UFL1 deficiency facilitates the fast-to-slow myofiber shift via the Ca2+/CaN/NFAT signaling pathway

Understanding the regulatory mechanisms that determine skeletal muscle fiber-type composition is fundamental to elucidating muscle function and enhancing exercise metabolism. Although UFMylation plays a critical role in myofiber development, the expression pattern and specific function of its core E3 ligase, Ubiquitin-fold modifier-conjugating enzyme 1 (UFL1), in this process remain largely unknown. Herein, we found that UFL1 is highly expressed in fast-twitch fibers. Ultrastructural analysis in UFL1 deficiency revealed mitochondrial clustering and lipid metabolism disorders. Further metabolic assays showed that UFL1 deficiency induced a shift from glycolysis toward oxidative phosphorylation. In vitro and in vivo experiments confirmed that UFL1 deletion significantly promoted the transition from fast- to slow-twitch fibers. Meanwhile, UFL1 knockout also attenuated Rotenone-induced fast-twitch fiber transformation. Mechanistically, activation of the Ca²⁺/calcineurin (CaN)/Nuclear Factor of Activated T-Cells (NFAT) pathway significantly promoted the fast-to‐slow shift in fully differentiated myotubes, which was restored by FK506, VIVIT or UFL1 overexpression. In conclusion, UFL1 deficiency promoted the fast‐to‐slow myofiber type transition through Ca²⁺/CaN/NFAT signaling pathway. These findings provide novel insights into metabolic remodeling in skeletal muscle.