ATP6AP2 dysregulation as a central hub in DMD pathogenesis: An integrated multi-omics and therapeutic study

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting and persistent chronic inflammation, yet the multi-lineage cellular drivers of its pathogenesis remain incomplete. In this study, single-cell RNA sequencing (scRNA-seq) identified Atp6ap2 as a profoundly upregulated gene across multiple skeletal muscle cell types-particularly endothelial cells, fibroblasts, and myoblasts-in both mdx and severe mdx mice. Weighted gene co-expression network analysis (WGCNA) linked Atp6ap2 expression to DMD progression, while enrichment analyses revealed that its dysregulation severely impairs vascular homeostasis and extracellular matrix integrity via the PI3K-Akt, focal adhesion, and cell cycle pathways. Utilizing Connectivity Map (CMap) analysis, we identified temozolomide (TMZ) as a top pharmacological candidate capable of reversing the ATP6AP2-associated gene signature. In vivo validation demonstrated that TMZ administration significantly enhanced motor coordination, balance, and grip strength in mdx mice, while markedly preserving dystrophic muscle architecture, reducing myofiber necrosis, and alleviating interstitial fibrosis. Mechanistically, integrated transcriptomic and metabolomic profiling revealed that TMZ induced profound metabolic and signaling shifts, modulating the Notch, MAPK, and Ras pathways, as well as autophagy and glycerophospholipid metabolism. Furthermore, scRNA-seq and cell-cell communication analyses indicated that TMZ dynamically reorganized multicellular networks, decreasing aberrant fibroblast and endothelial interactions. Crucially, immunofluorescence and Western blot validations confirmed that TMZ drastically attenuated the infiltration of F4/80-positive macrophages and suppressed their pro-inflammatory M1 polarization (indicated by reduced co-localization with iNOS and ATP6AP2), while successfully reversing the dysregulation of the ATP6AP2 axis and restoring its downstream targets MAP4K2, DGKE, and EFNA1. Collectively, our findings demonstrate that TMZ mitigates dystrophic pathology by targeting the ATP6AP2 signaling axis and dampening macrophage-mediated inflammatory responses, highlighting its potential as a novel immunopharmacological therapeutic strategy for DMD.