Background: Immune-mediated inflammatory myopathies (IIMs) are a diverse group of disorders characterized by chronic inflammation, muscle damage, and impaired regeneration, resulting in progressive muscle weakness. Muscle stem cells (MuSCs) are central to skeletal muscle regeneration. Previous studies have demonstrated heterogeneity among MuSCs and shown that their function is strongly shaped by the local microenvironment. However, how the MuSC microenvironment is perturbed in IIMs and how these alterations contribute to defective muscle regeneration remain poorly understood.

Methods: We utilized imaging mass cytometry (IMC) to examine the heterogeneity of MuSCs at the single-cell level and to explore cellular alterations in the MuSC niches within muscle biopsies from IIM patients. Muscle samples from patients with dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and control individuals (CO) were analyzed.

Results: A total of 69288 cells, including 57623 stromal cells, of which 14595 MuSCs were analyzed. Seventeen distinct stromal cell subpopulations were identified, including four MuSCs subpopulations. We observed subtype-specific alterations in the frequencies of MuSC subpopulations in IIMs. Specifically, CD56 ^hi ;CD90 ^hi ;PAX7 ^low MuSCs were particularly upregulated in PM and DM, while CD82 ^hi ;PAX7 ^low MuSCs were primarily upregulated in IBM biopsies. GLI1 ^hi ;PAX7 ^hi MuSCs were increased only in PM patients and CD82 ^neg ;PAX7 ^low MuSCs did not show significant changes in frequency compared with CO. Each MuSC subpopulation was associated with distinct spatial niches. Two MuSC niches (CD82 ^hi ;PAX7 ^low and CD82 ^neg ;PAX7 ^low ) were enriched for different stromal cells, while the other two (CD56 ^hi ;CD90 ^hi ;PAX7 ^low and GLI1 ^hi ;PAX7 ^hi ) for distinct subsets of immune cells. The CD82 ^hi ;PAX7 ^low MuSC niche was dominated by LECs, while the CD82 ^neg ;PAX7 ^low MuSC niche was composed of ECs and muscle fibers. The CD56 ^hi ;CD90 ^hi ;PAX7 ^low MuSC cellular neighborhood (CN) was enriched in NK cells, HLA-DR ^- M2 macrophages, and regenerating muscle fibers, while the GLI1 ^hi ;PAX7 ^hi MuSC CN was enriched in HLA-DR ⁺ M2 macrophages, HLA-DR ⁺ CD20 ⁺ B cells, HLA-DR ⁺ CD28 ^- T cells, and helper T cells. Additionally, we identified IIM-subtype-specific perturbations in the cellular interaction network of MuSC populations. The frequencies of MuSC subpopulations correlated with standard histopathological measures of muscle regeneration. The frequency of CD82 ^neg ;PAX7 ^low MuSCs was negatively associated with the degree of muscle regeneration, while the frequency of GLI1 ^hi ;PAX7 ^hi MuSCs was positively associated with the degree of perimysial or endomysial fibrosis or adipose degeneration.

Conclusions: Using IMC, we characterized the heterogeneity of MuSCs in IIMs and identified subtype-specific changes in MuSC frequencies, their local niches, and cellular interactions. These findings offer potential for targeted therapeutic modulation of MuSC subpopulations to improve muscle regeneration and address the disabling muscle weakness seen in IIMs.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Bilgesu Safak Tumerdem: None declared, Yi-Nan Li: None declared, Tim Filla: None declared, Rolf Schroeder: None declared, Anna Brunn: None declared, Alexandru Micu: None declared, Ayla Stütz: None declared, Laura-Marie Lahu: None declared, Aleix Rius Rigau: None declared, Christina Bergmann: None declared, Alexandru-Emil Matei: None declared, Jörg Distler J.H.W. Distler is CEO of 4D Science and scientific lead of FibroCure., J.H.W. Distler is CEO of 4D Science and scientific lead of FibroCure., J.H.W. Distler has consultancy relationships with Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, BMS, Callidatas, Calluna, Galapagos, GSK, Janssen, Kyverna, Novartis, Pfizer, Quell Therapeutics and UCB., J.H.W. Distler has received research funding from Anamar, ARXX, BMS, Boehringer Ingelheim, Cantargia, Celgene, CSL Behring, Exo Therapeutics, Galapagos, GSK, Incyte, Inventiva, Kiniksa, Kyverna, Lassen Therapeutics, Mestag, Sanofi-Aventis, SpicaTx, RedX, UCB and ZenasBio., Andrea-Hermina Györfi A.-H. Györfi has received lecture fees from Boehringer Ingelheim and AbbVie.