RESUMEN
Spasticity is the most common neurological disorder associated with increased muscle contraction causing impaired movement and gait. The aim of this study was to characterize the physical performance, skeletal muscle function, and phenotype of mice with a hereditary spastic mutation (B6.Cg-Glrbspa/J). Motor function, gait, and physical activity of juvenile and adult spastic mice and the morphological, histological, and mechanical characteristics of their soleus and gastrocnemius medialis muscles were compared with those of their wild-type (WT) littermates. Spastic mice showed attenuated growth, impaired motor function, and low physical activity. Gait of spastic mice was characterized by a typical hopping pattern. Spastic mice showed lower muscle forces, which were related to the smaller physiological cross-sectional area of spastic muscles. The muscle-tendon complex length-force relationship of adult gastrocnemius medialis was shifted toward shorter lengths, which was explained by attenuated longitudinal tibia growth. Spastic gastrocnemius medialis was more fatigue resistant than WT gastrocnemius medialis. This was largely explained by a higher mitochondrial content in muscle fibers and relatively higher percentage of slow-type muscle fibers. Muscles of juvenile spastic mice showed similar differences compared with WT juvenile mice, but these were less pronounced than between adult mice. This study shows that in spastic mice, disturbed motor function and gait is likely to be the result of hyperactivity of skeletal muscle and impaired skeletal muscle growth, which progress with age.
Asunto(s)
Parálisis Cerebral , Espasticidad Muscular , Animales , Parálisis Cerebral/patología , Ratones , Espasticidad Muscular/genética , Espasticidad Muscular/patología , Fuerza Muscular , Músculo Esquelético/fisiología , Rendimiento Físico Funcional , Receptores de GlicinaRESUMEN
Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.
Asunto(s)
Terapia Genética , Sistema de la Enzima Desramificadora del Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno Tipo III/genética , Enfermedad del Almacenamiento de Glucógeno Tipo III/metabolismo , Hígado/metabolismo , Músculo Esquelético/metabolismo , Fenotipo , Animales , Biomarcadores , Glucemia , Dependovirus/genética , Modelos Animales de Enfermedad , Activación Enzimática , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Glucógeno/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo III/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo III/terapia , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Especificidad de ÓrganosRESUMEN
Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca2+ release over the full range of voltage sensitivity of EC coupling. SR Ca2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca2+-gated ryanodine receptors to EC coupling. In addition, we found that SR Ca2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process.
Asunto(s)
Señalización del Calcio/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Androstadienos/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Acoplamiento Excitación-Contracción/efectos de los fármacos , Acoplamiento Excitación-Contracción/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Miopatías Estructurales Congénitas/tratamiento farmacológico , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/fisiopatología , Técnicas de Placa-Clamp , Proteínas Tirosina Fosfatasas no Receptoras/genética , WortmaninaRESUMEN
Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.
Asunto(s)
Músculo Esquelético/metabolismo , Mutagénesis Sitio-Dirigida , Miopatías Estructurales Congénitas/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Adenoviridae/genética , Animales , Desmina/genética , Desmina/metabolismo , Eliminación de Gen , Vectores Genéticos , Masculino , Ratones , Debilidad Muscular/genética , Debilidad Muscular/patología , Enfermedades Musculares , Miopatías Estructurales Congénitas/patología , Fenotipo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Análisis de Secuencia de ADNRESUMEN
Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparß, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.
Asunto(s)
Receptores de Activinas Tipo II/antagonistas & inhibidores , Fragmentos Fc de Inmunoglobulinas/farmacología , Músculos/fisiopatología , Distrofia Muscular Animal/fisiopatología , Animales , Línea Celular , Metabolismo Energético/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Porinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes.
Asunto(s)
Músculo Esquelético , Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/genética , Animales , Núcleo Celular/metabolismo , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiopatología , Distrofia Miotónica/genética , Distrofia Miotónica/fisiopatología , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The alpha1S subunit has a dual function in skeletal muscle: it forms the L-type Ca(2+) channel in T-tubules and is the voltage sensor of excitation-contraction coupling at the level of triads. It has been proposed that L-type Ca(2+) channels might also be voltage-gated sensors linked to transcriptional activity controlling differentiation. By using the U7-exon skipping strategy, we have achieved long-lasting downregulation of alpha1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of alpha1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy-related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of alpha1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Canales de Calcio/fisiología , Morfogénesis/genética , Músculo Esquelético/embriología , Animales , Autofagia/genética , Secuencia de Bases , Canales de Calcio/genética , Canales de Calcio Tipo L/genética , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fuerza Muscular/genética , Músculo Esquelético/anatomía & histología , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/patología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Tamaño de los Órganos/genética , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Distribución Tisular/genéticaRESUMEN
Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.
Asunto(s)
Metabolismo Energético , Contracción Muscular , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Resistencia Física , Animales , Genotipo , Glucólisis , Ácido Láctico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Musculares/metabolismo , Fatiga Muscular , Miostatina/deficiencia , Miostatina/genética , Consumo de Oxígeno , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Fenotipo , Fosfopiruvato Hidratasa/metabolismo , Carrera , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The anabolic effects of androgens on skeletal muscles are thought to be mediated predominantly through the androgen receptor (AR), a member of the ligand-dependent nuclear receptor superfamily. However, despite numerous studies performed in men and in rodents, these effects remain poorly understood. To characterize androgen signaling in skeletal muscles, we generated mice in which the AR is selectively ablated in myofibers. We show that myocytic AR controls androgen-induced insulin-like growth factor IEa (IGF-IEa) expression in the highly androgen-sensitive perineal muscles and that it mediates androgen-stimulated postnatal hypertrophy of these muscles. In contrast, androgen-dependent postnatal hypertrophy of limb muscle fibers is independent of myocytic AR. Thus, androgens control perineal and limb muscle mass in male mice through myocytic AR-dependent and -independent pathways, respectively. Importantly, we also show that AR deficiency in limb myocytes impairs myofibrillar organization of sarcomeres and decreases muscle strength, thus demonstrating that myocytic AR controls key pathways required for maximum force production. These distinct androgen signaling pathways in perineal and limb muscles may allow the design of screens to identify selective androgen modulators of muscle strength.
Asunto(s)
Extremidades/fisiología , Células Musculares/química , Fuerza Muscular , Músculo Esquelético/fisiología , Receptores Androgénicos/fisiología , Síndrome de Resistencia Androgénica/fisiopatología , Andrógenos/farmacología , Animales , Masculino , Ratones , Desarrollo de Músculos , SarcómerosRESUMEN
Autosomal dominant centronuclear myopathy (AD-CNM) is due to mutations in the gene encoding dynamin 2 (DNM2) involved in endocytosis and intracellular membrane trafficking. To understand the pathomechanisms resulting from a DNM2 mutation, we generated a knock-in mouse model expressing the most frequent AD-CNM mutation (KI-Dnm2(R465W)). Heterozygous (HTZ) mice developed a myopathy showing a specific spatial and temporal muscle involvement. In the primarily and prominently affected tibialis anterior muscle, impairment of the contractile properties was evidenced at weaning and was progressively associated with atrophy and histopathological abnormalities mainly affecting mitochondria and reticular network. Expression of genes involved in ubiquitin-proteosome and autophagy pathways was up-regulated during DNM2-induced atrophy. In isolated muscle fibers from wild-type and HTZ mice, Dnm2 localized in regions of intense membrane trafficking (I-band and perinuclear region), emphasizing the pathophysiological hypothesis in which DNM2-dependent trafficking would be altered. In addition, HTZ fibers showed an increased calcium concentration as well as an intracellular Dnm2 and dysferlin accumulation. A similar dysferlin retention, never reported so far in congenital myopathies, was also demonstrated in biopsies from DNM2-CNM patients and can be considered as a new marker to orientate direct genetic testing. Homozygous (HMZ) mice died during the first hours of life. Impairment of clathrin-mediated endocytosis, demonstrated in HMZ embryonic fibroblasts, could be the cause of lethality. Overall, this first mouse model of DNM2-related myopathy shows the crucial role of DNM2 in muscle homeostasis and will be a precious tool to study DNM2 functions in muscle, pathomechanisms of DNM2-CNM and developing therapeutic strategies.
Asunto(s)
Dinamina II/genética , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Mutación/genética , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/fisiopatología , Animales , Conducta Animal , Calcio/metabolismo , Disferlina , Embrión de Mamíferos/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Heterocigoto , Homocigoto , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Actividad Motora/fisiología , Contracción Muscular/fisiología , Proteínas Musculares/metabolismo , Debilidad Muscular/complicaciones , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Músculo Esquelético/ultraestructura , Atrofia Muscular/complicaciones , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Fenotipo , Transporte de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Glucógeno/biosíntesis , Músculo Esquelético/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Glucógeno Sintasa/genética , Glucógeno Sintasa/metabolismo , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismoRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.
Asunto(s)
Fibras Musculares de Contracción Rápida/metabolismo , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patología , Fenotipo , Análisis de Varianza , Animales , Western Blotting , Perfilación de la Expresión Génica , Glucólisis/fisiología , Inmunohistoquímica , Cuerpos de Inclusión Intranucleares/metabolismo , Cuerpos de Inclusión Intranucleares/patología , Ratones , Ratones Transgénicos , Modelos Biológicos , Contracción Muscular/fisiología , Fibras Musculares de Contracción Rápida/patología , Atrofia Muscular/etiología , Distrofia Muscular Oculofaríngea/complicaciones , Proteína I de Unión a Poli(A)/genética , Análisis de Componente Principal , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
INTRODUCTION: The effects of locomotor training (LT) on skeletal muscle after peripheral nerve injury and acetylcholinesterase deficiency are not well documented. METHODS: We determined the effects of LT on mouse soleus muscle performance after sciatic nerve transection with excision (full and permanent denervation), nerve transection (partial functional reinnervation), nerve crush (full denervation with full functional reinnervation), and acetylcholinesterase deficiency (alteration in neuromuscular junction functioning). RESULTS: We found no significant effect of LT on the recovery of soleus muscle weight, maximal force in response to muscle stimulation, and fatigue resistance after nerve transection with or without excision. However, LT significantly increased soleus muscle fatigue resistance after nerve crush and acetylcholinesterase deficiency. Moreover, hindlimb immobilization significantly aggravated the deficit in soleus muscle maximal force production and atrophy after nerve crush. CONCLUSIONS: LT is beneficial, and reduced muscle use is detrimental for intrinsic muscle performance in the context of disturbed nerve-muscle communication.
Asunto(s)
Terapia por Ejercicio , Locomoción/fisiología , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Enfermedades Neuromusculares/fisiopatología , Acetilcolinesterasa/deficiencia , Animales , Atrofia , Estimulación Eléctrica , Suspensión Trasera/fisiología , Técnicas In Vitro , Contracción Isométrica , Masculino , Ratones , Neuronas Motoras/patología , Contracción Muscular/fisiología , Desnervación Muscular , Fatiga Muscular/fisiología , Músculo Esquelético/patología , Compresión Nerviosa , Enfermedades Neuromusculares/patología , Tamaño de los Órganos , Resistencia Física/fisiología , Nervio Ciático/patologíaRESUMEN
INTRODUCTION: Duchenne Muscular Dystrophy (DMD) is characterized by the lack of dystrophin that leads to severe myofiber degeneration. We have shown that endomysial fibrosis is correlated with age at ambulation loss in DMD patients. However, the dystrophin-deficient mdx mouse does not have fibrotic lesions in adult limb muscles. Here, we describe a model of chronic mechanical muscle injury that triggers chronic lesions in mdx hindlimb muscle. METHODS: Micromechanical injuries were performed daily in tibialis anterior muscles for 2 weeks. RESULTS: Endomysial fibrosis appeared beginning 1 week post-injury, remained stable for 3 months and was associated with loss of specific maximal force. Fibrosis was associated with an increased expression of factors involved in fibrogenesis including α-smooth muscle actin, connective tissue growth factor, and lysyl oxidase, which colocalized with collagen deposits. CONCLUSIONS: This induced fibrotic dystrophic model may be useful to study mechanisms of fibrosis in dystrophinopathies and to evaluate antifibrotic treatments.
Asunto(s)
Modelos Animales de Enfermedad , Miembro Posterior , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Distrofias Musculares/patología , Distrofia Muscular de Duchenne/patología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Distrofina/deficiencia , Distrofina/genética , Distrofina/metabolismo , Fibrosis , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Duchenne muscular dystrophy is characterized by muscular atrophy, fibrosis, and fat accumulation. Several groups have demonstrated that in the mdx mouse, the exon-skipping strategy can restore a quasi-dystrophin in almost 100% of the muscle fibers. On the other hand, inhibition of the myostatin pathway in adult mice has been described to enhance muscle growth and improve muscle force. Our aim was to combine these two strategies to evaluate a possible additive effect. We have chosen to inhibit the myostatin pathway using the technique of RNA interference directed against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). The restoration of a quasi-dystrophin was mediated by the vectorized U7 exon-skipping technique (U7-DYS). Adeno-associated vectors carrying either the sh-AcvrIIb construct alone, the U7-DYS construct alone, or a combination of both constructs were injected in the tibialis anterior (TA) muscle of dystrophic mdx mice. We show that even if each separate approach has some effects on muscle physiology, the combination of the dystrophin rescue and the downregulation of the myostatin receptor is required to massively improve both the tetanic force and the specific force. This study provides a novel pharmacogenetic strategy for treatment of certain neuromuscular diseases associated with muscle wasting.
Asunto(s)
Distrofina/metabolismo , Distrofia Muscular de Duchenne/terapia , Miostatina/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Línea Celular , Dependovirus/genética , Distrofina/genética , Femenino , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos mdx , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Clinically used botulinum neurotoxins (BoNTs) are natural products of Clostridium botulinum. A novel, recombinant BoNT type A1 (rBoNT/A1; IPN10260) has been synthesized using the native amino acid sequence expressed in Escherichia coli and has previously been characterized in vitro and ex vivo. Here, we aimed to characterize rBoNT/A1 in vivo and evaluate its effects on skeletal muscle. The properties of rBoNT/A1 following single, intramuscular administration were evaluated in the mouse and rat digit abduction score (DAS) assays and compared with those of natural BoNT/A1 (nBoNT/A1). rBoNT/A1-injected tibialis anterior was assessed in the in situ muscle force test in rats. rBoNT/A1-injected gastrocnemius lateralis (GL) muscle was assessed in the compound muscle action potential (CMAP) test in rats. The rBoNT/A1-injected GL muscle was evaluated for muscle weight, volume, myofiber composition and immunohistochemical detection of cleaved SNAP25 (c-SNAP25). Results showed that rBoNT/A1 and nBoNT/A1 were equipotent and had similar onset and duration of action in both mouse and rat DAS assays. rBoNT/A1 caused a dose-dependent inhibition of muscle force and a rapid long-lasting reduction in CMAP amplitude that lasted for at least 30 days. Dose-dependent reductions in GL weight and volume and increases in myofiber atrophy were accompanied by immunohistochemical detection of c-SNAP25. Overall, rBoNT/A1 and nBoNT/A1 exhibited similar properties following intramuscular administration. rBoNT/A1 inhibited motoneurons neurotransmitter release, which was robust, long-lasting, and accompanied by cleavage of SNAP25. rBoNT/A1 is a useful tool molecule for comparison with current natural and future modified recombinant neurotoxins products.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Proteínas Recombinantes/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Inyecciones Intramusculares , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Tamaño de los Órganos , Ratas , Proteína 25 Asociada a Sinaptosomas/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.
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Acetilcolinesterasa/deficiencia , Acetilcolinesterasa/genética , Síndrome de Isaacs/enzimología , Síndrome de Isaacs/genética , Placa Motora/enzimología , Osteocondrodisplasias/enzimología , Osteocondrodisplasias/genética , Alelos , Animales , Modelos Animales de Enfermedad , Electromiografía , Femenino , Dosificación de Gen , Proteoglicanos de Heparán Sulfato/deficiencia , Proteoglicanos de Heparán Sulfato/genética , Humanos , Síndrome de Isaacs/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Placa Motora/fisiopatología , Contracción Muscular/genética , Contracción Muscular/fisiología , Mutación Missense , Osteocondrodisplasias/fisiopatología , FenotipoRESUMEN
Measuring the DNA telomere length of skeletal muscle in experienced endurance runners may contribute to our understanding of the effects of chronic exposure to endurance exercise on skeletal muscle. This study compared the minimum terminal restriction fragment (TRF) length in the vastus lateralis muscle of 18 experienced endurance runners (mean age: 42 +/- 7 years) to those of 19 sedentary individuals (mean age: 39 +/- 10 years). The runners had covered almost 50,000 km in training and racing over 15 years. Minimum TRF lengths measured in the muscle of both groups were similar (P = 0.805) and within the normal range. Minimum TRF length in the runners, however, was inversely related to their years spent running (r = -0.63, P = 0.007) and hours spent training (r = -0.52, P = 0.035). Therefore, since exposure to endurance running may influence minimum TRF length, and by implication, the proliferative potential of the satellite cells, chronic endurance running may be seen as a stressor to skeletal muscle.
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Atletas , Músculo Esquelético/metabolismo , Resistencia Física , Carrera/fisiología , Telómero/metabolismo , Adulto , Atletas/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Carrera/estadística & datos numéricosRESUMEN
Although acetylcholinesterase (AChE) knockout mice survive, they have abnormal neuromuscular function. We analysed further the effects of the mutation on hind limb muscle contractile properties. Tibialis anterior muscle from AChE KO mice is unable to maintain tension during a short period of repetitive nerve stimulation (tetanic fade) and has an increased twitch tension in response to a single nerve electric stimulation. In response to direct muscle stimulation, we found that maximal velocity of shortening of soleus muscle is increased and maximum tetanic force is decreased in AchE KO mice versus control animals. As the contractile properties of the soleus muscle were altered by AChE ablation, our results suggest cellular and molecular changes in AChE ablated muscle containing both fast and slow muscle fibres.
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Acetilcolinesterasa/metabolismo , Músculo Esquelético/fisiología , Acetilcolinesterasa/genética , Animales , Estimulación Eléctrica , Contracción Isométrica , Ratones , Ratones Noqueados , Músculo Esquelético/enzimologíaRESUMEN
X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and it is characterized by profound defects in T, B, and natural killer (NK) cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early murine leukemia retrovirus (MLV)-derived gammaretroviral vectors showed restoration of T cell immunity in patients, but it resulted in vector-induced insertional oncogenesis. We developed a self-inactivating (SIN) lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1α short promoter (EFS-IL2RG), and we tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin- HSPCs in an Il2rg-/-/Rag2-/- mouse model. The study showed restoration of T, B, and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile, and it showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase I/II clinical trial aimed at restoring both T and B cell immunity in SCID-X1 children upon non-myeloablative conditioning.