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EM.P.3.07 The fate of glycosaminoglycans (GAGs) during skeletal muscle regeneration and myoblast differentiation
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Abstracts / Neuromuscular Disorders 19 (2009) 543–660
differentiation and fusion of myoblasts, but also in the assembly of the ECM-myofiber and nerve-myofiber contacts. Supported by grants OTKA T049608, ETT 008/2006 and RET-08/2004. doi:10.1016/j.nmd.2009.06.103
EM.P.3.07 The fate of glycosaminoglycans (GAGs) during skeletal muscle regeneration and myoblast differentiation M. Oudghir 1, A. Duchesnay 2, T. van Kuppevelt 3, I. Martelly2 1 University of Marrakech, Marrakech, Morocco, 2 Université Paris ESTParis 12, Créteil, France, 3 Han University, Nijmegen, The Netherlands
Proteoglycans (PG) of the extracellular matrix and associated to cell membrane have emerged as main regulators of skeletal muscle development and repair. The known effects of PG are mainly due to their sulphated glycosaminoglycan (GAG) moieties, namely dermatan sulphate/chondroitin sulphate (DS/CS) and heparan sulphate (HS), that interact with proteins including growth factors and regulate their activity. It is therefore of special interest to get insight into the spatio-temporal pattern of GAG expressions during skeletal muscle regeneration. GAG amounts were quantitated in the regenerating fast (EDL) and slow (Soleus) rat skeletal muscles after whole muscle crush. Changes in GAG composition were studied using an ELISA competition test assay and different GAG epitopes were visualized using specific antibodies in the course of regeneration In both muscle types, total GAG amounts were dramatically collapsed one day after injury, then increased during muscle repair. HS that was less abundant than DS/CS in the first days after crush became the most important GAG species when muscle reconstruction took place. Time-dependent GAG alterations and specificities were revealed depending on muscle type. Similarly changes in GAG amount and composition were observed in vitro when satellite cells isolated from fast or slow muscles shifted from proliferation to differentiation state. Synthetic GAGs, added at the onset of regeneration, used to replace the lost natural GAGs, stimulated myoblast differentiation and accelerated muscle repair. These spatio-temporal changes of GAGs in the environment of myogenic cells may control growth factor bioavailability and signalling events associated to myogenesis after muscle injury. According to these results, the use synthetic GAGs may be suggested as therapeutic agent in some neuromuscular diseases and muscle injury. doi:10.1016/j.nmd.2009.06.104
EM.P.3.08 Matrix matters in man and mouse N.C. Voermans, B.G. van Engelen Radboud University Nijmegen Medical Centre, Neurology, Nijmegen, The Netherlands Objective: Ehlers–Danlos (EDS) syndrome and Marfan syndrome are heritable connective tissue disorder caused by mutations in genes encoding collagen, tenascin-X (TNX), and fibrillin-1. Primary muscle involvement in these disorders can be expected based on interactions between muscle and extracellular matrix (ECM), similarly as in collagen VI myopathies. We performed a clinical study on EDS and Marfan patients and investigated muscle function in a mice-model of EDS to investigate the role of the ECM on muscle function. Methods: Standardized questionnaires, physical examina-
tion, nerve conduction studies, electromyography, muscle ultrasound, and muscle biopsy were performed in 40 EDS and 10 Marfan patients. Clinical observations, muscle and peripheral nerve histology and gene expression profiling in muscle were performed in TNX knockout mice. Results: Most EDS patients had mild to moderate neuromuscular involvement consisting of myopathy and axonal polyneuropathy, with a dose-effect relation of residual TNX levels and degree of neuromuscular involvement. Most Marfan patients displayed mild myopathy or polyneuropathy, and signs of lumbosacral radiculopathy. TNX knockout mice showed reduced muscle strength with histological signs of myopathy and lower density of ECM fibrils in muscle and peripheral nerve. Gene expression profiling showed increased turnover of ECM components. Quantitative muscle force was reduced at lower length, and TNX deficiency was found to affect the mechanical interaction between muscles due to changes of series elastic components of muscle and tendon. Conclusion: Mild to moderate neuromuscular involvement is common in EDS and Marfan syndrome. The findings in EDS patients suggest a pathophysiological role for muscle and peripheral nerve ECM, which is supported by results of animal studies. This study should increase awareness of neuromuscular symptoms in Marfan and EDS patients and increase insight in the role of ECM in muscle and peripheral nerve function. doi:10.1016/j.nmd.2009.06.105
THERAPEUTIC APPROACHES USING RNA SURGERY AND RELATED TECHNOLOGIES; POSTER PRESENTATIONS T.P.1.01 Pre-trial antisense screening of myogenic cells from boys with Duchenne muscular dystrophy and genomic and transcriptomic biomarkers discovery for treatment monitoring M. Neri1, M.S. Falzarano 1, M. Fabris 1, M. Bovolenta 1, E. Bassi 1, D. Perrone 2, A. Medici 2, P. Sabatelli 1, L. Merlini 1, F. Gualandi 1, P. Rimessi 1, A. Ferlini 1 1
University of Ferrara, Dipartimento di medicina sperimentale e diagnostic, Ferrara, Italy, 2 University of Ferrara, Dipartimento di biologia evolutiva, Ferrara, Italy Antisense oligoribonucleotides (AONs) are able to induce exon skipping in the dystrophin hnRNA; a successful first-in-man trial has recently been completed on DMD and a multicentric trial is approaching .The objectives of our project are (i) pre-screening characterization of myogenic cells from DMD boys with dystrophin deletions eligible for either exon 44 or 51 skipping approach to test their suitability to the trial; (ii) identification of genomic and transcriptomic biomarkers to be possibly used to monitor the treatment efficacy and safety. We have set up myogenic cell cultures (both fibroblast and myoblasts) from 10 DMD patients and we forced fibroblasts into myogenic cells by using MyoD. Deletion mutations have been confirmed by MLPA. Intronic breakpoints have been defined by using our novel CGH-DMD array and rare SNPs within exon 44 and 51 have been excluded. Transcription studies on RNA from patients’ skeletal muscle confirmed the pathogenic nature of the deletion mutations as well as excluded inconsistent splicing behaviours. We are transfecting myogenic cells with PRO051 (Prosensa Therapeutics B.V.) for the following studies: (a) Real-Time RT-PCR in order to detect and quantify skipped transcript amount and to correlate it with the intronic breakpoints, (b) Real-Time RT-PCR by using a TaqManÒ Custom Array covering dystrophin exons in order to verify the correct dystrophin full mRNA composition after the treatment (c) functional effect of the AONs treatment by evaluating dystrophin
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
differentiation and fusion of myoblasts, but also in the assembly of the ECM-myofiber and nerve-myofiber contacts. Supported by grants OTKA T049608, ETT 008/2006 and RET-08/2004. doi:10.1016/j.nmd.2009.06.103
EM.P.3.07 The fate of glycosaminoglycans (GAGs) during skeletal muscle regeneration and myoblast differentiation M. Oudghir 1, A. Duchesnay 2, T. van Kuppevelt 3, I. Martelly2 1 University of Marrakech, Marrakech, Morocco, 2 Université Paris ESTParis 12, Créteil, France, 3 Han University, Nijmegen, The Netherlands
Proteoglycans (PG) of the extracellular matrix and associated to cell membrane have emerged as main regulators of skeletal muscle development and repair. The known effects of PG are mainly due to their sulphated glycosaminoglycan (GAG) moieties, namely dermatan sulphate/chondroitin sulphate (DS/CS) and heparan sulphate (HS), that interact with proteins including growth factors and regulate their activity. It is therefore of special interest to get insight into the spatio-temporal pattern of GAG expressions during skeletal muscle regeneration. GAG amounts were quantitated in the regenerating fast (EDL) and slow (Soleus) rat skeletal muscles after whole muscle crush. Changes in GAG composition were studied using an ELISA competition test assay and different GAG epitopes were visualized using specific antibodies in the course of regeneration In both muscle types, total GAG amounts were dramatically collapsed one day after injury, then increased during muscle repair. HS that was less abundant than DS/CS in the first days after crush became the most important GAG species when muscle reconstruction took place. Time-dependent GAG alterations and specificities were revealed depending on muscle type. Similarly changes in GAG amount and composition were observed in vitro when satellite cells isolated from fast or slow muscles shifted from proliferation to differentiation state. Synthetic GAGs, added at the onset of regeneration, used to replace the lost natural GAGs, stimulated myoblast differentiation and accelerated muscle repair. These spatio-temporal changes of GAGs in the environment of myogenic cells may control growth factor bioavailability and signalling events associated to myogenesis after muscle injury. According to these results, the use synthetic GAGs may be suggested as therapeutic agent in some neuromuscular diseases and muscle injury. doi:10.1016/j.nmd.2009.06.104
EM.P.3.08 Matrix matters in man and mouse N.C. Voermans, B.G. van Engelen Radboud University Nijmegen Medical Centre, Neurology, Nijmegen, The Netherlands Objective: Ehlers–Danlos (EDS) syndrome and Marfan syndrome are heritable connective tissue disorder caused by mutations in genes encoding collagen, tenascin-X (TNX), and fibrillin-1. Primary muscle involvement in these disorders can be expected based on interactions between muscle and extracellular matrix (ECM), similarly as in collagen VI myopathies. We performed a clinical study on EDS and Marfan patients and investigated muscle function in a mice-model of EDS to investigate the role of the ECM on muscle function. Methods: Standardized questionnaires, physical examina-
tion, nerve conduction studies, electromyography, muscle ultrasound, and muscle biopsy were performed in 40 EDS and 10 Marfan patients. Clinical observations, muscle and peripheral nerve histology and gene expression profiling in muscle were performed in TNX knockout mice. Results: Most EDS patients had mild to moderate neuromuscular involvement consisting of myopathy and axonal polyneuropathy, with a dose-effect relation of residual TNX levels and degree of neuromuscular involvement. Most Marfan patients displayed mild myopathy or polyneuropathy, and signs of lumbosacral radiculopathy. TNX knockout mice showed reduced muscle strength with histological signs of myopathy and lower density of ECM fibrils in muscle and peripheral nerve. Gene expression profiling showed increased turnover of ECM components. Quantitative muscle force was reduced at lower length, and TNX deficiency was found to affect the mechanical interaction between muscles due to changes of series elastic components of muscle and tendon. Conclusion: Mild to moderate neuromuscular involvement is common in EDS and Marfan syndrome. The findings in EDS patients suggest a pathophysiological role for muscle and peripheral nerve ECM, which is supported by results of animal studies. This study should increase awareness of neuromuscular symptoms in Marfan and EDS patients and increase insight in the role of ECM in muscle and peripheral nerve function. doi:10.1016/j.nmd.2009.06.105
THERAPEUTIC APPROACHES USING RNA SURGERY AND RELATED TECHNOLOGIES; POSTER PRESENTATIONS T.P.1.01 Pre-trial antisense screening of myogenic cells from boys with Duchenne muscular dystrophy and genomic and transcriptomic biomarkers discovery for treatment monitoring M. Neri1, M.S. Falzarano 1, M. Fabris 1, M. Bovolenta 1, E. Bassi 1, D. Perrone 2, A. Medici 2, P. Sabatelli 1, L. Merlini 1, F. Gualandi 1, P. Rimessi 1, A. Ferlini 1 1
University of Ferrara, Dipartimento di medicina sperimentale e diagnostic, Ferrara, Italy, 2 University of Ferrara, Dipartimento di biologia evolutiva, Ferrara, Italy Antisense oligoribonucleotides (AONs) are able to induce exon skipping in the dystrophin hnRNA; a successful first-in-man trial has recently been completed on DMD and a multicentric trial is approaching .The objectives of our project are (i) pre-screening characterization of myogenic cells from DMD boys with dystrophin deletions eligible for either exon 44 or 51 skipping approach to test their suitability to the trial; (ii) identification of genomic and transcriptomic biomarkers to be possibly used to monitor the treatment efficacy and safety. We have set up myogenic cell cultures (both fibroblast and myoblasts) from 10 DMD patients and we forced fibroblasts into myogenic cells by using MyoD. Deletion mutations have been confirmed by MLPA. Intronic breakpoints have been defined by using our novel CGH-DMD array and rare SNPs within exon 44 and 51 have been excluded. Transcription studies on RNA from patients’ skeletal muscle confirmed the pathogenic nature of the deletion mutations as well as excluded inconsistent splicing behaviours. We are transfecting myogenic cells with PRO051 (Prosensa Therapeutics B.V.) for the following studies: (a) Real-Time RT-PCR in order to detect and quantify skipped transcript amount and to correlate it with the intronic breakpoints, (b) Real-Time RT-PCR by using a TaqManÒ Custom Array covering dystrophin exons in order to verify the correct dystrophin full mRNA composition after the treatment (c) functional effect of the AONs treatment by evaluating dystrophin