Single nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue...

Single cell/nuclei technologies have revolutionized our understanding of the remodeling of complex multi-cellular tissue that accompanies injury, regeneration, and disease. Duchenne muscular dystrophy (DMD) is a fatal genetic disease of childhood characterized by progressive skeletal muscle weakness resulting from mutation of DMD and loss of functional dystrophin. Here we report, at single nuclei/cell resolution, on intramuscular cell and gene expression dynamics within a broad cohort of needle muscle biopsies obtained from DMD individuals with varying degrees of severity, including a subset with low levels of dystrophin. We report a strong negative correlation between expression of dystrophin and disease severity and report substantial differences in cellularity and cell type-specific gene expression in DMD severity groups versus healthy muscle. Expression signatures indicate that DMD myofibers become immunologically alert, upregulating innate and adaptive immune sensors, including TLR4, IL15, TNF family receptors and MHC and costimulators. In this cohort, dystrophic muscle was remodeled with 50% fewer myofibers with expansion and diversification of fibroblasts and myeloid cells. We identify a DMD-specific TNFα-responsive Thy-1⁺/C3⁺ fibroblast subpopulation which we propose are inflammatory tissue priming fibroblasts and three DMD-specific myeloid populations which express signatures of innate immune memory. There is an 8-fold increase in CD8⁺GZMK+/GZMB+ T cells (Tek), with characteristics of both adaptive and innate immune activity. We propose that these non-myofiber muscle resident cells interact and epigenetically instill long-term tissue memory to perpetuate and amplify a hyper-inflammatory state in DMD muscle, contributing to impaired regeneration, myofiber death and fibrosis. This compendium of single/cell nuclei serves as a valuable reference and has immediate impact for biomarker discovery, clinical trial design, identification of barriers to dystrophin replacement therapies and novel druggable cell mechanisms operating in DMD.