Normal cardiac lymphatics and their mimics

Significant lymphatic structural remodeling and dysfunction have been observed in preclinical models of cardiovascular disease. However, a detailed understanding of the normal structure and distribution of lymphatic vessels (LyVs) in the heart is still lacking. The goal of this study is to define the pattern of LyVs at various cardiac anatomical sites using Prox1-tdTomato lymphatic reporter mice. By light sheet microscopy, we first confirmed the presence of an extensive network of LyVs on the epicardial surface of the ventricles, while minimal signal was detected on the atria. We then evaluated LyV distribution within the heart using cryo- and vibratome sections. To ensure accurate identification of Prox1-tdTomato⁺ LyVs, we performed immunostaining of common lymphatic markers (LYVE1, podoplanin, and VEGFR3). In the ventricles, LyVs were enriched on the epicardium, subepicardial region, and endocardium of the right ventricular septum. We also detected LyVs on the subepicardial surface of the left atrium, within the mitral valve and interatrial septum and near the valves and atrioventricular node (AVN). In addition to LyVs, LYVE1 and PROX1 were expressed by other cell types. LYVE1 was expressed by tissue resident macrophages and a subset of endocardial cells lining the trabeculated regions of the atria and ventricles, and PROX1 was mainly expressed by valvular endothelial cells, endocardial cells lining the interatrial septum and a subset of cells within the AVN. Finally, single-cell RNA sequencing (scRNA-Seq) analysis revealed six subtypes of cardiac lymphatic endothelial cells. Our study serves as a comprehensive resource to facilitate the proper identification of LyVs in the mouse heart.<b>NEW & NOTEWORTHY</b> This is the first study detailing normal lymphatic vessel distribution at various cardiac anatomical sites using a lymphatic reporter mouse model, multiple markers, and modern imaging modalities, providing a blueprint for future studies. We also performed integrated single-cell RNA sequencing (scRNA-Seq) analysis to define the cellular and transcriptional heterogeneity of cardiac lymphatic endothelial cells. Finally, our study underscores the nonspecific nature of lymphatic markers and emphasizes the necessity of using at least two markers to identify lymphatic vessels.