Evaluation of the muscle development process in the limb-girdle muscular dystrophy type 2B

Author(s)

Lucas Santos e Souza

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2024-11-01

Examining board members

Mariz Vainzof; Ernesto da Silveira Goulart Guimarães; Elen Haruka Miyabara; Helga Cristina Almeida da Silva

• Advisor: Mariz Vainzof

Abstract

Skeletal muscle is an abundant and highly specialized tissue in the human body, performing functions such as locomotion, support, and energy storage. The regenerative capacity of this tissue after injuries is remarkable, and a complex and extensive network of mechanisms involving numerous genes and proteins is recruited to execute this function. Neuromuscular diseases, such as Muscular Dystrophies, result from mutations in genes that play essential roles in the proper functioning of muscle. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) arises from pathogenic mutations in the DYSF gene, which encodes the protein dysferlin, involved in muscle regeneration and membrane repair. Previous studies conducted by our group using human myoblasts from an LGMD2B patient revealed an impairment in myoblast differentiation, characterized by the formation of smaller myotubes with fewer nuclei. Additional results showed that, in dystrophic mice, the regenerative capacity is maintained, but the muscle fibers remain small and immature. Therefore, in this study, we investigated the effect of dysferlin absence on the differentiation and maturation process of muscle fibers in vitro by analyzing the expression of genes and proteins associated with the process in additional myoblast lines from three LGMD2B patients. We identified that cellular differentiation in these lines is indeed impaired, as evidenced by the significant reduction in myotube size and the predominance of small myotubes with few nuclei. We confirmed this effect through the silencing of the DYSF gene by CRISPR/Cas9 in normal myoblasts, highlighting the crucial role of dysferlin in myogenesis. We found that the absence of dysferlin does not significantly alter the expression of genes associated with muscle development and newly identified genes involved in the process. Furthermore, we demonstrated that LGMD2B muscle cells exhibit a global gene expression profile that differs from controls, especially in pathways and biological processes associated with cytoskeleton organization and regulation, which are intrinsically related to muscle development. Thus, we provided evidence indicating that dysferlin deficiency is related to a delay in the myogenic process, wherein the expression of some genes does not follow the same pattern observed in normal cells. Finally, we report the study of a family with LGMD2B-affected patients that has been extensively studied over time. In this study, we identified the pathogenic mutations in the DYSF gene causing the disease and correlated the presence of one of the mutations with a European genetic background, which may be associated with the significant clinical variability observed among the affected individuals in the family. (AU)

• FAPESP's process: 20/04744-8 - Evaluation of the muscle regeneration process in limb-girdle Muscular Dystrophy 2B
• Grantee: Lucas Santos e Souza
• Support Opportunities: Scholarships in Brazil - Doctorate