sciences, Institute of Biochemistry, Szeged, Hungary, 2 University of Szeged, Faculty of General Medicine, Department of Biochemistry, Szeged, Hungary, 3 University of Szeged, Faculty of Natural Sciences, Department of Biochemistry and Molecular Biology, Szeged, Hungary, 4 Biological Research Center of the Hungarian Academy of Sciences, Laboratory of Functional Genomics, Szeged, Hungary Adult skeletal muscle has a remarkable regenerative capacity in response to injuries. The regeneration is dependent on an orchestrated response between the inductive signals of cytokines, growth factors and extracellular matrix (ECM) components. Matrilin-2 is a modular, oligomeric protein deposited in the ECM of many cell types. Colocalization in human skin and in vitro interaction of matrilin-2 with collagen I, fibrillin-2, fibronectin and the laminin-1-nidogen-1 complex were previously shown. We used the notexininduced in vivo model of muscle regeneration. Elevated level of matrilin-2 mRNA was observed already on day 2 of regeneration and reached maximum level between 4 and 7 days, similarly to myogenin gene expression. Matrilin-2 was localized in the matrix surrounding proliferating myoblasts on day 2 and around differentiating myoblasts and myotubes on day 4 of regeneration. In regenerating and control muscle partial colocalization of matrilin-2 was observed with fibronectin, the basement membrane laminin 2 and the costameric marker dystrophin. During in vitro differentiation of C2/7 myoblasts quantitative real-time RT-PCR showed a similar transient increase for matrilin-2, fibronectin, laminin A2 and collagen 6A1 gene expression. Immunostaining demonstrated deposition of matrilin-2 into extracellular filamentous network around proliferating, migrating and fusing myoblasts. We localized matrilin-2 in the epi-, peri- and endoneurium of the motoneurons. We co-localized matrilin-2 with alpha-bungarotoxin in the neuromuscular junctions between days 4 and 7, when the motor endplates are reformed. Detection of the targeted inactivation of Matn2 gene in transgenic mice on muscle regeneration is under progress. The observations suggest a role for matrilin-2 not only in the proliferation, migration,
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
used to estimate mdx mouse respiratory impairment, systemic functional assessments compared with histopathological analysis have not been demonstrated. Here we report a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected during early insufficiency in the mdx mouse as well as an assessment of muscle tetanic force. In result, the dystrophic changes in the diaphragm lead to respiratory dysfunctions. Moreover, we propose gene therapy approaches targeting diaphragm with delivery of viral vectors. These total assessment system for rodent respiratory function, including whole-body plethysmography, may be useful to evaluate the therapeutic approaches for the neuro-muscular disease models. doi:10.1016/j.nmd.2009.06.097 INVESTIGATIONS OF THE EXTRACELLULAR MATRIX; POSTER PRESENTATIONS EM.P.3.01 The adult phenotype of congenital muscular dystrophy (MDC1A) due to mutation of LAMA2 N. Canki-Klain1, C. Béroud 2, N.F. Clarke 3, I. Kovac 4, S. Chambert 2, P. Guicheney 3 1 Zagreb University School of Medicine and University Hospital Centre Zagreb, Croatian Institute for Brain Research& Dept. Neurol., Zagreb, Croatia, 2 INSERM, U827, Université Montpellier 1, Montpellier, France, 3 Inserm UMR S956, CHU Pitié-Salpêtrière, Paris, France, 4 University Hospital Centre Zagreb,Institute for Rehabilitation and Orthopaedic Devices, Department of Physical Medicine and Rehabilitation, Zagreb, Croatia
Mutations in the laminin alpha 2-chain gene (LAMA2) were originally identified in a subset of patients with autosomal recessive merosin-deficient congenital muscular dystrophy linked to chromosome 6 (MDC1A). The clinical phenotype of these patients is characterized by severe muscle weakness at birth or in the first six months of life, delayed motor milestones, inability to walk, markedly raised creatine kinase, and abnormal white matter signal on brain MRI with normal intelligence. We report a 48-year-old man, born to unaffected parents who were second cousins. He had profound hypotonia at birth, started to walk at 6 years and was independently mobile until age 14 years. He could never fully elevate his arms and lost the ability to sit in vertical position from age 17 years. At his current age of 48 he has end-stage muscular atrophy, with his neck in fixed extreme extension, and a severe thoracolumbal scoliosis with torsion. His contractures made it impossible to perform brain MRI. The patient has normal intellect, progressive swallowing difficulties, signs of severe obstructive and restrictive pulmonary disease, and normal CK. Genome-wide SNP analysis of the proband, and close relatives using the Illumina 6000 SNP panel showed a large region of homozygosity around theLAMA2 gene. We confirmed homozygous linkage to LAMA2 by analysis of 8 flanking microsatellite markers and analysed LAMA2 for mutations from genomic DNA by sequencing the 65 exons and flanking intronic regions. We identified a novel homozygous mutation c.IVS15+3A>C (c.2208+3A>G) that is predicted to alter splicing of exon 15. The splicing defect induced by this mutation is unknown (no muscle available) but, as suggested by bioinformatics analysis and previous results in other genes, a reduced level of normal transcript is expected potentially resulting in partial laminin alpha 2 deficiency. This might explain why the patient achieved ambulation. doi:10.1016/j.nmd.2009.06.098
EM.P.3.02 Intracellular signaling pathway alterations in laminin a2 chain deficient skeletal muscle and brain V. Carmignac, M. Durbeej-Hjalt Biomedical Center B12, Division for Cell and Matrix Biology, University of Lund, Department of Experimental Medical Science, Lund, Sweden Laminin a2 chain, an extracellular matrix protein, is mainly expressed in the neuromuscular system. Homozygous mutations of the laminin a2 chain gene are associated with congenital muscular dystrophy 1A (MDC1A). Patients affected by MDC1A develop function abnormalities in skeletal muscle, peripheral nerve and central nervous system. To gain a greater insight into the molecular mechanisms by which loss of laminin a2 chain causes MDC1A, we have begun to characterize intracellular signaling cascades in muscle and brain. We found that expression of heat shock protein 70 and 90 is significantly reduced in skeletal muscle and brain from 4week-old dy3K/dy3K mice (a mouse model for MDC1A). Also, the global ubiquitination of proteins is also significantly raised in these tissues. Notably, the changes are detected before the dystrophic phenotype becomes evident (in 6-day-old mice). In skeletal muscle, the MAP kinase and AKT pathways remained intact, whereas the MAP kinase signaling pathway is upregulated in brain. Finally, all signaling abnormalities are restored in laminin a2 chain deficient mice overexpressing laminin a1 chain. In summary, we have started to assemble a map of the main components of the laminin a2 chain induced signal transduction pathways in skeletal muscle and brain in the hope that we can apply this knowledge to the treatment of MDC1A. doi:10.1016/j.nmd.2009.06.099
EM.P.3.03 Differential expression of genes involved in muscular degeneration in four dystrophic mouse models Onofre-Oliveira, P.C.M. Martins, V.L. Ferreira, P.C.G. D. Ayub-Guerrieri, M. Vainzof Human Genome Research Center – IB – USP, Genetics and Evolutive Biology, Sao Paulo, Brazil Muscle weakness in muscular dystrophies is the consequence of an imbalance between successive cycles of degeneration and regeneration, with further replacement of the degraded muscle fibers by adipose and connective tissues. One important marker for degeneration is the expression of TGFB1, which is an inflammatory cytokine with a possible role in the stimulation of fibrosis in the dystrophic muscle through the activation of genes related to the expression of collagen. Several mouse models for genetic muscular dystrophies myd are recognized. Among them, mdx, SJL/J, Lama2dy 2J/J and Large are models for Duchenne, LGMD2B, CMD1A and CMD1D muscular dystrophies, respectively, presenting variable histopathological and clinical findings. High expression of Tgfb1 and alpha 2 subunit of collagen 1 (Col1a2) genes has been found in the mdx mouse. Here we studied the expression of these two genes in three additional mouse models, carrying different defects in muscle proteins, to better understand the involved pathophysiological mechanisms, aiming future therapies. Using Real-Time PCR, we quantified expression of the dy Tgfb1 and Col1a2 genes in the mdx, SJL/J, Lama2 2J/J and Largemyd strains and normal controls. The expression data was compared to histopathological alterations in the respective muscles. Tgfb1 was found to be highly expressed in all four models. However, the Col1a2 gene was upregulated in mdx and SJL/J, both of which showed the most preserved muscle histology, but exhibited lower expression
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
in the more severely affected muscles from Lama2dy 2J/J and Largemyd strains. The results suggest that Tgfb1 gene is activated in the dystrophic process in all types/stages of degeneration while activation of Col1a2 gene possibly occurs in earliest stages of this process. Funding by FAPESP, CNPq-INCT, FINEP, ABDIM. doi:10.1016/j.nmd.2009.06.100
EM.P.3.04 Muscle-derived Duchenne muscular dystrophy fibroblasts show altered production of extra-cellular matrix components S. Zanotti, S. Gibertini, S. Saredi, F. Blasevich, R. Mantegazza, L. Morandi, M. Mora Fondazione IRCCS Istituto Neurologico C. Besta, Neuromuscular Diseases and Neuroimmunology Unit, Milano, Italy In order to increase understanding of pro-fibrotic mechanisms in dystrophic muscle, we isolated primary human fibroblasts from DMD and control muscle biopsies, and investigated their proliferating activity and soluble collagen production, as well as transcript and protein levels of decorin, myostatin, TGF-b1, MMP-1 (interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the MMP inhibitors TIMP-1, TIMP-2, TIMP-3 and TIMP-4. We found significantly greater proliferation rate and soluble collagen production in DMD fibroblasts than controls. Decorin, myostatin and MMP-7 transcripts and proteins were significantly upregulated, and MMP-1 and TIMP-3 were significantly downregulated in DMD fibroblasts compared to controls. The study has revealed several differences between fibroblasts from DMD muscle and those from normal muscle, particularly as regards factors likely to affect ECM turnover. Although the detailed significance of these differences is not clear, they show that DMD fibroblasts are characterized by a profibrotic phenotype. Dystrophin absence itself could exert a direct influence on ECM homeostasis by allowing leakage of cellular components to the extracellular space, or abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes. By such mechanisms dystrophin absence may influence muscle fibroblasts either directly by altering their adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. doi:10.1016/j.nmd.2009.06.101
EM.P.3.05 Expression of fibulins 1–5 during myogenesis in vitro and in skeletal muscle regenerating in vivo, and in dystrophic mdx muscles M.D. Grounds, T. Shavlakadze, J. Vukovic The University of Western Australia, School of Anatomy & human Biology, Perth, Australia Many myopathies are due to gene defects that result in absent or altered extracellular matrix (ECM) and numerous ECM molecules are in intimate contact with myofibres and with the quiescent muscle precursors (satellite cells). Complex interactions between ECM components and different cells play a crucial role in muscle function and all aspects of new muscle formation after damage. Fibulins (FBLN) are members of an adhesive glycoprotein family that are associated with fibrosis in some conditions. The role of FBLN 1–5 was investigated using immunostaining and qPCR in cultured myogenic C2C12 cells, in tissue sections of regenerating whole muscle grafts,
575
in dystrophic (mdx) quadriceps and diaphragm, and in normal adult (2 month) and geriatric (23 month) mouse muscle. Cultured myoblasts expressed all fibulins and a subset was differentially regulated during myogenesis in myotubes. Immunostaining of regenerating myogenic cells (especially myotubes) in whole muscle grafts (at 6 days) generally confirmed this myogenic expression pattern. Dystrophic mdx quadriceps muscle (at 3 month) showed increased immunostaining for FBLN-1, -2 and -4 proteins around regenerating (small diameter) myofibres. While FBLN-3 and -5 were absent from dystrophic quadriceps aged up to 6 month, they were present in 3-month mdx diaphragm where they co-localised with fibronectin. These novel results indicate that different fibulins are differentially expressed and are involved in myogenesis and myofibre regeneration. A significant finding is that FBLN-3 and -5 are associated with the severe dystrophic pathology in the mdx diaphragm. Since fibrosis is a major complication resulting from progressive myofibre damage in dystrophic muscle, it is of especial interest to fully understand the roles of all ECM molecules, including fibulins, associated with this process. doi:10.1016/j.nmd.2009.06.102
EM.P.3.06 Transient upregulation of matrilin-2 gene expression suggests a role in early steps of skeletal muscle regeneration É. Korpos 1, L. Mátés 1, L. Mendler 2, M. Kiricsi 3, Á. Zvara 4, F. Deák 1, Z. Rottenberger 1, A. Keller-Pintér 2, L. Puskás 4, L. Dux2, I. Kiss 1 1
Biological Research Center of the Hungarian Academy of Sciences, Institute of Biochemistry, Szeged, Hungary, 2 University of Szeged, Faculty of General Medicine, Department of Biochemistry, Szeged, Hungary, 3 University of Szeged, Faculty of Natural Sciences, Department of Biochemistry and Molecular Biology, Szeged, Hungary, 4 Biological Research Center of the Hungarian Academy of Sciences, Laboratory of Functional Genomics, Szeged, Hungary Adult skeletal muscle has a remarkable regenerative capacity in response to injuries. The regeneration is dependent on an orchestrated response between the inductive signals of cytokines, growth factors and extracellular matrix (ECM) components. Matrilin-2 is a modular, oligomeric protein deposited in the ECM of many cell types. Colocalization in human skin and in vitro interaction of matrilin-2 with collagen I, fibrillin-2, fibronectin and the laminin-1-nidogen-1 complex were previously shown. We used the notexininduced in vivo model of muscle regeneration. Elevated level of matrilin-2 mRNA was observed already on day 2 of regeneration and reached maximum level between 4 and 7 days, similarly to myogenin gene expression. Matrilin-2 was localized in the matrix surrounding proliferating myoblasts on day 2 and around differentiating myoblasts and myotubes on day 4 of regeneration. In regenerating and control muscle partial colocalization of matrilin-2 was observed with fibronectin, the basement membrane laminin 2 and the costameric marker dystrophin. During in vitro differentiation of C2/7 myoblasts quantitative real-time RT-PCR showed a similar transient increase for matrilin-2, fibronectin, laminin A2 and collagen 6A1 gene expression. Immunostaining demonstrated deposition of matrilin-2 into extracellular filamentous network around proliferating, migrating and fusing myoblasts. We localized matrilin-2 in the epi-, peri- and endoneurium of the motoneurons. We co-localized matrilin-2 with alpha-bungarotoxin in the neuromuscular junctions between days 4 and 7, when the motor endplates are reformed. Detection of the targeted inactivation of Matn2 gene in transgenic mice on muscle regeneration is under progress. The observations suggest a role for matrilin-2 not only in the proliferation, migration,