iPSC-Derived Myotubes from ME/CFS Patients Reveal Signature Genes, Isoforms and Pathways with Drug Repurposing Potential

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystem disorder. Its hallmark feature, post-exertional malaise (PEM), is a delayed and prolonged worsening of symptoms following even minor exertion. As PEM commonly includes muscle pain, weakness, exercise intolerance and impaired recovery, skeletal muscle dysfunction may contribute to its pathophysiology, but the underlying molecular processes remain poorly understood. We generated induced pluripotent stem cell-derived skeletal muscle myotubes from individuals with ME/CFS (n = 6) and healthy controls (n = 7) to investigate disease-associated transcriptomic alterations and identify potential therapeutic candidates. Myogenic progenitor cells from both groups differentiated into elongated, multinucleated myotubes expressing titin and myosin heavy chain, while qPCR profiling of myogenic differentiation markers further supported successful differentiation. Gene-level analysis identified three differentially expressed genes at an FDR-adjusted p < 0.05: ERVH48-1, CDX4 and SLC2A14. Isoform-switching analysis identified 16 genes with significant differential isoform usage, including CACNA1H, MIEF2, SMAD2 and CHD4. Gene set enrichment analysis identified broader pathway-level alterations involving RNA processing, protein synthesis, proteostasis, mitochondrial quality control, metabolism and muscle-related processes. Library of Integrated Network-based Cellular Signatures Phase 2 (LINCS2) analysis further prioritised 14 compounds associated with mitochondrial metabolism, glucose and lipid regulation, PPAR signalling, calcium and excitatory signalling, neuroimmune activity and cellular stress responses. Overall, ME/CFS iPSC-derived myotubes showed modest gene-level differences alongside broader isoform- and pathway-level alterations, supporting their use for investigating skeletal muscle biology and prioritising candidates for functional validation.