Exploring the Impact of Urolithins A and B on Muscle Health: A Transcriptomic Analysis in Human Myotubes

Urolithin A (UA) and urolithin B (UB) are gut microbiota-derived metabolites of ellagitannins reported to influence mitochondrial function, inflammation and muscle metabolism. Their comparative transcriptomic effects in human skeletal muscle cells remain undefined. We characterized UA- and UB-induced molecular responses in primary human myotubes.

Primary CD56⁺ satellite cells were isolated from vastus lateralis muscle of 9 donors (6 men, 3 women; age 55-96 years; mean 74.1 ± 13.8 years) and differentiated into myotubes. Cells were treated 24 h with UA or UB (5 μM). RNA sequencing generated ~20 million paired-end reads per sample. Differential expression analysis was performed using DESeq² (design = ~Patient + Treatment). Differentially expressed genes were defined as adjusted p value (FDR) < 0.01 and |Log2FoldChange| > 0.32. Pathway enrichment was assessed using Ingenuity Pathway Analysis. Selected targets were validated by RT-qPCR and ELISA in 4 donors from the RNA-seq cohort using UA and UB at 1, 5 and 10 μM.

UA and UB significantly modulated 1918 and 339 genes, respectively (FDR < 0.01; |log2FoldChange| > 0.32), demonstrating distinct transcriptomic reprogramming in human myotubes. Pathway analysis showed that UA predominantly affected oxidative phosphorylation, mitochondrial dysfunction, inositol phosphate metabolism and glycosylation pathways (N-linked glycosylation z score 2.11), whereas UB activated cholesterol biosynthesis (z score 2.45), mevalonate pathway (z score 2.00), adipogenesis (z score 1.41) and inhibited eicosanoid signalling (z score -2.71). At 5 μM (RNA-seq), UA increased NOTCH1 (+73%), MYMX (+70%), PANX1 (+50%) and MSTN (+64%), and decreased FGF9 (-75%), ICAM5 (-52%) and MRLN (-33%), whereas UB decreased IGFN1 (-75%), TGFBI (-60%) and STC2 (-35%) and increased TGM2 (+59%). UA increased LIF (+80%) and decreased PTGS1 (-41%) and IL17B (-49%), whereas UB decreased PTGS1 (-43%) and increased IL17B (+45%). RT-qPCR validation confirmed UA-induced increases in NOTCH1 (5 μM, p = 0.0471), MYMX (10 μM, p = 0.0363), and PANX1 (5 μM, p = 0.0012; 10 μM, p = 0.0065), dose-dependent reductions in FGF9 (r² = 0.9389) and ICAM5 (r² = 0.8804), and opposite regulation of IL17B (UA r² = 0.8309; UB 10 μM, p = 0.0498) and PTGS1. Both UA and UB reduced TGFBI protein levels dose-dependently (UA r² = 0.8582; UB r² = 0.7415).

UA and UB induce quantitatively and qualitatively distinct transcriptomic programmes in human myotubes. UA preferentially modulates mitochondrial and inflammatory pathways, whereas UB primarily affects lipid metabolism and muscle-related processes. These findings provide mechanistic insight into urolithin-mediated regulation of human muscle cell biology able Stro.