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− mice, a model of Ullrich congenital muscular dystrophy (UCMD)
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Abstracts / Neuromuscular Disorders 19 (2009) 543–660
MRE technique may provide interesting mechanical variables of skeletal muscles in vivo and could be used in the future for the follow-up of patients suffering from neuromuscular disorders, particularly during therapeutic trials. doi:10.1016/j.nmd.2009.06.267
COLLAGEN VI-RELATED MUSCULAR DYSTROPHY II: EXPERIMENTAL APPROACHES; POSTER PRESENTATIONS EM.P.5.01 Gene expression and proteome profiles in Col6a1 / mice, a model of Ullrich congenital muscular dystrophy (UCMD) M. Bovolenta 1, S. De Palma2, M. Vasso 1, N.M. Maraldi 3, F. Gualandi 1, L. Merlini 1, P. Sabatelli 1, T. Tiepolo 4, P. Bonaldo 4, P. Bernardi 5, C. Gelfi 2, A. Ferlini 1 1 University of Ferrara, Medical Genetics, Ferrara, Italy, 2 CNR, Bioimaging and Molecular Physiology, Milano, Italy, 3 CNR, Molecular Genetics (IGM), Bologna, Italy, 4 University of Padova, Histology Microbiology and Medical Biotechnologies, Padova, Italy, 5 University of Padova, Biomedical Sciences, Padova, Italy
Ullrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM) are congenital muscular dystrophies caused by abnormalities of Collagen VI due to COL6 gene mutations. The absence of Collagen VI has a major impact inside the fibres by triggering a short circuit in the cell’s energy generators, the mitochondria. This short circuit is caused by opening of a channel called the ‘‘Permeability Transition Pore” (PTP), which can be inhibited by the drug cyclosporin A (CsA). Muscle cells from patients affected by UCMD and BM also responded to CsA. A short-term pilot trial with CsA in patients affected by UCMD and BM has provided encouraging results. The muscle defects of Col6a1 / mice and UCMD patients indicate that ColVI must relay a set of signals regulating cell survival. To better understand the mechanism by which the muscle fibres are spared and respond to treatment we have focused our attentions to the whole mouse gene expression and proteome comparative studies of WT and Col6a1 / mice before and after treatment with CsA. A preliminary proteomic investigation of this mouse model by 2D-DIGE showed that 37 proteins are differentially expressed in diaphragm extracts of Col6a1 / mice vs. control mice indicating a rearrangement in contractile and metabolic proteins induced by ColVI deficiency. The systematic comparison of muscle transcriptome by expression profiles and muscle proteome performed on the same muscle extract will provide new information about how gene expression eventually translates into the specific phenotypes displayed by our animal model. This approach will identify deregulated genes and proteins involved in the recovery of muscle function after drug treatment as well as signalling pathways that are involved in disease pathogenesis. doi:10.1016/j.nmd.2009.06.268
EM.P.5.02 Role of mitochondria in the pathogenesis of muscular dystrophies E. Palma1, A. Angelin 1, T. Tiepolo 1, P. Sabatelli 2, N.M. Maraldi 2, L. Merlini 3, P. Bonaldo 1, P. Bernardi 2 University of Padova, Padova, Italy, 2 Istituto Ortopedico Rizzoli, Bologna, Italy, 3 University of Ferrara, Ferrara, Italy 1
Mutations of COL6A1-A3 genes, encoding the extracellular matrix protein collagen VI, cause muscular diseases, among which Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UCMD) are the mostly described. Studies on the mouse model of these disorders (Col6a1 / ) and the patients affected by UCMD have shown an increased rate of spontaneous apoptosis, ultrastructural alterations in mitochondria and sarcoplasmic reticulum and a latent mitochondrial dysfunction in skeletal muscle. We demonstrated that these dysfunctions were caused by inappropriate opening of the permeability transition pore (PTP), a mitochondrial inner membrane channel that plays a role in several forms of cell death and can be desensitized by cyclosporin (Cs) A after binding to cyclophilin (Cyp) D. Indeed, the mitochondrial phenotype could be rescued both by treatment with CsA or its non-immunosuppressive derivative, Debio 025, and by genetic inactivation of CypD in the Col6a1 / mice. Moreover, the treatment with CsA of UCMD patients led to rescue from mitochondrial dysfunction, normalization of apoptotic rates and increased muscle regeneration. Recent studies have shown that mitochondrial dysfunction is also present in mouse models of DMD and MDC1A, and in muscle-derived cells isolated from patients with LGMD2B. On the basis of all these data, we decided to investigate if mitochondria play a key pathogenic role in BM and in other muscular dystrophies, and to evaluate the role of the different muscle cell types (myoblasts versus fibroblasts) in the pathogenesis and the progression of the COL6 related diseases. doi:10.1016/j.nmd.2009.06.269
EM.P.5.03 The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-=- myopathic mice A. Angelin1, T. Tiepolo 1, E. Palma 1, P. Sabatelli 2, L. Merlini 3, L. Nicolosi 1, F. Finetti 4, P. Braghetta 1, G. Vuagniaux 5, J.M. Dumont 5, C.T. Baldari 4, P. Bonaldo 1, P. Bernardi 1 1 University of Padova, Padova, Italy, 2 Istituto Ortopedico Rizzoli, Bologna, Italy, 3 University of Ferrara, Ferrara, Italy, 4 University of Siena, Siena, Italy, 5 DebioPharm, Lausanne, Switzerland
Background and purpose: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D MeAla3 EtVal4 cyclosporin (Debio 025) in myopathic Col6a1 / mice, a model of muscular dystrophies due to defects of collagen VI. Experimental approach: We have studied calcineurin activity, T cell activation, propensity to open of the permeability transition pore in mitochondria and skeletal muscle fibres, muscle ultrastructure and apoptotic rates in Col6a1 / mice before after treatment with Debio 025. Key results: Debio 025 does not inhibit calcineurin, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevents mitochondrial dysfunction and normalizes the apoptotic rates and ultrastructural lesions of myopathic Col6a1 / mice. Conclusions and implications: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1 / myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025; and represent an essential step toward a therapy of Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression. doi:10.1016/j.nmd.2009.06.270
Abstracts / Neuromuscular Disorders 19 (2009) 543–660
MRE technique may provide interesting mechanical variables of skeletal muscles in vivo and could be used in the future for the follow-up of patients suffering from neuromuscular disorders, particularly during therapeutic trials. doi:10.1016/j.nmd.2009.06.267
COLLAGEN VI-RELATED MUSCULAR DYSTROPHY II: EXPERIMENTAL APPROACHES; POSTER PRESENTATIONS EM.P.5.01 Gene expression and proteome profiles in Col6a1 / mice, a model of Ullrich congenital muscular dystrophy (UCMD) M. Bovolenta 1, S. De Palma2, M. Vasso 1, N.M. Maraldi 3, F. Gualandi 1, L. Merlini 1, P. Sabatelli 1, T. Tiepolo 4, P. Bonaldo 4, P. Bernardi 5, C. Gelfi 2, A. Ferlini 1 1 University of Ferrara, Medical Genetics, Ferrara, Italy, 2 CNR, Bioimaging and Molecular Physiology, Milano, Italy, 3 CNR, Molecular Genetics (IGM), Bologna, Italy, 4 University of Padova, Histology Microbiology and Medical Biotechnologies, Padova, Italy, 5 University of Padova, Biomedical Sciences, Padova, Italy
Ullrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM) are congenital muscular dystrophies caused by abnormalities of Collagen VI due to COL6 gene mutations. The absence of Collagen VI has a major impact inside the fibres by triggering a short circuit in the cell’s energy generators, the mitochondria. This short circuit is caused by opening of a channel called the ‘‘Permeability Transition Pore” (PTP), which can be inhibited by the drug cyclosporin A (CsA). Muscle cells from patients affected by UCMD and BM also responded to CsA. A short-term pilot trial with CsA in patients affected by UCMD and BM has provided encouraging results. The muscle defects of Col6a1 / mice and UCMD patients indicate that ColVI must relay a set of signals regulating cell survival. To better understand the mechanism by which the muscle fibres are spared and respond to treatment we have focused our attentions to the whole mouse gene expression and proteome comparative studies of WT and Col6a1 / mice before and after treatment with CsA. A preliminary proteomic investigation of this mouse model by 2D-DIGE showed that 37 proteins are differentially expressed in diaphragm extracts of Col6a1 / mice vs. control mice indicating a rearrangement in contractile and metabolic proteins induced by ColVI deficiency. The systematic comparison of muscle transcriptome by expression profiles and muscle proteome performed on the same muscle extract will provide new information about how gene expression eventually translates into the specific phenotypes displayed by our animal model. This approach will identify deregulated genes and proteins involved in the recovery of muscle function after drug treatment as well as signalling pathways that are involved in disease pathogenesis. doi:10.1016/j.nmd.2009.06.268
EM.P.5.02 Role of mitochondria in the pathogenesis of muscular dystrophies E. Palma1, A. Angelin 1, T. Tiepolo 1, P. Sabatelli 2, N.M. Maraldi 2, L. Merlini 3, P. Bonaldo 1, P. Bernardi 2 University of Padova, Padova, Italy, 2 Istituto Ortopedico Rizzoli, Bologna, Italy, 3 University of Ferrara, Ferrara, Italy 1
Mutations of COL6A1-A3 genes, encoding the extracellular matrix protein collagen VI, cause muscular diseases, among which Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UCMD) are the mostly described. Studies on the mouse model of these disorders (Col6a1 / ) and the patients affected by UCMD have shown an increased rate of spontaneous apoptosis, ultrastructural alterations in mitochondria and sarcoplasmic reticulum and a latent mitochondrial dysfunction in skeletal muscle. We demonstrated that these dysfunctions were caused by inappropriate opening of the permeability transition pore (PTP), a mitochondrial inner membrane channel that plays a role in several forms of cell death and can be desensitized by cyclosporin (Cs) A after binding to cyclophilin (Cyp) D. Indeed, the mitochondrial phenotype could be rescued both by treatment with CsA or its non-immunosuppressive derivative, Debio 025, and by genetic inactivation of CypD in the Col6a1 / mice. Moreover, the treatment with CsA of UCMD patients led to rescue from mitochondrial dysfunction, normalization of apoptotic rates and increased muscle regeneration. Recent studies have shown that mitochondrial dysfunction is also present in mouse models of DMD and MDC1A, and in muscle-derived cells isolated from patients with LGMD2B. On the basis of all these data, we decided to investigate if mitochondria play a key pathogenic role in BM and in other muscular dystrophies, and to evaluate the role of the different muscle cell types (myoblasts versus fibroblasts) in the pathogenesis and the progression of the COL6 related diseases. doi:10.1016/j.nmd.2009.06.269
EM.P.5.03 The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-=- myopathic mice A. Angelin1, T. Tiepolo 1, E. Palma 1, P. Sabatelli 2, L. Merlini 3, L. Nicolosi 1, F. Finetti 4, P. Braghetta 1, G. Vuagniaux 5, J.M. Dumont 5, C.T. Baldari 4, P. Bonaldo 1, P. Bernardi 1 1 University of Padova, Padova, Italy, 2 Istituto Ortopedico Rizzoli, Bologna, Italy, 3 University of Ferrara, Ferrara, Italy, 4 University of Siena, Siena, Italy, 5 DebioPharm, Lausanne, Switzerland
Background and purpose: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D MeAla3 EtVal4 cyclosporin (Debio 025) in myopathic Col6a1 / mice, a model of muscular dystrophies due to defects of collagen VI. Experimental approach: We have studied calcineurin activity, T cell activation, propensity to open of the permeability transition pore in mitochondria and skeletal muscle fibres, muscle ultrastructure and apoptotic rates in Col6a1 / mice before after treatment with Debio 025. Key results: Debio 025 does not inhibit calcineurin, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevents mitochondrial dysfunction and normalizes the apoptotic rates and ultrastructural lesions of myopathic Col6a1 / mice. Conclusions and implications: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1 / myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025; and represent an essential step toward a therapy of Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression. doi:10.1016/j.nmd.2009.06.270