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Introduction Systemic sclerosis (SSc)-associated primary heart involvement (SSc-pHI) is a major cause of mortality in SSc patients, but its cellular and molecular mechanisms remain unclear. We investigated fibroblast (Fb) subpopulations, their niches, and cellular networks in SSc-pHI, and compared them with those in chronic (postviral myocardial fibrosis (PVMF), hypertrophic cardiomyopathy (HCM), ischemic cardiomyopathy (ICM)) and acute (acute myocardial infarction (AMI)) fibrotic remodeling using cyclic in situ hybridization.Material and Methods We employed the CosMx SMI platform to analyze FFPE myocardial biopsies from 15 SSc-pHI, 10 PVMF, 5 HCM, 5 ICM and 3 AMI patients, examining two 1.8mm 2 regions per sample. The Human Universal Cell Characterization RNA Core Panel (950 genes), supplemented with 19 Fb subset markers was used for gene expression profiling. Cell segmentation was performed on the CosMx SMI platform. The Banksy R package was used for the identification of cellular populations. Dimensionality reduction was performed by principal component analysis and Leiden clustering. The Delaunay triangulation method was used for the identification of cellular neighbors. The composition of neighboring cells was computed for each cell and K-means clustering was applied to define cellular local microenvironments.Results We identified 11672 Fb. Spatially informed clustering revealed six Fb populations: GSN/DCN+, AP1+, TIMP1+, COL1A1/POSTN+, NOTCH3+, and myofibroblasts ( figure 1A). GSN/DCN+ Fb and myofibroblasts were significantly enriched in SSc-pHI and other forms of chronic compared to acute fibrotic remodeling, while TIMP1+, COL1A1/POSTN+, and NOTCH3+ Fb were significantly upregulated in acute compared to chronic conditions. GSN/DCN+ Fb and AP1+ Fb cellular neighborhoods (CNs) were enriched and colocalized in areas of perivascular and subendocardial fibrosis in chronic conditions (figure 1B), while the NOTCH3+ Fb/COL1A1/POSTN+ Fb CN was enriched in the infarct associated fibrotic area in AMI. Furthermore, we observed significant disease-specific changes in the composition of the Fb niches (figure 1C). Functional analyses revealed distinct functions of the Fb populations while spatial interaction analyses showed profound differences in their signaling network with other fibroblasts, immune cells and vascular cells in SSc-pHI (figure 1D) compared to both other forms of chronic fibrotic remodeling and AMI.Conclusions We show marked alterations in Fb composition, interactions, and niche signaling in SSc-pHI compared with other myocardial diseases featuring acute or chronic fibrotic remodeling. We further identify functionally distinct Fb populations, including disease-specific subsets with unique topologies. These findings shed new light on the pathophysiology of fibrotic myocardial remodeling and highlight potential therapeutic targets.Abstract OC.46 Figure 1