Repulsive Guidance Molecules a, b and c Are Skeletal Muscle Proteins, and Repulsive Guidance Molecule a Promotes Cellular Hypertrophy and Is Necessary for Myotube Fusion

Abstract

Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. 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.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy

“…Mef2 can also act as a nodal point for remodeling programs in metabolic gene expression, fiber-type switching, and skeletal muscle regeneration [58,59,61]. Mef2a upregulation can also contribute to terminal differentiation and myoblast fusion, which is also consistent with the present GO term analysis and with the RGMa muscle phenotype [25,38]. Mef2a, Mef2c, and Mef2d deleted in combination in satellite cells abolished skeletal muscle regeneration after cardiotoxin injury [59].…”

“…In previous works, we found RGMa transcripts in the myogenic and satellite cell precursors in the somites during chicken embryonic development [24] and at the sarcolemma and in the sarcoplasm of adult mice muscle cells [25]. RGMa overexpression in C2C12 cells induced the formation of larger myotubes (hypertrophy) with an increased number of myonuclei (nuclei accretion), while its knockdown resulted in the appearance of smaller cells, with a deficient ability to form multinucleated myotubes [25].…”

“…RGMa transcripts could be found in chicken somites at the origin site of the muscle and satellite cell precursors [24]. In adult muscle, RGMa was found in regions of the sarcolemma and sarcoplasm, with an expression pattern similar to sarcomeric proteins [25]. Initial functional studies revealed that RGMa can induce myonuclear accretion and hypertrophy of myotubes, suggesting that this axon guidance molecule might be involved with the mechanisms that modulate skeletal muscle cell size [25].…”

SAP30 Gene Is a Probable Regulator of Muscle Hypertrophy in Chickens

“…A stage micrometer was used for standardized analysis. Myotube area was determined by length × width, and the fusion index was determined by the ratio of the number of nuclei inside myotube cells over the total nucleus number 100, after 3 days in DM, following Martins et al (2014) and Costa et al (2021) .…”