Physiological Reference Mapping Reveals Context-Dependent Dysregulation of Regenerative Programs in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) involves chronic degeneration and regeneration, causing dystrophic muscle stem cells (MuSCs) to differ from uninjured wild-type tissue. Conventional disease-versus-resting-control comparisons may therefore conflate dysregulation with regeneration. We developed a physiological reference-mapping framework integrating normal regeneration trajectories with replicated dystrophic datasets to estimate disease-associated deviations relative to regenerative state and spatial context. We applied it across single-cell/single-nucleus, bulk, and spatial RNA-sequencing datasets. In a replicated genotype-by-state MuSC dataset comprising wild-type (WT) and dystrophin-deficient mdx mouse samples across four lineage states, with three biological replicates per group, activated mdx MuSCs showed excessive extracellular matrix induction (standardized mdx-WT difference = 1.431; FDR = 3.69 × 10−9) and an attenuated MYC-associated response (−0.731; FDR = 2.57 × 10−4) among five prespecified programs. Spatial decomposition indicated substantial within-niche remodeling beyond altered niche composition. Physiological calibration identified extracellular matrix induction as a normal transient program that becomes excessive in dystrophy and suggested that reduced oxidative phosphorylation transcription relative to resting muscle may represent insufficient suppression relative to normal regeneration. An independent human dystrophinopathy spatial cohort supported increased whole-biopsy extracellular matrix transcription. Physiological reference mapping therefore helps distinguish regenerative adaptation from context-dependent disease dysregulation in DMD.