Single‐Nucleus RNA Sequencing Unravels Early Mechanisms of Human Becker Muscular Dystrophy

OBJECTIVE: The transcriptional heterogeneity at a single-nucleus level in human Becker muscular dystrophy (BMD) dystrophic muscle has not been explored. Here, we aimed to understand the transcriptional heterogeneity associated with myonuclei, as well as other mononucleated cell types that underly BMD pathogenesis by performing single-nucleus RNA sequencing. METHODS: We profiled single-nucleus transcriptional profiles of skeletal muscle samples from 7 BMD patients and 3 normal controls. RESULTS: A total of 17,216 nuclei (12,879 from BMD patients and 4,337 from controls) were classified into 13 known cell types, including 9 myogenic lineages and 4 non-myogenic lineages, and 1 unclassified nuclear type according to their cell identities. Among them, type IIx myonuclei were the first to degenerate in response to dystrophin reduction. Differential expression analysis revealed that the fibro-adipogenic progenitors (FAPs) population had the largest transcriptional changes among all cell types. Sub-clustering analysis identified a significantly compositional increase in the activated FAPs (aFAPs) subpopulation in BMD muscles. Pseudotime analysis, regulon inference, and deconvolution analysis of bulk RNA-sequencing data derived from 29 BMD patients revealed that the aFAPs subpopulation, a distinctive and previously unrecognized mononuclear subtype, was profibrogenic and expanded in BMD patients. Muscle quantitative real-time polymerase chain reaction and immunofluorescence analysis confirmed that the mRNA and protein levels of the aFAPs markers including LUM, DCN, and COL1A1 in BMD patients were significantly higher than those in controls, respectively. INTERPRETATION: Our results provide insights into the transcriptional diversity of human BMD muscle at a single-nucleus resolution and new potential targets for anti-fibrosis therapies in BMD. ANN NEUROL 2024;96:1070-1085.