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Influence of mesenchymal stem cell on STAT signalling pathway in muscle satellite cell
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316 muscle fibre number between the two genotypes. The frequency distribution of minimal muscle fibre diameter in EDL muscles differed between genotypes, with a skew towards smaller fibres and increased kurtosis in PAR2-null. Furthermore, NADH histochemistry and myosin heavy chain immunostaining demonstrated that PAR2-null mice had significantly greater proportions of oxidative muscle fibres, and type 1 and 2a myosin heavy chain positive fibres in the EDL, and a significantly greater proportion of type 1 myosin heavy chain positive fibres in the Sol, than wildtype mice. These results suggest that PAR2 does influence the development and biochemical properties of EDL and Sol muscles, possibly due to its effects on myoblast proliferation and differentiation. Future work is planned to test the role of PAR2 in skeletal muscle regeneration in a mouse model of muscle injury. http://dx.doi.org/10.1016/j.nmd.2015.06.125
G.P.111 Influence of mesenchymal stem cell on STAT signalling pathway in muscle satellite cell Y. Nakajou *,1, Y. Maeda 2, Y. Yonemochi 1, T. Doki 1, N. Tawara 1, S. Yamashita 1, Y. Ando 1 1 Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2 Kumamoto Saishunso National Hospital, Kumamoto, Japan In skeletal muscle regeneration mutual effects between muscle satellite cell and muscle resident mesenchymal stem cell (m-MSC) are quite important. Instead of the m-MSC we tentatively administrated bone marrow derived MSC (Bm-MSC) to the Duchenne muscle dystrophy (DMD) model mouse. Interestingly, significant improvements were found not just in muscle mass, life span, and locomotor activity, but in histology. These results may indicate that MSCs from any tissue intensify muscle regenerative environment. In this presentation, we show the following findings: Bm-MSC gets Pax7-positive cells increased in the coculture with primarily isolated satellite cells carrying integrin alpha-7 and CD34; Bm-MSC strongly expresses some soluble factors; among the factors, CXCL12 induces complete shut-down of p-STAT3 expression in the cultured satellite cells; CXCL12 concentration in serum from DMD patients is significantly higher than from healthy controls. Previous reports showed the activation of JAK-STAT signalling pathway in satellite cells inhibited their capacity to undergo symmetric stem cell expansion and vice versa in STATsuppression by chemical antagonist. Our results show that addition of CXCL12 to satellite cell can completely suppress p-STAT3 expression and results in increment of Pax7-positive cell in vitro. CXCL12 has been hitherto known as a critical factor for maintaining the stemness of hematopoietic stem cell through CXCR4, a specific receptor for CXCL12, expressed on them. Intriguingly, CXCR4 is also on the muscle satellite cell. However, the specific role of the receptor on satellite cell is still unclear. Here, we propose that CXCL12 is a key molecule to maintain muscle satellite cell pool. http://dx.doi.org/10.1016/j.nmd.2015.06.126
MCARDLE DISEASE AND RELATED GLYCOGENOSES G.P.112 Molecular, morphological and physiological studies in a mouse model of McArdle disease: Similarities to the human disease T. Krag *,1, T. Pinos 2, T. Nielsen 1, A. Brull 2, A. Andreu 2, J. Vissing 1 1 Rigshospitalet, Copenhagen Neuromuscular Center, Copenhagen, Denmark; 2 Vall d’Hebron Research Institute, Mitochondrial Pathology and Neuromuscular Disorder, Barcelona, Spain McArdle disease (muscle glycogenosis type V) is a rare disease caused by myophosphorylase deficiency leading to blocked glycogen breakdown. A recently
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developed mouse model of McArdle disease with the p.R50X nonsense mutation shares some of the key features of patients with McArdle disease, i.e. glycogen accumulation and exercise intolerance. In this study, we have investigated how the myophosphorylase deficiency affects muscle physiology, morphology and glucose metabolism in 20-week old McArdle mice, and compared some of these findings to those in patients with McArdle disease. We found that contractile properties in the homozygous McArdle mouse was significantly affected by structural degeneration due to massive glycogen accumulation, and that glycolytic muscle fatigued significantly faster compared to wild type muscle. Histology demonstrated significant structural damage, massive glycogen accumulation and ongoing muscle regeneration. Interestingly, the glycogen accumulation and ensuing damage was differentiated among similar glycolytic muscles in homozygous mice, suggesting that underlying metabolic requirements may be a deciding factor. Western blot analyses of signalling factors involved in carbohydrate metabolism revealed that while glycogen synthase is inhibited, the mouse adapts poorly to the inability to convert glycogen to glucose. In contrast, McArdle patients are less affected morphologically, have less glycogen accumulation and adapts to the energy crisis by increasing the expression of GLUT4 and glycolytic enzyme levels to be able to capture glucose and metabolize it. Our study shows that despite differences in muscle damage and strength, glycogen accumulation and glucose metabolism response, the mouse model of McArdle disease shares fundamental physiological and clinical properties with the human disease and therefore could be a suitable model for further studies of disease mechanism and development of therapies. http://dx.doi.org/10.1016/j.nmd.2015.06.127
G.P.113 Clinical and molecular features of a large cohort of Italian McArdle patients D. Cassandrini 1, P. Tonin 2, L. Morandi 3, O. Musumeci 4, M. Filosto 5, G. Siciliano 6, E. Pegoraro 7, L. Santoro 8, R. Massa 9, T. Mongini 10, M. Sacchini 11, E. Bertini 12, G. Marrosu 13, M. Rigoldi 14, A. Burlina 15, A. Pini 16, S. Previtali 17, F. Santorelli 1, A. Toscano 4, C. Bruno *,18 1 Fondazione Stella Maris, Pisa, Italy; 2 Univ. Dept. Neurology, Verona, Italy; 3 Istituto C. Besta, Milano, Italy; 4 Univ. Dept. Neurology, Messina, Italy; 5 Dept. Neurology, Brescia, Italy; 6 Univ. Dept. Neurology, Pisa, Italy; 7 Univ. Dept. Neurology, Padova, Italy; 8 Univ. Dept. Neurology, Napoli, Italy; 9 Neurology-Tor Vergata, Roma, Italy; 10 Univ. Dept. Neurology, Torino, Italy; 11 Ospedale Meyer, Firenze, Italy; 12 Osp. Bambin Gesù, Roma, Italy; 13 Univ. Dept. Neurology, Cagliari, Italy; 14 Osp. San Gerardo, Monza, Italy; 15 Univ. Dept. Pediatrics, Padova, Italy; 16 Dept. Pediatrics, Bologna, Italy; 17 Neurology-Ist. S. Raffaele, Milano, Italy; 18 Istituto Giannina Gaslini, Italy McArdle disease is caused by deficiency of the muscle glycogen phosphorylase (myophosphorylase, GMP) due to mutations in the PYGM gene. The main clinical features are generally homogeneous, being characterized by acute crises of exercise intolerance with episodic myoglobinuria, and increased CK level at rest. In muscle biopsy the GMP deficiency is demonstrated by histochemical or biochemical analysis. We have collected a total of 77 McArdle patients (37 males and 40 females), ranging from 4 to 72 years, from 70 different families. Onset of symptoms started in the first two decades of life in 66% of patients, and high basal CK level was present in 99% of cases. Muscle biopsy was performed in 64 patients. Sixty-one patients had a history of acute crises of exercise intolerance during life, and 15 of these experienced myoglobinuria. 46% of patients experienced the second-wind phenomenon. PYGM gene analysis identified 33 different mutations, 12 of which were not previously reported (3 missense, 2 nonsense, 4 deletion, and 3 splice-junctions). Seventy-one patients had both alleles mutated (32 homozygotes and 39 compound heterozygotes). Our data confirm the relative homogeneity of the clinical feature of McArdle disease. The R50X resulted to be the most common mutation, accounting for 50% of alleles. http://dx.doi.org/10.1016/j.nmd.2015.06.128
G.P.111 Influence of mesenchymal stem cell on STAT signalling pathway in muscle satellite cell Y. Nakajou *,1, Y. Maeda 2, Y. Yonemochi 1, T. Doki 1, N. Tawara 1, S. Yamashita 1, Y. Ando 1 1 Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2 Kumamoto Saishunso National Hospital, Kumamoto, Japan In skeletal muscle regeneration mutual effects between muscle satellite cell and muscle resident mesenchymal stem cell (m-MSC) are quite important. Instead of the m-MSC we tentatively administrated bone marrow derived MSC (Bm-MSC) to the Duchenne muscle dystrophy (DMD) model mouse. Interestingly, significant improvements were found not just in muscle mass, life span, and locomotor activity, but in histology. These results may indicate that MSCs from any tissue intensify muscle regenerative environment. In this presentation, we show the following findings: Bm-MSC gets Pax7-positive cells increased in the coculture with primarily isolated satellite cells carrying integrin alpha-7 and CD34; Bm-MSC strongly expresses some soluble factors; among the factors, CXCL12 induces complete shut-down of p-STAT3 expression in the cultured satellite cells; CXCL12 concentration in serum from DMD patients is significantly higher than from healthy controls. Previous reports showed the activation of JAK-STAT signalling pathway in satellite cells inhibited their capacity to undergo symmetric stem cell expansion and vice versa in STATsuppression by chemical antagonist. Our results show that addition of CXCL12 to satellite cell can completely suppress p-STAT3 expression and results in increment of Pax7-positive cell in vitro. CXCL12 has been hitherto known as a critical factor for maintaining the stemness of hematopoietic stem cell through CXCR4, a specific receptor for CXCL12, expressed on them. Intriguingly, CXCR4 is also on the muscle satellite cell. However, the specific role of the receptor on satellite cell is still unclear. Here, we propose that CXCL12 is a key molecule to maintain muscle satellite cell pool. http://dx.doi.org/10.1016/j.nmd.2015.06.126
MCARDLE DISEASE AND RELATED GLYCOGENOSES G.P.112 Molecular, morphological and physiological studies in a mouse model of McArdle disease: Similarities to the human disease T. Krag *,1, T. Pinos 2, T. Nielsen 1, A. Brull 2, A. Andreu 2, J. Vissing 1 1 Rigshospitalet, Copenhagen Neuromuscular Center, Copenhagen, Denmark; 2 Vall d’Hebron Research Institute, Mitochondrial Pathology and Neuromuscular Disorder, Barcelona, Spain McArdle disease (muscle glycogenosis type V) is a rare disease caused by myophosphorylase deficiency leading to blocked glycogen breakdown. A recently
S219
developed mouse model of McArdle disease with the p.R50X nonsense mutation shares some of the key features of patients with McArdle disease, i.e. glycogen accumulation and exercise intolerance. In this study, we have investigated how the myophosphorylase deficiency affects muscle physiology, morphology and glucose metabolism in 20-week old McArdle mice, and compared some of these findings to those in patients with McArdle disease. We found that contractile properties in the homozygous McArdle mouse was significantly affected by structural degeneration due to massive glycogen accumulation, and that glycolytic muscle fatigued significantly faster compared to wild type muscle. Histology demonstrated significant structural damage, massive glycogen accumulation and ongoing muscle regeneration. Interestingly, the glycogen accumulation and ensuing damage was differentiated among similar glycolytic muscles in homozygous mice, suggesting that underlying metabolic requirements may be a deciding factor. Western blot analyses of signalling factors involved in carbohydrate metabolism revealed that while glycogen synthase is inhibited, the mouse adapts poorly to the inability to convert glycogen to glucose. In contrast, McArdle patients are less affected morphologically, have less glycogen accumulation and adapts to the energy crisis by increasing the expression of GLUT4 and glycolytic enzyme levels to be able to capture glucose and metabolize it. Our study shows that despite differences in muscle damage and strength, glycogen accumulation and glucose metabolism response, the mouse model of McArdle disease shares fundamental physiological and clinical properties with the human disease and therefore could be a suitable model for further studies of disease mechanism and development of therapies. http://dx.doi.org/10.1016/j.nmd.2015.06.127
G.P.113 Clinical and molecular features of a large cohort of Italian McArdle patients D. Cassandrini 1, P. Tonin 2, L. Morandi 3, O. Musumeci 4, M. Filosto 5, G. Siciliano 6, E. Pegoraro 7, L. Santoro 8, R. Massa 9, T. Mongini 10, M. Sacchini 11, E. Bertini 12, G. Marrosu 13, M. Rigoldi 14, A. Burlina 15, A. Pini 16, S. Previtali 17, F. Santorelli 1, A. Toscano 4, C. Bruno *,18 1 Fondazione Stella Maris, Pisa, Italy; 2 Univ. Dept. Neurology, Verona, Italy; 3 Istituto C. Besta, Milano, Italy; 4 Univ. Dept. Neurology, Messina, Italy; 5 Dept. Neurology, Brescia, Italy; 6 Univ. Dept. Neurology, Pisa, Italy; 7 Univ. Dept. Neurology, Padova, Italy; 8 Univ. Dept. Neurology, Napoli, Italy; 9 Neurology-Tor Vergata, Roma, Italy; 10 Univ. Dept. Neurology, Torino, Italy; 11 Ospedale Meyer, Firenze, Italy; 12 Osp. Bambin Gesù, Roma, Italy; 13 Univ. Dept. Neurology, Cagliari, Italy; 14 Osp. San Gerardo, Monza, Italy; 15 Univ. Dept. Pediatrics, Padova, Italy; 16 Dept. Pediatrics, Bologna, Italy; 17 Neurology-Ist. S. Raffaele, Milano, Italy; 18 Istituto Giannina Gaslini, Italy McArdle disease is caused by deficiency of the muscle glycogen phosphorylase (myophosphorylase, GMP) due to mutations in the PYGM gene. The main clinical features are generally homogeneous, being characterized by acute crises of exercise intolerance with episodic myoglobinuria, and increased CK level at rest. In muscle biopsy the GMP deficiency is demonstrated by histochemical or biochemical analysis. We have collected a total of 77 McArdle patients (37 males and 40 females), ranging from 4 to 72 years, from 70 different families. Onset of symptoms started in the first two decades of life in 66% of patients, and high basal CK level was present in 99% of cases. Muscle biopsy was performed in 64 patients. Sixty-one patients had a history of acute crises of exercise intolerance during life, and 15 of these experienced myoglobinuria. 46% of patients experienced the second-wind phenomenon. PYGM gene analysis identified 33 different mutations, 12 of which were not previously reported (3 missense, 2 nonsense, 4 deletion, and 3 splice-junctions). Seventy-one patients had both alleles mutated (32 homozygotes and 39 compound heterozygotes). Our data confirm the relative homogeneity of the clinical feature of McArdle disease. The R50X resulted to be the most common mutation, accounting for 50% of alleles. http://dx.doi.org/10.1016/j.nmd.2015.06.128