Physiological medium and 3-hydroxybutyrate modulate autophagy-linked organelle remodeling in human external urethral sphincter myoblasts

Abstract Autophagy-linked organelle remodeling is essential for skeletal muscle differentiation and is closely linked to the metabolic environment. The ketone body 3-hydroxybutyrate (3HB) serves as an alternative energy substrate and signaling molecule that modulates organelle function and myogenic programs. This study investigated how extracellular metabolic conditions and 3HB regulate autophagy-linked organelle remodeling in human external urethral sphincter (hEUS) myoblasts. Immortalized hEUS myoblasts (US2-KD) were differentiated under four conditions combining high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM) or low-glucose physiologically formulated minimum essential medium (LG-MEM) with or without 3HB. Metabolomic profiling revealed that the medium composition predominantly shaped energy and amino acid pathways, whereas 3HB induced subtler, context-dependent shifts in metabolites related to autophagy and mitochondrial function. At the cellular level, LG-MEM accelerated myogenic differentiation compared to HG-DMEM, with earlier induction of MYOG and MYH7 and faster maturation of myotubes. LG-MEM also altered LC3B expression patterns, while transmission electron microscopy showed fewer excess autophagosomes and autolysosomes along with more prominent myofibril-like ultrastructure, consistent with more efficient autophagic activity and organelle remodeling. These findings suggest that a physiological metabolic environment facilitates autophagy-linked organelle remodeling in hEUS myoblasts and that 3HB acts as a fine-tuning signal, potentially supporting metabolism-oriented approaches for stress urinary incontinence.