06 | Pharmacological AMPK activation promotes higher levels of Pax7 expression in human muscle stem cells

D. Michel¹, A. Bouche^1|2, R. Mounier³, D. Hannouche^1|2, Thomas Laumonier^1|2 | ¹Cell Therapy and Musculoskeletal Disorders Laboratory, Department of Orthopedic Surgery, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland; ²Department of Cell Physiology and Metabolism, Faculty of Medicine, Geneva, Switzerland; ³Institut Neuromyogène, CNRS 5261 – INSERM U1315 - UCBL1, Lyon, France.

Abstract

Muscle stem cells (MuSCs) have gained attention as a promising therapeutic intervention for degenerative muscle diseases. Previously, we demonstrated that human muscle reserve cells (MuRCs) closely resemble quiescent MuSCs and exhibit enhanced regenerative capacity in vivo. Transcriptomic analyses revealed heterogeneity in Pax7 expression among human MuRCs, identifying a Pax7-High subpopulation characterised by a deeper quiescent state and reduced myogenic priming. This subpopulation exhibits a metabolic preference for fatty acid uptake and mitochondrial β-oxidation, which are pathways closely linked to AMPK signaling. In this study, we examined the impact of compound 991, an allosteric AMPK activator, on the differentiation of human myoblasts and the subsequent fate of human MuRCs. Treatment with compound 991 did not alter the differentiation capacity of human myoblasts, as indicated by comparable percentages of MEF2C-positive cells and of Pax7-positive MuRCs. Western blot analysis revealed that treatment with compound 991 led to a 70% increase in the pACC/ACC ratio in human MuRCs, indicating enhanced AMPK activation. Flow cytometry analyses showed that compound 991 increased Pax7 expression in human MuRCs by 1.4-fold. Interestingly, Pax7 upregulation in human MuRCs was reversed by etomoxir, an inhibitor of mitochondrial fatty acid uptake. This suggests a functional link between fatty acid metabolism and Pax7 expression in human MuRCs. Our findings suggest that AMPK activation by compound 991 does not impair the differentiation capacity of human myoblasts but rather promotes the in vitro generation of human MuRCs with increased Pax7 expression. Ongoing experiments are assessing the kinetics of human MuRC reactivation, as measured by EdU incorporation, following 6, 12 or 24 hours in growth medium.