Introduction: Skeletal muscle possesses remarkable regenerative capacities, mainly driven by the resident Muscle Stem Cells (MuSCs), quiescent within resting muscle tissue. Upon injury, MuSCs become activated, proliferate, differentiate and eventually part themselves between self-renewal and fusion, to allow muscle repair. This regenerative process is highly regulated by different molecular pathways, including Transforming Growth Factor β (TGFβ), a cytokine known to inhibit muscle cell fusion (Girardi et al., 2021; Melendez et al., 2021), and to control the extracellular matrix (ECM) remodelling. In multiple muscle pathologies, TGFβ dysregulation leads to aberrant ECM modeling and ultimately fibrosis.

Objective: Despite its known fibrotic effects, the role of TGFβ in the early regenerative steps remains poorly understood. We aim at deciphering TGFβ functions on quiescent and proliferating MuSCs, particularly.

Methods: To address these questions, we performed a transcriptome analysis of proliferating MuSCs treated with either TGFβ1 or the TGFβ receptor inhibitor SB431542. In parallel, we used a mouse muscle injury model and locally injected TGFβ1 to study its impact in vivo. Mechanistic studies are made on primary myoblasts in vitro.

Results: Differential gene expression analysis revealed that TGFβ1 dysregulates genes involved in ECM organization and in the Notch signalling pathway, but not cell cycle genes. In vitro experiments confirmed no effect of TGFβ1 on myoblast proliferation. These findings suggest that TGFβ modulates the cellular environment and signaling landscape without impairing the proliferative capacity of MuSCs.

Interestingly, injection of TGFβ1 in the muscle in vivo increases the number of MuSC, although further phenotypic characterization is needed to determine their state.

Conclusion: We are now further investigating these findings with two complementary strategies: (1) an ex vivo analysis of single isolated myofibers to investigate MuSC return to quiescence and distribution along the fiber; and (2) development of an in vitro 3D culture model that mimics muscle architecture, allowing for the study of MuSC behavior in a controlled, animal-free system.

Overall, our work aims to elucidate TGFβ-driven mechanisms that control MuSCs’ function along the regeneration process.