A high-resolution single-nucleus RNA sequencing reveals conserved macrophage activation trajectories across muscle regeneration and dystrophy.

Macrophages are dominant leukocytes in skeletal muscle, yet their functional heterogeneity, particularly in muscular dystrophies, remains poorly defined. To reconcile this, we generated a high-resolution single-nucleus RNA sequencing (snRNA-seq) atlas of skeletal muscle. By bypassing dissociation-induced stress, we achieved superior resolution over standard single-cell clustering, capturing multinucleated myocytes and resolving cell-cell interactomes. In regenerating muscle, we crisply delineated macrophage trajectories, identifying three parallel and co-existing activation states, along with a G2/M proliferative population. Integrating these trajectories with parabiosis-based fate mapping, we provide the first evidence that recruited monocytes undergo synchronous differentiation without local proliferation, exhibiting pyroptosis before population contraction. While this recruited inflammatory population is prominent during muscular dystrophy, it surprisingly retains transcriptional programs conserved with regeneration. In parallel, tissue-resident macrophages adapt to the local environment without initiating de novo pathogenic programs. These findings suggest that at the onset of muscular dystrophy, macrophages act primarily as adaptive bystanders rather than destroyers, providing a framework for immune regulation in muscle degeneration.