RESUMEN
Duchene muscular dystrophy leads to progressive muscle structural and functional decline due to chronic degenerative-regenerative cycles. Enhancing the regenerative capacity of dystrophic muscle provides potential therapeutic options. We previously demonstrated that the circadian clock repressor Rev-erbα inhibited myogenesis and Rev-erbα ablation enhanced muscle regeneration. Here we show that Rev-erbα deficiency in the dystrophin-deficient mdx mice promotes regenerative myogenic response to ameliorate muscle damage. Loss of Rev-erbα in mdx mice improved dystrophic pathology and muscle wasting. Rev-erbα-deficient dystrophic muscle exhibit augmented myogenic response, enhanced neo-myofiber formation and attenuated inflammatory response. In mdx myoblasts devoid of Rev-erbα, myogenic differentiation was augmented together with up-regulation of Wnt signaling and proliferative pathways, suggesting that loss of Rev-erbα inhibition of these processes contributed to the improvement in regenerative myogenesis. Collectively, our findings revealed that the loss of Rev-erbα function protects dystrophic muscle from injury by promoting myogenic repair, and inhibition of its activity may have therapeutic utilities for muscular dystrophy.
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Diferenciación Celular , Músculo Esquelético/citología , Distrofia Muscular Animal/prevención & control , Distrofia Muscular de Duchenne/prevención & control , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Regeneración , Animales , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/etiología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/etiología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Vía de Señalización WntRESUMEN
Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.
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Distrofina/genética , Edición Génica , Vectores Genéticos/administración & dosificación , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/prevención & control , Distrofia Muscular de Duchenne/prevención & control , Miocardio/metabolismo , Animales , Sistemas CRISPR-Cas , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Mutación , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Guía de KinetoplastidaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene. Absence of dystrophin protein leads to progressive degradation of skeletal and cardiac function and leads to premature death. Over the years, zebrafish have been increasingly used for studying DMD and are a powerful tool for drug discovery and therapeutic development. In our study, a birefringence screening assay led to identification of phosphodiesterase 10A (PDE10A) inhibitors that reduced the manifestation of dystrophic muscle phenotype in dystrophin-deficient sapje-like zebrafish larvae. PDE10A has been validated as a therapeutic target by pde10a morpholino-mediated reduction in muscle pathology and improvement in locomotion, muscle, and vascular function as well as long-term survival in sapje-like larvae. PDE10A inhibition in zebrafish and DMD patient-derived myoblasts were also associated with reduction of PITPNA expression that has been previously identified as a protective genetic modifier in two exceptional dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs that escaped the dystrophic phenotype. The combination of a phenotypic assay and relevant functional assessments in the sapje-like zebrafish enhances the potential for the prospective discovery of DMD therapeutics. Indeed, our results suggest a new application for a PDE10A inhibitor as a potential DMD therapeutic to be investigated in a mouse model of DMD.
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Distrofina/metabolismo , Distrofia Muscular Animal/prevención & control , Distrofia Muscular de Duchenne/prevención & control , Mioblastos/efectos de los fármacos , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Hidrolasas Diéster Fosfóricas/química , Pirazoles/farmacología , Quinolinas/farmacología , Animales , Perros , Distrofina/genética , Humanos , Larva/efectos de los fármacos , Larva/genética , Larva/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Mioblastos/metabolismo , Mioblastos/patología , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pez CebraRESUMEN
The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMDmdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases.
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Factores de Transcripción ARNTL/metabolismo , Modelos Animales de Enfermedad , Desarrollo de Músculos , Músculo Esquelético/citología , Distrofia Muscular Animal/prevención & control , Distrofia Muscular de Duchenne/prevención & control , Regeneración , Factores de Transcripción ARNTL/genética , Animales , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologíaRESUMEN
Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.
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Anexina A6/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Músculo Esquelético/lesiones , Distrofia Muscular Animal/prevención & control , Animales , Anexina A6/genética , Membrana Celular/patología , Femenino , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Dystrophin deficiency in mdx mice, a model for Duchenne muscular dystrophy, leads to muscle weakness revealed by a reduced specific maximal force as well as fragility (ie, higher susceptibility to contraction-induced injury, as shown by a greater force decrease after lengthening contractions). Both symptoms could be improved with dystrophin restoration-based therapies and long-term (months) voluntary exercise. Herein, we evaluated the effect of short-term (1-week) voluntary wheel running. We found that running improved fragility of tibialis anterior muscle (TA), but not plantaris muscle, independently of utrophin up-regulation, without affecting weakness. Moreover, TA muscle excitability was also preserved by running, as shown by compound muscle action potential measurements after lengthening contractions. Of interest, the calcineurin inhibitor cyclosporin A prevented the effect of running on both muscle fragility and excitability. Cyclosporin also prevented the running-induced changes in expression of genes involved in excitability (Scn4a and Cacna1s) and slower contractile phenotype (Myh2 and Tnni1) in TA muscle. In conclusion, short-term voluntary exercise improves TA muscle fragility in mdx mice, without worsening weakness. Its effect was related to preserved excitability, calcineurin pathway activation, and changes in the program of genes involved in excitability and slower contractile phenotype. Thus, remediation of muscle fragility of Duchenne muscular dystrophy patients through appropriate exercise training deserves to be explored in more detail.
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Calcineurina/metabolismo , Distrofia Muscular Animal/prevención & control , Condicionamiento Físico Animal , Animales , Ratones , Ratones Endogámicos mdx , Actividad Motora , Contracción Muscular , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologíaRESUMEN
Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq-/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Quimiotaxis de Leucocito/efectos de los fármacos , Diafragma/efectos de los fármacos , Distrofia Muscular Animal/prevención & control , Proteína Quinasa C-theta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T/efectos de los fármacos , Animales , Diafragma/enzimología , Diafragma/inmunología , Diafragma/patología , Modelos Animales de Enfermedad , Fibrosis , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Distrofia Muscular Animal/enzimología , Distrofia Muscular Animal/inmunología , Distrofia Muscular Animal/patología , Necrosis , Proteína Quinasa C-theta/deficiencia , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.
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Deferoxamina/farmacología , Inflamación/prevención & control , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/prevención & control , Estrés Oxidativo/efectos de los fármacos , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Deferoxamina/administración & dosificación , Diafragma/efectos de los fármacos , Diafragma/metabolismo , Femenino , Inflamación/metabolismo , Inyecciones Intraperitoneales , Quelantes del Hierro/administración & dosificación , Quelantes del Hierro/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/prevención & control , Distrofia Muscular de Duchenne/metabolismo , FN-kappa B/metabolismoRESUMEN
Nutritional muscular dystrophy (NMD) of chicks is induced by dietary selenium (Se)/vitamin E (Vit. E) deficiencies and may be associated with oxidative cell damage. To reveal the underlying mechanisms related to the presumed oxidative cell damage, we fed four groups of 1-day-old broiler chicks (n = 40/group) with a basal diet (BD; 10 µg Se/kg; no Vit. E added, -Se -Vit. E) or the BD plus all-rac-α-tocopheryl acetate at 50mg/kg (-Se +Vit. E), Se (as sodium selenite) at 0.3mg/kg (+Se -Vit. E), or both of these nutrients (+Se +Vit. E) for 6 weeks. High incidences of NMD (93%) and mortality (36%) of the chicks were induced by the BD, starting at week 3. Dietary Se deficiency alone also induced muscle fiber rupture and coagulation necrosis in the pectoral muscle of chicks at week 3 and thereafter, with increased (P < 0.05) malondialdehyde, decreased (P < 0.05) total antioxidant capacity, and diminished (P < 0.05) glutathione peroxidase activities in the muscle. To link these oxidative damages of the muscle cells to the Se-deficiency-induced NMD, we first determined gene expression of the potential 26 selenoproteins in the muscle of the chicks at week 2 before the onset of symptoms. Compared with the +Se chicks, the -Se chicks had lower (P < 0.05) muscle mRNA levels of Gpx1, Gpx3, Gpx4, Sepp1, Selo, Selk, Selu, Selh, Selm, Sepw1, and Sep15. The -Se chicks also had decreased (P < 0.05) production of 6 selenoproteins (long-form selenoprotein P (SelP-L), GPx1, GPx4, Sep15, SelW, and SelN), but increased levels (P < 0.05) of the short-form selenoprotein P in muscle at weeks 2 and 4. Dietary Se deficiency elevated (P < 0.05) muscle p53, cleaved caspase 3, cleaved caspase 9, cyclooxygenase 2 (COX2), focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), phospho-Akt, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, phospho-JNK, and phospho-ERK and decreased (P < 0.05) muscle procaspase 3, procaspase 9, and NF-κB inhibitor α. In conclusion, the downregulation of SelP-L, GPx1, GPx4, Sep15, SelW, and SelN by dietary Se deficiency might account for induced oxidative stress and the subsequent peroxidative damage of chick muscle cells via the activation of the p53/caspase 9/caspase 3, COX2/FAK/PI3K/Akt/NF-κB, and p38 MAPK/JNK/ERK signaling pathways. Metabolism of peroxides and redox regulation are likely to be the mechanisms whereby these selenoproteins prevented the onset of NMD in chicks.
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Apoptosis , Dieta/efectos adversos , Distrofia Muscular Animal/prevención & control , Peróxidos/metabolismo , Selenoproteínas/metabolismo , Animales , Antioxidantes , Western Blotting , Proliferación Celular , Células Cultivadas , Pollos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Técnicas para Inmunoenzimas , Masculino , Distrofia Muscular Animal/etiología , Distrofia Muscular Animal/metabolismo , Oxidación-Reducción , Estrés Oxidativo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Selenio/metabolismo , Selenoproteínas/genética , Glutatión Peroxidasa GPX1RESUMEN
Receptor-activator of NF-κB, its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology still is elusive. Here, we show that muscle cells can produce and secrete osteoprotegerin and pharmacologic treatment of dystrophic mdx mice with recombinant osteoprotegerin muscles. (Recombinant osteoprotegerin-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in fast-twitch extensor digitorum longus.) Our data identify osteoprotegerin as a novel protector of muscle integrity, and it potentially represents a new therapeutic avenue for both muscular diseases and osteoporosis.
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Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/prevención & control , Osteoprotegerina/metabolismo , Animales , Línea Celular , Fragmentos Fc de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Inflamación/patología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculos/efectos de los fármacos , Músculos/metabolismo , Músculos/patología , Músculos/fisiopatología , Distrofia Muscular Animal/fisiopatologíaRESUMEN
INTRODUCTION: The dystrophic features in hindlimb skeletal muscles of female mdx mice are unclear. METHODS: We analyzed force-generating capacity and force decline after lengthening contraction-induced damage (fragility). RESULTS: Young (6-month-old) female mdx mice displayed reduced force-generating capacity (-18%) and higher fragility (23% force decline) compared with female age-matched wild-type mice. These 2 dystrophic features were less accentuated in young female than in young male mdx mice (-32% and 42% force drop). With advancing age, force-generating capacity decreased and fragility increased in old (20 month) female mdx mice (-21% and 57% force decline), but they were unchanged in old male mdx mice. Moreover, estradiol treatment had no effect in old female mdx mice. CONCLUSIONS: Female gender-related factors mitigate dystrophic features in young but not old mdx mice. Further studies are warranted to identify the beneficial gender-related factor in dystrophic muscle.
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Contracción Muscular/fisiología , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Distrofia Muscular Animal/prevención & control , Distrofia Muscular Animal/fisiopatología , Caracteres Sexuales , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdxRESUMEN
Duchenne muscular dystrophy is a lethal genetic disease characterized by the loss of muscle integrity and function over time. Using Drosophila, we show that dystrophic muscle phenotypes can be significantly suppressed by a reduction of wunen, a homolog of lipid phosphate phosphatase 3, which in higher animals can dephosphorylate a range of phospholipids. Our suppression analyses include assessing the localization of Projectin protein, a titin homolog, in sarcomeres as well as muscle morphology and functional movement assays. We hypothesize that wunen-based suppression is through the elevation of the bioactive lipid Sphingosine 1-phosphate (S1P), which promotes cell proliferation and differentiation in many tissues, including muscle. We confirm the role of S1P in suppression by genetically altering S1P levels via reduction of S1P lyase (Sply) and by upregulating the serine palmitoyl-CoA transferase catalytic subunit gene lace, the first gene in the de novo sphingolipid biosynthetic pathway and find that these manipulations also reduce muscle degeneration. Furthermore, we show that reduction of spinster (which encodes a major facilitator family transporter, homologs of which in higher animals have been shown to transport S1P) can also suppress dystrophic muscle degeneration. Finally, administration to adult flies of pharmacological agents reported to elevate S1P signaling significantly suppresses dystrophic muscle phenotypes. Our data suggest that localized intracellular S1P elevation promotes the suppression of muscle wasting in flies.
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Regulación hacia Abajo/genética , Drosophila melanogaster/genética , Lisofosfolípidos/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/prevención & control , Fenotipo , Esfingosina/análogos & derivados , Regulación hacia Arriba/genética , Animales , Lisofosfolípidos/biosíntesis , Distrofia Muscular Animal/diagnóstico , Mutación , Miofibrillas/genética , Miofibrillas/metabolismo , Miofibrillas/patología , Transducción de Señal/genética , Esfingosina/biosíntesis , Esfingosina/genéticaRESUMEN
Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype. Dp116:mdx:utrn(-/-) transgenic mice had dramatic improvements in growth, mobility and lifespan compared with controls. This was associated with increased muscle mass and force generating capacity of limb muscles, although myofiber size and specific force were unchanged. Conversely, Dp116 had no effect on dystrophic injury as determined by muscle histopathology and serum creatine kinase levels. Dp116 also failed to restore normal fiber-type distribution or the post-synaptic architecture of the neuromuscular junction. These data demonstrate that the DGC is critical for growth and maintenance of muscle mass, a function that is independent of the ability to prevent dystrophic pathophysiology. Likewise, this is the first demonstration in skeletal muscle of a positive functional role for a dystrophin protein that lacks actin-binding domains. We conclude that both mechanical and non-mechanical functions of dystrophin are important for its role in skeletal muscle.
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Distrofina/metabolismo , Longevidad , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/prevención & control , Animales , Fenómenos Biomecánicos , Creatina Quinasa/sangre , Distrofina/química , Esófago/patología , Femenino , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Contracción Muscular , Músculo Esquelético/ultraestructura , Distrofia Muscular Animal/sangre , Distrofia Muscular Animal/fisiopatología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Unión Neuromuscular/ultraestructura , Tamaño de los Órganos , Isoformas de Proteínas/metabolismo , Análisis de Supervivencia , Utrofina/deficiencia , Utrofina/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4(+) and CD8(+) T cells in skeletal muscle tissue, while Foxp3(+) regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.
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Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular Animal/prevención & control , Sirolimus/farmacología , Animales , Western Blotting , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Diafragma/efectos de los fármacos , Diafragma/metabolismo , Diafragma/patología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/prevención & control , Fosforilación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.
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Inhibidores de Anhidrasa Carbónica/farmacología , Modelos Animales de Enfermedad , Distrofina/deficiencia , Distrofia Muscular Animal/prevención & control , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Diclorfenamida/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Distrofina/genética , Humanos , Metazolamida/farmacología , Ratones , Ratones Endogámicos mdx , Actividad Motora , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Interferencia de ARN , Factores de TiempoRESUMEN
Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.
Asunto(s)
Modelos Animales de Enfermedad , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Lipocalinas/antagonistas & inhibidores , Distrofia Muscular Animal/prevención & control , Anestésicos Locales/toxicidad , Animales , Western Blotting , Bupivacaína/toxicidad , Citocinas/genética , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Distrofia Muscular Animal/inducido químicamente , Distrofia Muscular Animal/enzimología , Necrosis , Piperidinas/farmacología , Prostaglandina D2/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The lack of dystrophin in mdx mice and in Duchenne muscular dystrophy causes sarcolemmal breakdown and increased calcium influx followed by myonecrosis. We examined whether the calcium channel blockers diltiazem and verapamil protect dystrophic muscles from degeneration. Mdx mice received daily intraperitoneal injections of diltiazem or verapamil for 18 days, followed by removal of the sternomastoid, diaphragm, tibialis anterior, and cardiac muscles. Control mdx mice were injected with saline. Both drugs significantly decreased blood creatine kinase levels. Total calcium content was significantly higher in mdx muscles than in control C57Bl/10. Verapamil and diltiazem reduced total calcium content only in diaphragm and cardiac muscle. Histological analysis showed that diltiazem significantly attenuated myonecrosis in diaphragm. Immunoblots showed a significant increase of calsequestrin and beta-dystroglycan levels in some diltiazem- and verapamil-treated muscles. Possible interactions of these drugs with the sarcoplasmic reticulum and sarcolemma may also contribute to the improvement of the dystrophic phenotype.
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Bloqueadores de los Canales de Calcio/uso terapéutico , Diltiazem/uso terapéutico , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/prevención & control , Sarcolema/efectos de los fármacos , Verapamilo/uso terapéutico , Animales , Calcio/metabolismo , Calsecuestrina/metabolismo , Creatina Quinasa/sangre , Modelos Animales de Enfermedad , Distroglicanos/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Sarcolema/metabolismoAsunto(s)
Distrofina/genética , Terapia Genética/métodos , Distrofia Muscular Animal/genética , Interferencia de ARN , Animales , Distrofina/deficiencia , Distrofina/metabolismo , Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/prevención & control , ARN Interferente Pequeño/genética , Factores de Tiempo , TransfecciónAsunto(s)
Acetilcisteína/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/prevención & control , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Caveolina 3/metabolismo , Modelos Animales de Enfermedad , Distroglicanos/metabolismo , Distrofina/genética , Distrofina/metabolismo , Depuradores de Radicales Libres/farmacología , Ratones , Ratones Endogámicos mdx , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Distrofia Muscular Animal/etiología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Distrofia Muscular de Duchenne/prevención & control , Mutación/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three-week-old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-kappaB, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of beta-dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients.