Caracterização in situ dos orientadores de axônio por repulsão (RGMs) na musculatura esquelética, e caracterização funcional do membro A (RGMa) durante a miogênese em camundongos

Abstract

Repulsive Guidance Molecules (RGMs) are negative cues that, during development, guide axonal growth in order to allow the proper innervation of the tissues and organs. RGMs compose a family of four members comprised by: RGMa, RGMb, RGMc and RGMd. New evidence has suggested possible roles for RGMs during skeletal muscle development and maintenance in vertebrates. RGMa and RGMb were found in somites, specially in domains known to be the origin site of skeletal muscle pioneer and stem cells. Evidences also suggested the presence of these molecules in adult mice skeletal muscle cells. In the present study, we aimed to (1) determine the localization of RGMa, RGMb, and RGMc in adult mice skeletal muscle cells; and (2) investigate the effects of RGMa over-expression and knockdown in skeletal muscle cells in vitro. RGMa, RGMb e RGMc were found in the sarcolemma. RGMa was also observed in the sarcoplasm with a striated pattern of labeling similar to the sarcomeric proteins. We were able to colocalize RGMa and known sarcoplasmic proteins suggesting a role for this axon guidance molecule as a sarcoplamisc protein in skeletal muscle cells. Western blot analysis revealed the presence of two RGMa peptides in adult skeletal muscle samples, a 60 kDa and a 33 kDa fragment. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa over-expression produced larger, hypertrophic cells, mainly myotubes, whereas RGMa knockdown resulted in the appearance of athrophic cells, with a typical morphology of undifferentiated myoblasts. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types since it interferes in myoblast fusion. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation in vitro.