G Demenego (1), S Puccio (3, 4), GA Cassanmagnago (1, 2), P Carullo (1), G De Simone (5), FS Colombo (5), G Basso (6), CM Greco (1, 2), C Peano (2, 3) and G Condorelli (1, 2)*

1 Department of Cardiovascular Medicine, IRCCS Humanitas Research Hospital, via Manzoni 56, 20089 Rozzano (MI), Italy; 2 Department of Biomedical Sciences, Humanitas University, via Rita Levi Montalcini 4, 20072 Pieve Emanuele, Milan, Italy; 3 Institute of Genetics and Biomedical Research, UoS Milan, National Research Council, via Manzoni 56, 20089 Rozzano, Milan, Italy; 4 Laboratory of Translational Immunology, IRCCS Humanitas Research Hospital, via Manzoni 56, 20089 Rozzano, Milan, Italy; 5 Flow Cytometry Core, IRCCS Humanitas Research Hospital, via Manzoni 56, 20089, Rozzano, Milan, Italy; 6 Genomic Unit, Humanitas Research Hospital, Rozzano, Milan, Italy

* Corresponding: gianluigi.condorelli@hunimed.eu

Cardiac endothelial cells (cECs) play a central role in maintaining myocardial homeostasis and adapting to stress, yet their phenotypic heterogeneity and molecular responses during pressure overload-induced hypertrophy remain incompletely understood. In this study, we employed an integrative multiomics approach combining single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to comprehensively map the transcriptional and epigenetic landscape of cECs in a murine model of transverse aortic constriction (TAC). We identified 15 transcriptionally distinct cEC subpopulations - including capillary, endocardial, lymphatic, and proliferative endothelial clusters - with significant shifts in composition under pressure overload, notably an increase in proliferative and endocardial ECs indicative of early adaptive angiogenic responses. Chromatin accessibility profiling revealed TAC-specific enrichment of AP-1 family transcription factor motifs, linked to upregulation of proangiogenic and stress-response genes, while sham hearts exhibited motif signatures consistent with endothelial quiescence. Gene ontology analysis highlighted shared and unique pathways across cEC subtypes, with particular enrichment of mitochondrial translation and chromatin remodeling processes in capillary and endocardial clusters, suggesting a metabolic and epigenetic reprogramming during cardiac stress adaptation. To histologically validate the spatial identity of key endothelial populations, we performed RNA scope in situ hybridization targeting cluster-specific markers, which confirmed the localization patterns and distribution predicted by scRNA-seq. Together, our findings provide a detailed atlas of cEC heterogeneity in the hypertrophic heart and uncover novel molecular programs that may serve as potential targets for modulating endothelial plasticity and improving cardiac adaptation to pressure overload.