Integrated Single-Nucleus and Spatial Transcriptomic Analysis of Cellular and Spatial Heterogeneity of Ferroptosis-Related Transcriptional Programs in Hypertrophic Cardiomyopathy

Objective: Pathological remodeling in hypertrophic cardiomyopathy (HCM) involves multiple cardiac cell populations and their local microenvironments.It remains uncertain which cells exhibit prominent ferroptosis-related transcriptional programs, how these programs vary with cardiomyocyte state transitions, and where they are distributed within the myocardium.This study integrated single-nucleus RNA sequencing and spatial transcriptomics to characterize these transcriptional features across cell types, cell states, and tissue space.Methods: Publicly available single-nucleus RNA-sequencing datasets from human HCM and non-failing hearts underwent quality control, integration, and cell-type annotation.Cell composition and pseudobulk differential expression were evaluated at the donor level.Separate module scores were calculated for ferroptosis drivers, defense genes, and markers, and UCell was used for independent validation.Cardiomyocytes were reclustered and ordered along a pseudotemporal trajectory to characterize ferroptosis-related transcriptional features across cell states.Spatial transcriptomic data were then used to localize these signals and define associated cellular niches.CellChat and NicheNet analyses were used to prioritize candidate ligand-receptor interactions potentially involved in local regulation.Results: HCM myocardium showed changes in both cellular composition and transcriptional state, involving cardiomyocytes, fibroblasts, endothelial cells, pericytes, macrophages, and other vascular-associated populations.Ferroptosis-related transcriptional scores were heterogeneous across cell types, with relatively strong signals in macrophages, epicardial cells, adipocytes, mast cells, and selected fibroblast populations.Cardiomyocytes did not form a discrete ferroptosis-high cluster; instead, score and gene-expression changes accompanied transitions between cardiomyocyte states.Spatial analysis localized these transcriptional signals to selected myocardial regions associated with mixedcell niches rather than a uniform distribution throughout the tissue.FN1-CD44, LTBP1-ITGB5, and NAMPT-INSR emerged as candidate communication axes, although spatial support varied among tissue sections.Conclusion: Ferroptosis-related transcriptional features in HCM show marked celltype, cell-state, and spatial heterogeneity and are associated with multicellular remodeling and the local tissue environment.These results provide computational evidence of ferroptosis-related transcriptional programs, but they do not establish functional ferroptotic cell death or causal signaling relationships.