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G.P.201
Abstracts / Neuromuscular Disorders 24 (2014) 791–924 dystrophies, 10 patients with motor neuron disease, 10 young and 10 elderly age matched control patients were selected. Immunohistological staining using following antibodies was performed: PAX7, N-CAM, MyoD1, myogenin, neonatal myosin heavy chain, developmental myosin heavy chain and spectrin. The same area in adjacent sections was photographed, followed by counting of fibers and satellite cells. The quotient of myogenin and PAX7 was used as an index for satellite cell activation, and the expressions of the variants of myosin heavy chain were compared between the groups. All biopsies were from the anterior tibial muscle. We found a significantly higher index of activated satellite cells in neurogenic myopathy than in dystrophic myopathy or controls. The higher index of satellite cell activation was paralleled by a higher expression of MyoD1 (indicating satellite cell proliferation). Embryonic myosin was more frequent in dystrophic compared to neurogenic myopathy. The parameters were low and did not differ between the young and elderly control biopsies. The results indicate that there is satellite cell activation in neurogenic myopathy exceeding the activation in dystrophies, and that regenerating fibers in this disease group may by pass the stage of expressing embryonic myosin. http://dx.doi:10.1016/j.nmd.2014.06.276
G.P.201 Aberrant mechanical–metabolic coupling in muscular dystrophy: Gene expression and functional studies in mdx mouse muscle in relation to age and exercise G.M. Camerino 1, R.F. Capogrosso 1, M. Cannone 1, P. Mantuano 1, A. Giustino 1, A.M. Massari 1, A. Cozzoli 1, R.W. Grange 2, A. De Luca 1 1 University of Bari, Bari, Italy; 2 Virginia Polytechnic and State University, Blacksburg, VA 24061, USA Weakness and fatigability, typical features of Duchenne muscular dystrophy, are aggravated in mdx mice by a chronic exercise on horizontal treadmill (30 min running at 12 m/min twice a week). This protocol leads to a significant decrease in limb force vs. non-exercised mdx mice by grip test; preliminary results by torque recordings confirm this functional impairment. Parallel ex vivo studies show that the exercise increases the resistance to eccentric contraction in C57BL/10 wild-type (wt) extensor digitorum longus (EDL) muscles, while such an adaptation is not observed in mdx ones, which remain weaker than controls. In order to understand the molecular mechanisms underlying exercise susceptibility of mdx mice we investigated by quantitative realtime PCR the outcome of 4 (T4, 8 weeks of age) and 12 (T12; 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in gastrocnemius muscle of mdx and wt mice. Basal expression of peroxisome-proliferator receptor c coactivator 1a´ (PGC-1a´) and sirtuin1 is higher in mdx vs. wt at both ages. Exercise increases PGC-1a´ expression in wt, while in mdx mice T12 exercise down-regulates PGC1a´, sirtuin-1, PPARc and the autophagy marker BNIP3. 16 week-old mdx mice show a basal over-expression of the slow-phenotype genes; T12 exercise fully contrasts this basal adaptation and the high expression of follistatin and myogenin, being ineffective in wt mice. Damage- and inflammation-related genes, such as NADPH-oxidase, TGFaˆ and TNF-a´ are overexpressed in mdx muscle in all conditions. In parallel the anti-inflammatory adiponectin is lower in T12 exercised mdx muscle. Then a chronic exercise with minor adaptive effects in wt muscle, contrasts compensatory changes in the benign mdx phenotype leading to a disequilibrium between protective and damaging signals and disclosing potential drug targets. http://dx.doi:10.1016/j.nmd.2014.06.277
877
G.P.202 Characterization of skeletal muscle and heart during aging in rats, and response to formoterol, a selective b2 adrenoceptor agonist, in aged rats C. Lambert, J. Boesch, M. Obrecht, J. Grosjean, P.R. Allegrini, S. Hatakeyama Novartis Pharma AG, Basel, Switzerland Loss of skeletal muscle function and mass occurs during aging as well as under various disease conditions, such as cancer, COPD, liver disease, and congestive heart failure. Despite the high prevalence of such conditions, no effective treatment for muscle wasting diseases exists. In particular, sarcopenia, under the concept of frailty frequently observed in elderly people, is a geriatric syndrome defined by an excessive reduction in muscle mass and a concurrent decline in physical function, leading to mobility limitations, reduced quality of life, increased risk of falls, fractures and hospitalizations, and ultimately loss of independence. Selective b2 adrenoceptor agonists elicit skeletal muscle hypertrophy associated with increased force producing capacity. Several studies have reported efficacy of b2 adrenoceptor agonists in improving muscle wasting in many animal species and humans. Here, skeletal muscle and heart in aged rats were characterized with regard to mass, function and gene expression changes. Progressive loss of mass and force production capacity in skeletal muscle was observed; in contrast cardiac mass was increased and its function was decreased. In skeletal muscles, these phenotypic changes were associated with dramatic changes in genes known to be involved in atrophy, differentiation and in particular neuromuscular junction. In the heart, gene expression changes indicated pathological remodeling including up-regulation of fetal gene markers. Furthermore, the effect of formoterol on skeletal muscle and heart of aged sarcopenic rats was evaluated. The treatment spared the age-related loss of skeletal muscle mass and force production capacity in rats, while increasing heart weight. Hence, although b2 adrenoceptor agonists might be a potential therapeutic option for treating age-related loss of skeletal muscle, development of skeletal muscle selective agents is awaited since any detrimental cardiac effects may not be tolerated in elderly population. http://dx.doi:10.1016/j.nmd.2014.06.278
G.P.203 IL-6- and calcineurin-mediated but not IGF-1-mediated mechanisms contribute to the upregulation of MHC I and HSP70 mRNA levels in C2C12 cells Y. Mori 1, J. Yamaji 1, R. Hiroshima 1, T. Nakano 1, A. Miyazaki 2, M. Watanabe 1 1 Kansai Univ. of Welfare Sciences, 582-0026, Japan; 2 Osaka Medical College, Takatsuki, Japan Skeletal muscle has been known to cause disuse atrophy during long-term recumbence and to recover by exercise. Although there are many studies about skeletal muscle regulatory factors, such as IGF-1 or IL-6, that are known to affect skeletal muscle mass, mechanism of muscle cell hypertrophy is still unclear. Our previous study using rat soleus muscle indicated that myosin heavy chain (MHC) and HSP70 proteins were significantly increased by the exercise after disuse atrophy, therefore we examined effects of IGF-1, IL-6, and La3+ on MHC I, HSP70, and IL-6 mRNA expression level using real-time PCR method in C2C12 skeletal myoblasts. The C2C12 cells which were differentiated into myocyte in D-MEM containing 2% FCS was incubated with chemicals in 24 h. First, we examined the effect of IGF-1 on MHC I, HSP70, and IL-6 mRNA expression levels. MHC I and HSP70 mRNA levels were significantly decreased by the incubation with IGF-1, although IL-6 mRNA level was increased. Second, we examined the
G.P.201 Aberrant mechanical–metabolic coupling in muscular dystrophy: Gene expression and functional studies in mdx mouse muscle in relation to age and exercise G.M. Camerino 1, R.F. Capogrosso 1, M. Cannone 1, P. Mantuano 1, A. Giustino 1, A.M. Massari 1, A. Cozzoli 1, R.W. Grange 2, A. De Luca 1 1 University of Bari, Bari, Italy; 2 Virginia Polytechnic and State University, Blacksburg, VA 24061, USA Weakness and fatigability, typical features of Duchenne muscular dystrophy, are aggravated in mdx mice by a chronic exercise on horizontal treadmill (30 min running at 12 m/min twice a week). This protocol leads to a significant decrease in limb force vs. non-exercised mdx mice by grip test; preliminary results by torque recordings confirm this functional impairment. Parallel ex vivo studies show that the exercise increases the resistance to eccentric contraction in C57BL/10 wild-type (wt) extensor digitorum longus (EDL) muscles, while such an adaptation is not observed in mdx ones, which remain weaker than controls. In order to understand the molecular mechanisms underlying exercise susceptibility of mdx mice we investigated by quantitative realtime PCR the outcome of 4 (T4, 8 weeks of age) and 12 (T12; 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in gastrocnemius muscle of mdx and wt mice. Basal expression of peroxisome-proliferator receptor c coactivator 1a´ (PGC-1a´) and sirtuin1 is higher in mdx vs. wt at both ages. Exercise increases PGC-1a´ expression in wt, while in mdx mice T12 exercise down-regulates PGC1a´, sirtuin-1, PPARc and the autophagy marker BNIP3. 16 week-old mdx mice show a basal over-expression of the slow-phenotype genes; T12 exercise fully contrasts this basal adaptation and the high expression of follistatin and myogenin, being ineffective in wt mice. Damage- and inflammation-related genes, such as NADPH-oxidase, TGFaˆ and TNF-a´ are overexpressed in mdx muscle in all conditions. In parallel the anti-inflammatory adiponectin is lower in T12 exercised mdx muscle. Then a chronic exercise with minor adaptive effects in wt muscle, contrasts compensatory changes in the benign mdx phenotype leading to a disequilibrium between protective and damaging signals and disclosing potential drug targets. http://dx.doi:10.1016/j.nmd.2014.06.277
877
G.P.202 Characterization of skeletal muscle and heart during aging in rats, and response to formoterol, a selective b2 adrenoceptor agonist, in aged rats C. Lambert, J. Boesch, M. Obrecht, J. Grosjean, P.R. Allegrini, S. Hatakeyama Novartis Pharma AG, Basel, Switzerland Loss of skeletal muscle function and mass occurs during aging as well as under various disease conditions, such as cancer, COPD, liver disease, and congestive heart failure. Despite the high prevalence of such conditions, no effective treatment for muscle wasting diseases exists. In particular, sarcopenia, under the concept of frailty frequently observed in elderly people, is a geriatric syndrome defined by an excessive reduction in muscle mass and a concurrent decline in physical function, leading to mobility limitations, reduced quality of life, increased risk of falls, fractures and hospitalizations, and ultimately loss of independence. Selective b2 adrenoceptor agonists elicit skeletal muscle hypertrophy associated with increased force producing capacity. Several studies have reported efficacy of b2 adrenoceptor agonists in improving muscle wasting in many animal species and humans. Here, skeletal muscle and heart in aged rats were characterized with regard to mass, function and gene expression changes. Progressive loss of mass and force production capacity in skeletal muscle was observed; in contrast cardiac mass was increased and its function was decreased. In skeletal muscles, these phenotypic changes were associated with dramatic changes in genes known to be involved in atrophy, differentiation and in particular neuromuscular junction. In the heart, gene expression changes indicated pathological remodeling including up-regulation of fetal gene markers. Furthermore, the effect of formoterol on skeletal muscle and heart of aged sarcopenic rats was evaluated. The treatment spared the age-related loss of skeletal muscle mass and force production capacity in rats, while increasing heart weight. Hence, although b2 adrenoceptor agonists might be a potential therapeutic option for treating age-related loss of skeletal muscle, development of skeletal muscle selective agents is awaited since any detrimental cardiac effects may not be tolerated in elderly population. http://dx.doi:10.1016/j.nmd.2014.06.278
G.P.203 IL-6- and calcineurin-mediated but not IGF-1-mediated mechanisms contribute to the upregulation of MHC I and HSP70 mRNA levels in C2C12 cells Y. Mori 1, J. Yamaji 1, R. Hiroshima 1, T. Nakano 1, A. Miyazaki 2, M. Watanabe 1 1 Kansai Univ. of Welfare Sciences, 582-0026, Japan; 2 Osaka Medical College, Takatsuki, Japan Skeletal muscle has been known to cause disuse atrophy during long-term recumbence and to recover by exercise. Although there are many studies about skeletal muscle regulatory factors, such as IGF-1 or IL-6, that are known to affect skeletal muscle mass, mechanism of muscle cell hypertrophy is still unclear. Our previous study using rat soleus muscle indicated that myosin heavy chain (MHC) and HSP70 proteins were significantly increased by the exercise after disuse atrophy, therefore we examined effects of IGF-1, IL-6, and La3+ on MHC I, HSP70, and IL-6 mRNA expression level using real-time PCR method in C2C12 skeletal myoblasts. The C2C12 cells which were differentiated into myocyte in D-MEM containing 2% FCS was incubated with chemicals in 24 h. First, we examined the effect of IGF-1 on MHC I, HSP70, and IL-6 mRNA expression levels. MHC I and HSP70 mRNA levels were significantly decreased by the incubation with IGF-1, although IL-6 mRNA level was increased. Second, we examined the