RESUMEN
INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.
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Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Distrofia Miotónica/genética , Adulto , Animales , Línea Celular , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Masculino , Persona de Mediana Edad , Mioblastos/patología , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología , Pez CebraRESUMEN
Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.
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Proteínas Quinasas Activadas por AMP/metabolismo , Disferlina/química , Disferlina/metabolismo , Metformina/administración & dosificación , Músculo Esquelético/lesiones , Distrofia Muscular de Cinturas/tratamiento farmacológico , Animales , Línea Celular , Modelos Animales de Enfermedad , Disferlina/genética , Humanos , Rayos Láser/efectos adversos , Metformina/farmacología , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/metabolismo , Mutación , Fosforilación , Dominios Proteicos , Sarcolema/metabolismo , Pez CebraRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.
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Distrofia Muscular de Duchenne/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Perros , Distrofina/genética , Distrofina/metabolismo , Humanos , Células Musculares/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Pez Cebra/metabolismoRESUMEN
Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening.
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Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Pez Cebra/fisiología , Animales , Modelos Animales de Enfermedad , Cinética , Contracción Muscular , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares de Contracción Lenta/patología , Análisis de Regresión , Sarcómeros/metabolismo , Tetania/fisiopatologíaRESUMEN
Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Distrofina/genética , Hemo-Oxigenasa 1/biosíntesis , Distrofia Muscular de Duchenne/tratamiento farmacológico , Animales , GMP Cíclico/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Distrofina/deficiencia , Hemo-Oxigenasa 1/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Purinas/farmacología , ARN Mensajero/genética , Transducción de Señal/genética , Citrato de Sildenafil , Sulfonas/farmacología , Regulación hacia Arriba , Pez Cebra/genéticaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with α-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.
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Proteínas de Homeodominio/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Pez Cebra/genética , Actinas/genética , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Microscopía Electrónica de Transmisión , Músculo Esquelético/anomalías , Distrofia Muscular Facioescapulohumeral/patología , Óvulo/crecimiento & desarrollo , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hombro/anomalíasRESUMEN
Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.
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Proteínas Sustrato del Receptor de Insulina/metabolismo , Insulina/metabolismo , MicroARNs/metabolismo , Desarrollo de Músculos/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Diferenciación Celular/genética , Procesos de Crecimiento Celular/fisiología , Células Cultivadas , Femenino , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patología , Insulina/genética , Proteínas Sustrato del Receptor de Insulina/genética , Ratones , MicroARNs/genética , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Mioblastos/metabolismo , Mioblastos/fisiología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.
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Diferenciación Celular/genética , Histona Desacetilasas/metabolismo , Proteínas de la Membrana/metabolismo , Células Musculares/metabolismo , Desarrollo de Músculos/genética , Proteínas Musculares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular , Células Cultivadas , Regulación hacia Abajo/genética , Disferlina , Histona Desacetilasa 6 , Histona Desacetilasas/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Unión Proteica/genética , Alineación de Secuencia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Pez CebraRESUMEN
Two known zebrafish dystrophin mutants, sapje and sapje-like (sap(c/100)), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of seven candidate chemicals restored normal birefringence and increased survival of dystrophin-null fish. One chemical, aminophylline, which is known to be a nonselective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and up-regulate the cAMP-dependent PKA pathway in treated dystrophin-deficient fish. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy.
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Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/fisiopatología , Preparaciones Farmacéuticas , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Distrofina/metabolismo , Humanos , Distrofia Muscular de Duchenne/patología , Oligonucleótidos Antisentido , Fenotipo , Bibliotecas de Moléculas Pequeñas , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
In a forward genetic approach to identify novel genes for congenital muscle diseases, a zebrafish mutant, designated patchytail, was identified that exhibits degenerating muscle fibers with impaired motility behavior. Genetic mapping identified a genomic locus containing the zebrafish ortholog of the dystroglycan gene (DAG1). Patchytail fish contain a point mutation (c.1700T>A) in dag1, resulting in a missense change p.V567D. This change is associated with reduced transcripts and a complete absence of protein. The absence of α-dystroglycan and ß-dystroglycan caused destabilization of dystroglycan complex, resulting in membrane damages. Membrane damage was localized on the extracellular matrix at myosepta as well as basement membrane between adjacent myofibers. These studies also identified structural abnormalities in triads at 3 days post fertilization (dpf) of dystroglycan-deficient muscles, significantly preceding sarcolemmal damage that becomes evident at 7 dpf. Immunofluorescence studies identified a subpopulation of dystroglycan that is expressed at t-tubules in normal skeletal muscles. In dag1-mutated fish, smaller and irregular-shaped t-tubule vesicles, as well as highly disorganized terminal cisternae of sarcoplasmic reticulum, were common. In addition to skeletal muscle defects, dag1-mutated fish have brain abnormalities and ocular defects in posterior as well as anterior chambers. These phenotypes of dystroglycan-deficient fish are highly reminiscent of the phenotypes observed in the human conditions muscle-eye-brain disease and Walker-Warburg syndrome. This animal model will provide unique opportunities in the understanding of biological functions of dystroglycan in a wide range of dystroglycanopathies, as disruption of this gene in higher vertebrates results in early embryonic lethality.
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Modelos Animales de Enfermedad , Distroglicanos/genética , Distrofias Musculares/genética , Mutación Missense , Proteínas de Pez Cebra/genética , Pez Cebra , Animales , Secuencia de Bases , Distroglicanos/metabolismo , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Actividad Motora , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/fisiopatología , Mutación Puntual , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Recently, a number of chemical and drug screens using zebrafish embryos have been published. Using zebrafish dystrophin mutants, we screened a chemical library for small molecules that modulate the muscle phenotype and identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish. One chemical, aminophylline, which is known to be a non-selective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and to up-regulate cAMP-dependent protein kinase (PKA) in treated dystrophin deficient fish. Our methodologies, which combine drug screening with assessment of the chemical effects by genotyping and staining with anti-dystrophin, provide a powerful means to identify template structures potentially relevant to the development of novel human muscular dystrophies therapeutics.
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Modelos Animales de Enfermedad , Distrofina/genética , Distrofia Muscular de Duchenne/tratamiento farmacológico , Pez Cebra/genética , Animales , Evaluación Preclínica de Medicamentos , Distrofina/metabolismo , Embrión no Mamífero , Músculos/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Bibliotecas de Moléculas Pequeñas , Pez Cebra/anomalíasRESUMEN
Recently, a number of chemical and drug screens using zebrafish embryos have been published. Using zebrafish dystrophin mutants, we screened a chemical library for small molecules that modulate the muscle phenotype and identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish. One chemical, aminophylline, which is known to be a non-selective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and to up-regulate cAMP-dependent protein kinase (PKA) in treated dystrophin deficient fish. Our methodologies, which combine drug screening with assessment of the chemical effects by genotyping and staining with anti-dystrophin, provide a powerful means to identify template structures potentially relevant to the development of novel human muscular dystrophies therapeutics.
RESUMEN
We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.
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Proteínas de la Membrana/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/congénito , Enfermedades Musculares/genética , Mutación , Células Satélite del Músculo Esquelético/metabolismo , Animales , Femenino , Genes Recesivos , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Músculo Esquelético/patología , Linaje , Fenotipo , Pez CebraRESUMEN
Various muscular dystrophies are associated with the defective glycosylation of alpha-dystroglycan and are known to result from mutations in genes encoding glycosyltransferases. Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukuyama-type congenital muscular dystrophy (FCMD), that is thought to function as a glycosyltransferase. Mutations in FKRP have been linked to a variety of phenotypes including Walker-Warburg syndrome (WWS), limb girdle muscular dystrophy (LGMD) 2I and congenital muscular dystrophy 1C (MDC1C). Zebrafish are a useful animal model to reveal the mechanism of these diseases caused by mutations in FKRP gene. Downregulating FKRP expression in zebrafish by two different morpholinos resulted in embryos which had developmental defects similar to those observed in human muscular dystrophies associated with mutations in FKRP. The FKRP morphants showed phenotypes involving alterations in somitic structure and muscle fiber organization, as well as defects in developing eye morphology. Additionally, they were found to have a reduction in alpha-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, alpha-dystroglycan glycosylation and laminin binding activity of alpha-dystroglycan in the morphants. Co-injection of the human FKRP mRNA containing causative mutations found in human patients of WWS, MDC1C and LGMD2I could not restore their phenotypes significantly. Interestingly, these morphant fish having human FKRP mutations showed a wide phenotypic range similar to that seen in humans.
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Modelos Animales de Enfermedad , Glicosiltransferasas/metabolismo , Distrofias Musculares/metabolismo , Proteínas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Distroglicanos/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación de la Expresión Génica , Glicosilación , Glicosiltransferasas/genética , Humanos , Laminina/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/congénito , Distrofias Musculares/genética , Pentosiltransferasa , Unión Proteica , Proteínas/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Skeletal muscle is formed via fusion of myoblasts, a well-studied process in Drosophila. In vertebrates however, this process is less well understood, and whether there is evolutionary conservation with the proteins studied in flies is under investigation. Sticks and stones (Sns), a cell surface protein found on Drosophila myoblasts, has structural homology to nephrin. Nephrin is a protein expressed in kidney that is part of the filtration barrier formed by podocytes. No previous study has established any role for nephrin in skeletal muscle. We show, using two models, zebrafish and mice, that the absence of nephrin results in poorly developed muscles and incompletely fused myotubes, respectively. Although nephrin-knockout (nephrin(KO)) myoblasts exhibit prolonged activation of MAPK/ERK pathway during myogenic differentiation, expression of myogenin does not seem to be altered. Nevertheless, MAPK pathway blockade does not rescue myoblast fusion. Co-cultures of unaffected human fetal myoblasts with nephrin(KO) myoblasts or myotubes restore the formation of mature myotubes; however, the contribution of nephrin(KO) myoblasts is minimal. These studies suggest that nephrin plays a role in secondary fusion of myoblasts into nascent myotubes, thus establishing a possible functional conservation with Drosophila Sns.
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Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animales , Fusión Celular , Técnicas de Cocultivo , Humanos , Ratones , Músculo Esquelético/citología , Pez CebraRESUMEN
Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.
Asunto(s)
Fucosiltransferasas , Pez Cebra , Humanos , Animales , Ratones , Pez Cebra/genética , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Glicosilación , Desarrollo de Músculos/genéticaRESUMEN
Sapje-like (sap(cl100)) was one of eight potential zebrafish muscle mutants isolated as part of an early-pressure screen of 500 families. This mutant shows a muscle tearing phenotype similar to sapje (dys-/-) and both mutants fail to genetically complement suggesting they have a mutation in the same gene. Protein analysis confirms a lack of dystrophin in developing sapje-like embryos. Sequence analysis of the sapje-like dystrophin mRNA shows that exon 62 is missing in the dystrophin transcript causing exon 63 to be translated out of frame terminating translation at a premature stop codon at the end of exon 63. Sequence analysis of sapje-like genomic DNA identified a mutation in the donor splice junction at the end of dystrophin exon 62. This mutation is similar to splicing mutations associated with human forms of Duchenne Muscular Dystrophy. Sapje-like is the first zebrafish dystrophin splicing mutant identified to date and represents a novel disease model which can be used in future studies to identify therapeutic compounds for treating diseases caused by splicing defects.
Asunto(s)
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutación , Empalme del ARN , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Modelos Animales de Enfermedad , Distrofina/química , Distrofina/metabolismo , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Fenotipo , Alineación de Secuencia , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, ß-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.
Asunto(s)
Proteínas de la Membrana/metabolismo , Músculo Esquelético/anomalías , Músculo Esquelético/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Distroglicanos/metabolismo , Distrofina/metabolismo , Técnicas de Silenciamiento del Gen , Laminina/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Morfolinos/genética , Mutación , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
INTRODUCTION: Over the past 10 years, the use of zebrafish for scientific research in the area of muscle development has increased dramatically. Although several protocols exist for the isolation of adult myoblast progenitors from larger fish, no standardized protocol exists for the isolation of myogenic progenitors from adult zebrafish muscle. METHODS: Using a variant of a mammalian myoblast isolation protocol, zebrafish muscle progenitors have been isolated from the total dorsal myotome. These zebrafish myoblast progenitors can be cultured for several passages and then differentiated into multinucleated, mature myotubes. RESULTS: Transcriptome analysis of these cells during myogenic differentiation revealed a strong downregulation of pluripotency genes, while, conversely, showing an upregulation of myogenic signaling and structural genes. CONCLUSIONS: Together these studies provide a simple, yet detailed method for the isolation and culture of myogenic progenitors from adult zebrafish, while further promoting their therapeutic potential for the study of muscle disease and drug screening.