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Introduction Primary heart involvement (pHI) is one of the leading causes of death in systemic sclerosis (SSc). However, its cellular and molecular pathomechanisms are largely unknown. We employed imaging mass cytometry (IMC), a spatial proteomic technique with cellular resolution, to characterize the fibroblast populations and their microenvironment in SSc-pHI and compare it to other forms of virus-negative, autoimmune myocarditis (aVNM) and isolated VNM (iVNM).Material and Methods Endomyocardial biopsies were obtained from the right ventricle of ten SSc-pHI, nine iVNM, and seven aVNM patients. We established a panel of 40 metal-labeled antibodies for the identification of fibroblast populations and other stromal and immune cell subsets. Data analysis was performed as previously published by our group.Results We detected a total of 17985 fibroblasts and 6444 immune cells. We identified seven distinct fibroblast populations ( figure 1A): PI16high/POSTNhigh, PI16low/POSTNhigh, FAPhigh/POSTNhigh, TCF21+, CD90+/ADAM12+/FSP1+, and SOX9+ fibroblasts, and myofibroblasts. We observed a significant upregulation of PI16high/POSTNhigh or SOX9+ fibroblasts and CD34+ stromal cells in SSc-pHI compared with the other forms of myocarditis. PI16low/POSTNhigh fibroblasts were associated with aVNM. The expression of FAPhigh/POSTNhigh fibroblasts was increased in aVNM and iVNM, compared with SSc-pHI (figure 1B). Furthermore, PI16high/POSTNhigh or SOX9+ fibroblasts were particularly enriched in highly fibrotic areas (figure 1C). CD163high/HLA-DR+/CD68+ or CD163+/HLA-DR-/CD68- macrophages and HLA-DRhigh monocytes were significantly upregulated in SSc-pHI compared with the other two forms of myocarditis. Cytotoxic and helper T cells were less frequent in SSc-pHI than in aVNM. The microenvironment of PI16high/POSTNhigh fibroblast niche was enriched in TCF21+ fibroblasts, CD163+/HLA-DR-/CD68- macrophages, HLA-DRhigh monocytes, and CD20+ B cells, while the cellular neighborhood of PI16low/POSTNhigh fibroblasts was enriched in helper T cells and FAPhigh/POSTNhigh fibroblasts. The microenvironment of SOX9+ fibroblasts was enriched in several subsets of immune cells and FAPhigh/POSTNhigh fibroblasts. The CD34+ stromal cell niche was mainly composed of CD68+ mast cells, CD163high/HLA-DR+/CD68+ macrophages, TCF21+ or CD90+/ADAM12+/FSP1+ fibroblasts (figure 1D). The SOX9+ fibroblast and CD34+ stromal cell niches were upregulated in SSc-pHI compared to aVNM and iVNM. 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.Conclusions We identify seven distinct fibroblast populations in SSc-pHI, as well as profound changes in their frequencies, their microenvironment and cellular interactions compared with other forms of myocarditis. Our findings provide new insights into the pathophysiology of SSc-pHI and may offer a rationale for specific targeting of individual fibroblast populations in cardiac fibrosis.Abstract OC.17 Figure 1