RESUMEN
The tropism of Zika virus (ZIKV) has been described in the nervous system, blood, placenta, thymus, and skeletal muscle. We investigated the mechanisms of skeletal muscle susceptibility to ZIKV using an in vitro model of human skeletal muscle myogenesis, in which myoblasts differentiate into myotubes. Myoblasts were permissive to ZIKV infection, generating productive viral particles, while myotubes controlled ZIKV replication. To investigate the underlying mechanisms, we used gene expression profiling. First, we assessed gene changes in myotubes compared with myoblasts in the model without infection. As expected, we observed an increase in genes and pathways related to the contractile muscle system in the myotubes, a reduction in processes linked to proliferation, migration and cytokine production, among others, confirming the myogenic capacity of our system in vitro. A comparison between non-infected and infected myoblasts revealed more than 500 differentially expressed genes (DEGs). In contrast, infected myotubes showed almost 2,000 DEGs, among which we detected genes and pathways highly or exclusively expressed in myotubes, including those related to antiviral and innate immune responses. Such gene modulation could explain our findings showing that ZIKV also invades myotubes but does not replicate in these differentiated cells. In conclusion, we showed that ZIKV largely (but differentially) disrupts gene expression in human myoblasts and myotubes. Identifying genes involved in myotube resistance can shed light on potential antiviral mechanisms against ZIKV infection.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Antivirales/metabolismo , Femenino , Expresión Génica , Humanos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Embarazo , Virus Zika/fisiología , Infección por el Virus Zika/genéticaRESUMEN
Growing evidence demonstrates a continuous interaction between the immune system and the skeletal muscle in inflammatory diseases of different pathogenetic origins, in dystrophic conditions such as Duchenne Muscular Dystrophy as well as during normal muscle regeneration. Although one component of the innate immunity, the macrophage, has been extensively studied both in disease conditions and during cell or gene therapy strategies aiming at restoring muscular functions, much less is known about dendritic cells and their primary immunological targets, the T lymphocytes. This review will focus on the dendritic cells and T lymphocytes (including effector and regulatory T-cells), emphasizing the potential cross talk between these cell types and their influence on the structure and function of skeletal muscle.
RESUMEN
X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Diferenciación Celular , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Músculo Esquelético/patología , Enfermedades Musculares/patología , Autofagia , Secuencia de Bases , Biopsia , Brasil , Proliferación Celular , Preescolar , Femenino , Regulación de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Recién Nacido , Lisosomas/metabolismo , Masculino , Fusión de Membrana , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Enfermedades Musculares/genética , Mioblastos/metabolismo , Mioblastos/patología , Linaje , ARN Mensajero/genética , ARN Mensajero/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-ß1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-ß failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/ß-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to ß-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients.
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Interacciones Huésped-Patógeno , Desarrollo de Músculos , Factores Reguladores Miogénicos/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Línea Celular , Proliferación Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Ratones , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/parasitología , Factores Reguladores Miogénicos/genética , Toxoplasmosis/parasitología , Vía de Señalización WntRESUMEN
Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.
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Actinas/metabolismo , Disferlina/química , Distrofia Muscular de Cinturas/metabolismo , Mioblastos/citología , Citoesqueleto de Actina/metabolismo , Actinas/genética , Adolescente , Adulto , Línea Celular , Disferlina/genética , Disferlina/metabolismo , Femenino , Humanos , Masculino , Distrofia Muscular de Cinturas/genética , Mioblastos/metabolismo , Dominios ProteicosRESUMEN
Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.
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Diferenciación Celular , Proliferación Celular , Células Dendríticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Adulto , Antígenos de Diferenciación/biosíntesis , Células Dendríticas/citología , Femenino , Humanos , Recién Nacido , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Mioblastos Esqueléticos/citologíaRESUMEN
Duchenne muscular dystrophy (DMD) affects 1:3500-1:5000 male births, and is caused by X-linked mutations in the dystrophin gene, manifested by progressive muscle weakness and wasting due to the absence of dystrophin protein, leading to degeneration of skeletal muscle. DMD patients are clinically heterogeneous and the functional phenotype often cannot be correlated with the genotype. Therefore, defined reliable noninvasive biomarkers aiming at predicting if a given DMD child will progress more or less rapidly will be instrumental to better design inclusion of defined patients for future therapeutic assays. We recently showed that CD49d expression levels in blood-derived T-cell subsets can predict disease progression in DMD patients. Herein we describe in detail the methodology to be applied for defining, through four-color flow cytometry, the membrane expression levels of the CD49d (the α4 chain of the integrins α4ß1 and α4ß7) in circulating CD4+ and CD8+ T cell subsets. Since we have also shown that this molecule can also be placed as a potential target for therapeutics in DMD, we also describe the cell migration functional assay that can be applied to test potential CD49d inhibitors that can modulate their ability to cross endothelial or extracellular matrix (ECM) barriers.
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Biomarcadores/sangre , Citometría de Flujo/métodos , Integrina alfa4/sangre , Distrofia Muscular de Duchenne/sangre , Progresión de la Enfermedad , Distrofina/genética , Regulación de la Expresión Génica , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Fenotipo , Subgrupos de Linfocitos TRESUMEN
BACKGROUND: The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. METHODS: We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. RESULTS: We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. CONCLUSIONS: We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.
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Movimiento Celular , Matriz Extracelular/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Sistema de Señalización de MAP Quinasas , Metaloproteinasas de la Matriz/metabolismo , Mioblastos/metabolismo , Células Cultivadas , Humanos , Integrina alfa5beta1/metabolismo , Metaloproteinasas de la Matriz/genética , Mioblastos/efectos de los fármacos , Mioblastos/fisiología , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Laminina/metabolismoRESUMEN
BACKGROUND: Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies. RESULTS: Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X7 receptor and the transient receptor potential channel, TRPV2, another Ca(2+) permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca(2+) level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor. CONCLUSIONS: The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca(2+) levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.
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Conexinas/metabolismo , Proteínas de la Membrana/deficiencia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/deficiencia , Biopsia , Señalización del Calcio , Línea Celular , Disferlina , Humanos , Espacio Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación/genética , Receptores Purinérgicos P2X7/metabolismo , Sarcolema/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.