RESUMEN
The study of the population of muscle satellite cells (SC) is important to understand muscle regeneration and its involvement in the different dystrophic processes. We studied two dystrophic mouse models, Largemyd and Lama2dy2j/J, that show an intense and very similar pattern of muscle degeneration, but with differences in the expression of genes involved in the regeneration cascade. They are, therefore, interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this chapter are to describe the isolation and characterization of SC populations, evaluating the presence of myogenic and pluripotent stem cells markers in normal and dystrophic muscles.
Asunto(s)
Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/patología , Regeneración/fisiología , Células Satélite del Músculo Esquelético/citología , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Distrofias Musculares/patologíaRESUMEN
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
Satellite cells (SCs) are the main muscle stem cells responsible for its regenerative capacity. In muscular dystrophies, however, a failure of the regenerative process results in muscle degeneration and weakness. To analyze the effect of different degrees of muscle degeneration in SCs behavior, we studied adult muscle of the dystrophic strains: DMDmdx, Largemyd, DMDmdx/Largemyd, with variable histopathological alterations. Similar results were observed in the dystrophic models, which maintained normal levels of PAX7 expression, retained the Pax7-positive SCs pool, and their proliferation capacity. Moreover, elevated expression of MYOG, an important myogenic factor, was also observed. The ability to form new fibers was verified by the presence of dMyHC positive regenerating fibers. However, those fibers had incomplete maturation characteristics, such as small and homogenous fiber caliber, which could contribute to their dysfunction. We concluded that dystrophic muscles, independently of their degeneration degree, retain their SCs pool with proliferating and regenerative capacities. Nonetheless, the maturation of these new fibers is incomplete and do not prevent muscle degeneration. Taken together, these results suggest that the improvement of late muscle regeneration should better contribute to therapeutic approaches.
Asunto(s)
Distrofias Musculares/patología , Distrofias Musculares/fisiopatología , Regeneración , Células Satélite del Músculo Esquelético/patología , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Factor de Transcripción PAX7/metabolismo , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.
Asunto(s)
Ratones de la Cepa 129 , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Mutación , Fenotipo , Animales , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Factores Protectores , TranscriptomaRESUMEN
Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmd(mdx) mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Large(myd), harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmd(mdx)/Large(myd) mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.