Spatial transcriptomics reveals early and progressive molecular changes in late-onset Pompe disease muscle

Abstract Late-onset Pompe disease (LOPD) is caused by acid alpha-glucosidase deficiency, leading to lysosomal glycogen accumulation and progressive muscle pathology. Muscle biopsies show marked fibre heterogeneity, including non-vacuolated and vacuolated fibres. This study aimed to define transcriptomic alterations across these fibre states using spatial transcriptomics. Spatial transcriptomics was performed on muscle sections using the GeoMx Digital Spatial Profiler with laminin and LC3B staining. A total of 76 areas of interest were analysed, including 9 control, 35 non-vacuolated Pompe (Pompe-NV), and 32 vacuolated Pompe (Pompe-V) regions. Genes expressed in at least 5% of regions in at least one condition were retained for downstream analysis. Differential expression, pathway enrichment, and patient-level analyses were performed. Pompe-NV regions already showed marked transcriptional alterations compared with controls, with enrichment of pathways related to mitochondrial function, redox homeostasis, stress-associated pro-apoptotic signalling, MAPK signalling, and structural remodelling. Pompe-V regions displayed a more advanced transcriptional state, with stronger cytokine-responsive, tissue injury/remodelling, and neuromuscular-associated programmes. Pathway-level analyses indicated that mitochondrial dysfunction, oxidative stress responses, pro-apoptotic stress signalling, and p38-associated stress-response signalling are early features of Pompe muscle. With increasing pathological severity, extracellular matrix, fibronectin matrix, vasculature-related, muscle structural remodelling, cytokine production, and tissue injury inflammatory remodelling programmes became more prominent. Regeneration-associated changes were detected across Pompe muscle but were most evident in vacuolated fibres, which showed increased central nucleation and NCAM1/LC3B co-localisation. Patient-level PCA further identified a severity-associated transcriptomic continuum. LOPD muscle follows a progressive molecular remodelling trajectory across disease states. Early disease is marked by stress-associated transcriptional remodelling, whereas more advanced pathology is associated with increasingly prominent extracellular, inflammatory, regenerative, and structural remodelling programmes.