Evaluation of the muscle regeneration process in limb-girdle Muscular Dystrophy 2B

• Grant number: 20/04744-8
• Support type: Scholarships in Brazil - Doctorate
• Effective date (Start): January 01, 2021
• Effective date (End): January 31, 2023
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal researcher: Mariz Vainzof
• Grantee: Lucas Santos e Souza
• Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is caused by pathogenic mutations in the gene that codes for dysferlin, a protein that acts in muscle regeneration and membrane repair. Previous studies carried out by our group using human myoblasts from a LGMD2B patient showed a loss in the differentiation of myoblasts to myotubes by the formation of smaller myotubes and containing fewer nuclei, compared to a normal myoblast cell line. Additional results from our group also showed that, in strains of dystrophic mice, there was a deficiency in muscle maturation. Recent studies have identified and reported the role of new proteins involved in the fusion of myoblasts and in the late stages of muscle differentiation, such as Myomaker, Myomixer, Palmdelphin and Obestatin/PR39. Objectives: to investigate the effect of the absence of the protein dysferlin in the process of differentiation and maturation of muscle fibers through the analysis of the expression of genes and proteins associated with the process. Methodology: immortalized human myoblast cell lines carrying different mutations in the DYSF gene and control cell lines will be used. These strains are part of an international consortium of studies on patients with DMC2B. Cultured cells will be proliferated and differentiated, and the analyzes will be carried out at different times of differentiation. The samples will be used for RNA and proteins extraction, aiming the analysis of gene expression by qPCR and protein detection/quantification by western blot. Immunofluorescence studies will be used for the qualitative and localization analysis of these same protein products. In the cells, morphometric analyzes will also be carried out to evaluate muscle differentiation parameters, such as myotube diameter and fusion index. In addition, the formation of protein complexes involving dysferlin and other proteins involved in myogenesis will be investigated by the co-immunoprecipitation technique during different moments of differentiation. If possible, an analysis of gene expression modulation of proteins associated with myogenesis will also be carried out to evaluate a possible therapeutic effect in LGMD2B, and analysis of transcriptome and secretome at different moments of differentiation, in an attempt to identify the expression profile of deficient cells in dysferlin during the differentiation process. We hope to be able to add evidence to better understand the importance and involvement of the protein dysferlin in the path of muscle regeneration, aiming at therapeutic possibilities to be developed. (AU)