Background: Primary heart involvement (pHI) is one of the leading causes of death in systemic sclerosis (SSc), however, the cellular and molecular pathomechanisms of SSc-pHI are largely unknown [1, 2]. Compared with other forms of autoimmune or idiopathic myocarditis associated with fibrotic remodeling, SSc-myocarditis presents a higher degree of myocardial fibrosis, which is associated with increased mortality [3]. Imaging mass cytometry (IMC) is a spatial proteomic technique that relies on antibodies labeled with rare metal isotopes.

Objectives: We aimed to characterize the fibroblast subpopulations, their microenvironment and cellular networks in SSc-pHI and compare it to other, non-SSc forms of virus-negative, autoimmune-related myocarditis (aVNM) and isolated VNM (iVNM) using IMC.

Methods: Endomyocardial biopsies (EMB) were obtained from the right ventricle of 10 SSc-pHI-, 9 iVNM, and 7 aVNM patients. An area of 1.5 mm ² of each EMB was analyzed. We designed and validated a panel of 40 metal-labeled antibodies for the identification of fibroblast subpopulations and other types of stromal cells, as well as immune cell subsets. Data analysis was performed as described previously [4–7].

Results: We analyzed 13318 fibroblasts and 6444 immune cells. We identified seven fibroblast subpopulations: PI16 ^- /POSTN ^high -, PI16 ^high /POSTN ^high -, FAP ^high /POSTN ^high -, Tcf21 ⁺ -, CD90 ⁺ /ADAM12 ⁺ /FSP1 ⁺ -, SOX9 ⁺ fibroblasts, and myofibroblasts. We observed a significant upregulation of SOX9 ⁺ - and PI16 ^high /POSTN ^high fibroblasts in SSc-pHI compared with the other forms of myocarditis. PI16 ^- /POSTN ^high fibroblasts were associated with aVNM. FAP ^high /POSTN ^high fibroblasts were downregulated in SSc-pHI compared with aVNM and iVNM. CD163 ^high /HLA-DR ⁺ macrophages and HLA-DR ^high monocytes were the predominant immune cell subsets in SSc-pHI and were significantly upregulated in SSc-pHI compared with aVNM and iVNM. Cytotoxic T cells and HLA-DR ^- macrophages were lower in SSc than in aVNM. No SSc-specific changes in the vascular cell subpopulations were observed. The microenvironment of PI16 ^- /POSTN ^high fibroblasts was enriched in helper T cells and FAP ^high /POSTN ^high fibroblasts, while the PI16 ^high /POSTN ^high fibroblast niche was enriched in Tcf21 ⁺ fibroblasts, CD163 ^high /HLA-DR ⁺ macrophages, HLA-DR ^high monocytes, and B cells. The microenvironment of SOX9 ⁺ fibroblasts was enriched in several subsets of immune cells and FAP ^high /POSTN ^high fibroblasts. The SOX9 ⁺ fibroblast- and CD34 ⁺ stromal cell niches were upregulated in SSc-pHI compared to aVNM and iVNM. We detected two myofibroblast niches, which were enriched in CD163 ⁺ /HLA-DR ^- macrophages, or CD90 ⁺ /ADAM12 ⁺ /FSP1 ⁺ fibroblasts and CD68 ⁺ mast cells, respectively. These cellular changes were associated with profound differences in the cellular interaction network of the fibroblast subsets in SSc-pHI compared to aVNM and iVNM.

Conclusion: We demonstrate the presence of multiple phenotypically-distinct fibroblast populations in SSc-pHI, as well as profound changes in their frequencies, their microenvironment and cellular interactions compared with other forms of non-viral myocarditis. Our findings provide new insights into the pathophysiology of SSc-pHI and may provide a rationale for specific targeting of individual fibroblast populations in cardiac fibrosis.

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[2] Györfi AH, Filla T, Polzin A, Tascila K, Buch M, Tröbs M, et al. Evaluation of systemic sclerosis primary heart involvement and chronic heart failure in the European Scleroderma Trials and Research (EUSTAR) cohort. JAHA. 2025.

[3] De Luca G, Campochiaro C, De Santis M, Sartorelli S, Peretto G, Sala S, et al. Systemic sclerosis myocarditis has unique clinical, histological and prognostic features: a comparative histological analysis. Rheumatology (Oxford). 2020;59(9):2523-33.

[4] Rius Rigau A, Li YN, Matei AE, Gyorfi AH, Bruch PM, Koziel S, et al. Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics. Circ Res. 2024;134(7):875-91.

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[6] Gronberg C, Rattik S, Tran-Manh C, Zhou X, Rius Rigau A, Li YN, et al. Combined inhibition of IL-1, IL-33 and IL-36 signalling by targeting IL1RAP ameliorates skin and lung fibrosis in preclinical models of systemic sclerosis. Ann Rheum Dis. 2024;83(9):1156-68.

[7] Gyorfi AH, Matei AE, Fuchs M, Liang C, Rigau AR, Hong X, et al. Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation. J Exp Med. 2021;218(9).

Acknowledgements: NIL.

Disclosure of Interests: Ayla Stütz: None declared , Giacomo De Luca: None declared , Alexandru-Emil Matei: None declared , Yi-Nan Li: None declared , Veronica Batani: None declared , Tim Filla: None declared , Aleix Rius Rigau: None declared , Bilgesu Safak Tuemerdem: None declared , Dr. Cosimo Bruni Boehringer Ingelheim, Novartis Foundation for medical-biological research, EMDO Foundation, Iten-Kohaut Foundation. Educational grants from Wellcome Trust,Congress support from Boehringer-Ingelheim, Hartmann-Muller Foundation, Markus Eckstein: None declared , Georg Schett: None declared , Cristina Basso: None declared , Jörg Distler see below, CEO of 4D Science and scientific lead of FibroCure, Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Callidatas, Celgene, Galapagos, GSK, Inventiva, Janssen, Kyverna, Novartis, Pfizer, Quell Therapeutics and UCB, Anamar, ARXX, BMS, Bayer Pharma, Boehringer Ingelheim, Cantargia, Celgene, CSL Behring, Exo Therapeutics, Galapagos, GSK, Incyte, Inventiva, Kiniksa, Kyverna, Lassen Therapeutics, Mestag, Sanofi-Aventis, RedX, UCB and ZenasBio, Marco Matucci-Cerinic: None declared , Andrea-Hermina Györfi: None declared .

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.