Endothelial plasticity in atrial fibrosis by integrating single-cell sequencing and genetic lineage tracing

Background and aims: Atrial fibrosis represents a critical determinant in atrial fibrillation (AF) pathogenesis. Although endothelial dysfunction is a hallmark feature of AF, the precise mechanisms by which endothelial cells (ECs) contribute to atrial fibrosis remain incompletely understood. Methods: This study employed single-cell RNA sequencing (scRNA-seq) to analyze intercellular communication networks in atrial tissues from both sinus rhythm (SR) and AF patients. Cdh5-CreERT2;RFP mice were generated to track endothelial plasticity following transverse aortic constriction (TAC). Cell-cell interactions were investigated using isolated human primary atrial ECs and fibroblasts (FBs) in vitro. To elucidate the regulatory role of endothelial-derived TGF-β1 on FB function, endothelial-specific Tgf-β1 knockout (Tgf-β1ECKO) mice were generated. Results: scRNA-seq analysis identified ECs as predominant signal-sending cells with extensive FB connectivity in both SR and AF atrial tissues. During fibrosis progression, ECs displayed significant mesenchymal activation at the transcriptional level. However, immunofluorescence and high-content screening revealed minimal complete endothelial-to-FB transition. Cell-cell communication analysis and in vitro studies identified TGF-β1 as the key mediator through which mesenchymal-activated ECs (EndoMA) promoted FB proliferation and collagen production. Notably, endothelial-specific Tgf-β1 deletion attenuated TAC-induced atrial fibrosis and reduced AF susceptibility. Conclusions: Our findings demonstrate that EndoMA-derived TGF-β1 critically regulates FB function and drives atrial fibrosis progression. Targeting endothelial-specific pathways represents a promising therapeutic strategy for attenuating atrial fibrosis in AF pathogenesis. Human: Single cell sequencing from heart donors with sinus rhythm and patients with atrial fibrillation; Mice: Single cell sequencing from C57BL/6 J Cdh5-CreERT2/RFP mice performed transverse aortic constriction or sham surgery. ***Raw data for human samples will be submitted to the Genome Sequence Archive for Human due to patient privacy concerns***