RESUMO
BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.
Assuntos
Proteínas do Tecido Nervoso , Transcriptoma , Proteínas Ligadas por GPI , Humanos , Hipertrofia , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
The muscle regeneration process requires a properly assembled extracellular matrix (ECM). Its homeostasis depends on the activity of different matrix-metalloproteinases (MMPs). The reversion-inducing-cysteine-rich protein with kazal motifs (RECK) is a membrane-anchored protein that negatively regulates the activity of different MMPs. However, the role of RECK in the process of skeletal muscle differentiation, regeneration, and fibrosis has not been elucidated. Here, we show that during skeletal muscle differentiation of C2C12 myoblasts and in satellite cells on isolated muscle fibers, RECK is transiently up regulated. C2C12 myoblasts with reduced RECK levels are more prone to enter the differentiation program, showing an accelerated differentiation process. Notch-1 signaling was reduced, while p38 and AKT signaling were augmented in myoblasts with decreased RECK levels. Overexpression of RECK restores the normal differentiation process but diminished the ability to form myotubes. Transient up-regulation of RECK occurs during skeletal muscle regeneration, which was accelerated in RECK-deficient mice (Reck±). RECK, MMPs and ECM proteins augmented in chronically damaged WT muscle, a model of muscle fibrosis. In this model, RECK ± mice showed diminished fibrosis compared to WT. These results strongly suggest that RECK is acting as a potential myogenic repressor during muscle formation and regeneration, emerging as a new player in these processes, and as a potential target to treat individuals with the muscle-wasting disease.
Assuntos
Diferenciação Celular , Matriz Extracelular/metabolismo , Fibrose/prevenção & controle , Proteínas Ligadas por GPI/antagonistas & inibidores , Desenvolvimento Muscular , Músculo Esquelético/citologia , Regeneração , Animais , Fibrose/metabolismo , Fibrose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
Therapeutic ultrasound (TU) has been used for the last 50 y in rehabilitation, including treatment of soft tissues. Ultrasound waves can be employed in two different modes of operation, continuous and pulsed, which produce both thermal and non-thermal effects. Despite the large-scale use of TU, there are few scientific studies on its biologic effects during skeletal muscle differentiation. To better analyze the cellular effects of TU, we decided to follow cells in vitro. The main purpose of this study was to evaluate the effects of TU in primary chick myogenic cell cultures using phase contrast optical microscopy and immunofluorescence microscopy, followed by image analysis and quantification. Our results indicate that TU can stimulate the differentiation of skeletal muscle cells in vitro, as measured by the thickness of multinucleated myotubes, the ratio of mononucleated cells to multinucleated cells and expression of the muscle-specific protein desmin. This study is a first step toward a metrologic and science-based protocol for cell treatment under different ultrasound field exposures.