Anti-Diabetic Drug Canagliflozin Hinders Skeletal Muscle Regeneration in Mice

Aim/hypothesis: Gliflozins are a new class of oral antidiabetic drugs that decrease urinary glucose reabsorption by inhibiting sodium-glucose cotransporter 2 in proximal tubules. However, canagliflozin (CANA) was recently reported to increase the risk of lower extremity amputation while the underlying mechanism remains largely unexplored.Methods: Diabetic and non-diabetic mice were treated with gliflozins and subjected to femoral artery ligation. In situ muscle contractile recovery, fatigue properties, as well as morphological and histological regeneration after hindlimb ischemia were evaluated. The effects of gliflozins on myoblast differentiation and cellular mitochondrial activities were assessed in vitro. Ischemic tibialis anterior (TA) 3 days post injury were analysed using single-cell RNA sequencing. The sequencing results were further confirmed by PCR and western blotting. The effects of CANA on leucyl-tRNA synthetase 2 (LARS2), and the effects of LARS2 on myoblasts differentiation were confirmed by siRNA knock-down and plasmid/AAV-based gene delivery methods both in vivo and in vitro.Results: Both in vivo and in vitro data indicate that CANA, rather than dapagliflozin or empagliflozin, impairs intrinsic myogenic regeneration and differentiation, thus hindering ischemic limb muscle contractile properties, fatigue resistance recovery and tissue regeneration. Mitochondrial structure and activity are both disrupted by CANA in myoblasts. Single-cell RNA sequencing of ischemic TA reveals a decrease in LARS2 in muscle stem cells (MuSCs) attributable to CANA. Further investigation explicates the noncanonical function of LARS2, which plays pivotal roles in regulating myoblast differentiation and muscle regeneration by affecting mitochondrial structure and activity. Enhanced expression of LARS2 directly stimulates myoblasts to form large multinucleated syncytial myotubes, restores the differentiation of CANA-treated myoblasts, and accelerates ischemic skeletal muscle regeneration in CANA-treated mice.Conclusion/interpretation: CANA delays ischemic limb function recovery and tissue regeneration. Such impairment is due to the downregulation of LARS2 in MuSCs, which attenuates intrinsic myogenic regeneration and differentiation. Further investigation revealed that LARS2, as an essential factor for mitochondrial maintenance, plays pivotal roles in myoblast differentiation regulation.