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

BACKGROUND

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.

METHODS

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 DESeq 2 (design = ~Patient + Treatment).

Differentially expressed genes were defined as adjusted p value (FDR)  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.

RESULTS

UA and UB significantly modulated 1918 and 339 genes, respectively (FDR  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 (r 2 = 0.9389) and ICAM5 (r 2 = 0.8804), and opposite regulation of IL17B (UA r 2 = 0.8309; UB 10 μM, p = 0.0498) and PTGS1. Both UA and UB reduced TGFBI protein levels dose-dependently (UA r 2 = 0.8582; UB r 2 = 0.7415).

CONCLUSIONS

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.