This is the first study to investigate metabolic changes during the differentiation of human external urethral sphincter (hEUS) myoblasts and evaluates the impact of tricarboxylic acid (TCA) cycle inhibition on muscle-specific gene expression and energy metabolism. Additionally, we aim to develop novel therapeutic strategies for stress urinary incontinence (SUI), a condition associated with aging or post-prostatectomy that significantly impacts quality of life.

US2-KD cells, immortalized hEUS myoblasts, were cultured in differentiation medium for 192 hours. Metabolomic profiling of culture supernatants was performed using gas chromatography-mass spectrometry (GC/MS), followed by pathway enrichment analysis. The effects of UK5099, a mitochondrial pyruvate transport inhibitor, on differentiation markers (MYH1 gene expression) and ATP production were evaluated using RT-PCR, Western blotting, and luminescence assays. Enrichment ratios and statistical significance were calculated for key metabolic pathways.

Metabolomic analysis revealed significant changes in metabolic pathways during hEUS myoblast differentiation. Pathway enrichment analysis identified the following:
•	Early stage (Pre-48h): Pyruvaldehyde degradation exhibited the highest enrichment ratio (4.5, p < 0.001), followed by cysteine metabolism (4.0, p < 0.001) and urea cycle (3.5, p < 0.001).
•	Mid-stage (48-96h): Lipid metabolism became prominent with glycerolipid metabolism showing an enrichment ratio of 4.0 (p < 0.001) and ketone body metabolism at 3.8 (p < 0.001).
•	Late stage (96-144h): Mitochondrial electron transport chain pathways were enriched, with ketone body metabolism maintaining a high ratio of 3.8 (p < 0.001).
•	Final stage (144-192h): Purine metabolism showed the highest enrichment ratio (3.4, p < 0.001), followed by cardiolipin biosynthesis (3.2, p < 0.01).
UK5099 treatment significantly suppressed TCA cycle intermediates such as citrate (p < 0.01), α-ketoglutarate (p < 0.05), and malate (p < 0.001). MYH1 gene expression was reduced by more than 50% under TCA cycle inhibition (p < 0.01). Intracellular ATP levels also decreased significantly at all time points compared to untreated cells (p < 0.001).

Pathway enrichment analysis during the differentiation of hEUS myoblasts revealed that metabolic pathways could be broadly categorized into six major groups: Energy Metabolism, Amino Acid Metabolism, Lipid Metabolism, Coenzyme and Vitamin Metabolism, Nucleic Acid Metabolism, and Others. Among these, the TCA cycle emerged as a central pathway, playing a pivotal role in supporting energy metabolism and muscle-specific gene expression.
Inhibition of the TCA cycle disrupted mitochondrial function, leading to reduced ATP production and impaired differentiation. Despite compensatory activation of glutamate and GABA-related pathways, these mechanisms were insufficient to restore normal differentiation or energy production. This highlights the indispensable role of the TCA cycle in coordinating metabolic processes essential for hEUS myoblast differentiation.

This study highlights the critical importance of the TCA cycle in hEUS myoblast differentiation and its potential as a therapeutic target for SUI treatment. Combining regenerative therapies with interventions aimed at restoring mitochondrial function may enhance therapeutic outcomes for SUI patients.