Rho GTPases control cytoskeletal dynamics in muscle cells. Cytoskeletal dynamics in turn regulate myonuclear arrangement and GLUT4-mediated glucose uptake. Tight control of Rho GTPase activity is thus crucial to uphold myocellular function. The activity of Rho GTPases is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Yet, little is known about the specific GEFs and GAPs responsible for this regulation in skeletal muscle. Additionally, whether cytoskeletal modifications driven by Rho GTPase activity alter myonuclear arrangement and ultimately impair muscle fibre function is unexplored. Thus, we aimed to unravel new mechanisms of control for Rho GTPases in muscle via GEFs and GAPs.

We initially identified >30 GEF and GAP candidates for further study based on published phosphoproteomics data from human muscle biopsies in response to insulin, in insulin-resistant states, and exercise. Muscle-specific knockdown of several GEFs and GAPs, as well as the Rho GTPase CDC42, resulted in reduced motor function measured by climbing ability in vivo in Drosophila (P < 0.0001; one-way ANOVA; n = 3-17). We hypothesise that cytoskeletal dynamics drive the compromised physiological function by disrupting myonuclear positioning and thus the localisation of mRNA transcripts, which will be investigated in muscle-specific Rho GTPase knockout mice. 

Insight into the role of GEFs and GAPs in GLUT4 dynamics was gained through siRNA-mediated knockdown in mouse adipocytes, which demonstrated up to 35% reductions in insulin-stimulated GLUT4 translocation (two-way ANOVA; n = 4 per siRNA knockdown condition). Based on our results in Drosophila and mouse adipocytes, and further by cross-species similarity and siRNA knockdown efficiency, we have selected five candidates for detailed analyses in muscle cells. These analyses will include depletion of selected GEFs and GAPs using siRNA in L6-GLUT4myc tagged and C2C12 muscle cells, and characterisation of Rho GTPase activity alongside cytoskeletal and myonuclear dynamics. These findings hold the potential to enhance our understanding of the complex molecular mechanisms underlying skeletal muscle function and homeostasis, ultimately paving the way for novel therapeutic interventions for human muscle-related diseases.

All mouse and Drosophila experiments accorded with ethical standards set by current Danish, Finnish, and UK legislation.