Single‐cell RNA sequencing reveals a mechanism underlying the susceptibility of the left atrial appendage to intracardiac thrombogenesis during atrial fibrillation

FIGURE 1. Gene expression and cell atlas of atrial appendage samples from patients with atrial fibrillation. (A) Schematic representation of the single‐cell study process. Samples obtained from the left atrial appendage (LAA) (n = 3) and right atrial appendage (RAA) (n = 3) of three patients were processed using Chromium 10× chemistry and analysed using single‐cell RNA‐sequencing (scRNA‐seq). (B and C) Visualization of t‐distributed stochastic neighbour embedding (t‐SNE) of 32 326 cells and 11 major cardiac cell types. Each dot represents a single cell and is coloured according to the cell type designation (B) and sample source (C). The numbers correspond to cell cluster labels. (D) Violin plots of the expression of specific marker genes in each cell type. (E) Distribution of the identified cells according to cell type (left) and their proportions in the LAA and RAA (right). The colour code is shown in (B). (F) Distribution of each cell type between the LAA and RAA. The dashed vertical line indicates the expected proportions (the total number of LAA cells divided by the total number of cells from all specimens). (G) Left: Heat map showing the 20 most up‐regulated genes in each cell type;…

FIGURE 2. Subpopulations of endothelial cells and characteristics of the atrial appendages of patients with atrial fibrillation (AF). (A) t‐Distributed stochastic neighbour embedding (t‐SNE) plots showing eight endothelial cell (EC) subclusters in the left atrial appendage (LAA) and right atrial appendage (RAA). Each dot represents an individual cell. (B) Proportions of the eight EC subclusters in the LAA and RAA. The colour code is shown in (A). (C) Dot plot shows the percentage of cells that express the indicated marker genes. The size of the dot corresponds to the proportion of cells detected and the intensity of the colour, to the expression (mean log10 p‐value). (D) Volcano plot illustrating differential gene expression in endocardial endothelial cells (EECs) from the LAA and RAA. Genes down‐regulated and up‐regulated in the EECs of the LAA are coloured pink and blue, respectively. (E) Violin plots showing the expression of genes up‐regulated in the RAA (TFPI, TFPI2, A2M and THBD) and LAA (C3, NNMT, ADAMTS1 and SLCO2A1). Boxes indicate the lower and upper quartiles (from left to right), the line within each box indicates the median value, and the whiskers show 1.5× interquartile range…

FIGURE 3. Fibroblast (FB) distribution and transformation in the atrial appendages of patients with atrial fibrillation (AF). (A) t‐Distributed stochastic neighbour embedding (t‐SNE) plots showing FBs in the left atrial appendage (LAA) and right atrial appendage (RAA). Each dot represents an individual cell. (B) Percentage of FB among identified cells (left) and their distribution between the LAA and RAA (right). (C) Cardiac fibrosis in the LAA and RAA analysed using Masson trichrome staining (n = 3 per group); images to the left show boxed areas at higher magnification. Scale bars: 2 mm (left panels) and 100 μm (middle and right panels). The graph shows the percentages of fibrotic areas in each appendage. (D) Relative expression of differentially expressed genes (DEGs) up‐regulated in FBs of the LAA and RAA. Dot size and colour intensity correspond to the expression level. (E) t‐SNE plots are coloured according to the expression of marker genes for activated FBs in cells from the LAA and RAA. (F–H) Pseudotemporal order of endocardial endothelial cells (EECs), unknown cells and FBs presented according to cell clusters (F), pseudotime (G) and cell state (H). (I) Split view of the trajectory…

FIGURE 4. Classification and characteristics in the atrial appendages of patients with atrial fibrillation (AF). (A) t‐Distributed stochastic neighbour embedding (t‐SNE) plot of the indicated immune cell subclusters superimposed on the global t‐SNE plot. Each dot represents an individual cell. (B) Dot plot showing the percentage of cells expressing the indicated subcluster marker genes. The size of the dot corresponds to the proportion of cells detected and the intensity of the colour, to the expression level (mean log10 p‐value). (C) Distribution of the four types of immune cells between the left atrial appendage (LAA) and right atrial appendage (RAA). (D) Violin plot of the expression of LYVE1, MRC1 and CCR2 in macrophages from the LAA and RAA. (E) Heat map of differentially expressed genes (DEGs) in macrophages of the LAA and RAA. (F) Distribution of seven macrophage subclusters between the LAA and RAA. (G) Heat map of DEGs in seven macrophage subclusters. (H) Violin plots showing the expression of representative marker genes in each subcluster. Boxes indicate the lower and upper quartiles (from left to right), the line within each box indicates the median value, and the whiskers show…

FIGURE 5. Establishment of the model of atrial fibrillation (AF) mice and expression of tissue factor pathway inhibitor (TFPI) and tissue factor pathway inhibitor 2 (TFPI2). (A) Experimental schema of the model of AF mice. Male CMV‐Cre mice and Tbx5 +/− mice (Tbx5fl/+ ; CMV‐Cre) were used as sinus rhythm (SR) and AF mice, respectively. (B) Representative electrocardiogram (ECG) of SR mice and AF mice (left), and regular fibrillatory (F) waves indicating atrial flutter are zoomed‐in on the right. (C) Histological images of hearts by Masson trichrome staining (scale bar = 100 μm). (D) Relative fibrotic area in sections as shown in (C). (E) Representative images stained with wheat germ agglutinin (WGA) (red) and DAPI (blue). Scale bar = 20 μm. (F) Quantification of the cross‐sectional area (CSA) of atrial cardiomyocytes shown in (D). (G) Expression of select genes related to endothelial to mesenchymal transition (EndMT) in tissues of the atrial appendages of SR and AF mice. (H) Immunostaining for the TFPI (red) and endocardial endothelial cell (EEC) marker natriuretic peptide receptor 3 (NPR3) (green) in clarified heart sections of AF mice (scale bar = 1000 μm) and zoomed‐in details (small…

FIGURE 6. Isolation and localization of endocardial endothelial cells (EECs) for the expression of tissue factor pathway inhibitor (TFPI), tissue factor pathway inhibitor 2 (TFPI2) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) in atrial fibrillation (AF) and sinus rhythm (SR) mice. (A) Diagram of digestion, sorting and subsequent verification of EECs isolated from the left atrial appendage (LAA) and right atrial appendage (RAA). (B) FACS data verifying the isolation of EECs with natriuretic peptide receptor 3 (NPR3) and cadherin 11 (CDH11). (C) Western blots (WB) data verifying increased expression of NPR3 and CDH11 in EECs compared to human umbilical vein endothelial cells (HUVECs) (up) and human pulmonary microvascular endothelial cells (HPMECs) (down). (D) Immunostaining data verifying the colocalization of CDH11 (green) and NPR3 (red) in the EECs. (E) WB data showed the expression of TFPI, TFPI2 and ADAMTS1 in the LAA and RAA of AF and SR mice. (F) Quantitative analysis verified that TFPI and TFPI2 decreased, but ADAMTS1 increased in AF of the LAA compared to that of the RAA. (G) TFPI, TFPI2 and ADAMTS1 expression did not change significantly in Tbx5…

FIGURE 7. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) cleaves both tissue factor pathway inhibitor (TFPI) and tissue factor pathway inhibitor 2 (TFPI2) through direct interaction. (A) Interaction between ADAMTS1 and TFPI in endocardial endothelial cells (EECs) of mice by Co‐IP. Mice EECs were subjected to immunoprecipitation (IP) with anti‐TFPI, anti‐ADAMTS1 or anti‐IgG antibodies, followed by western blots (WB) with anti‐TFPI or anti‐ADAMTS1 antibodies. (B) Interaction between ADAMTS1 and TFPI2 in mice EECs by Co‐IP. Mice EECs were subjected to IP with anti‐TFPI2, anti‐ADAMTS1 or anti‐IgG antibodies, followed by WB with anti‐TFPI2 or anti‐ADAMTS1 antibodies. (C) Schematic representation of the fusion proteins used to perform pull‐down and cleavage assays. (D and E) Interaction of ADAMTS1 with TFPI (D) or TFPI2 (E) in vitro by glutathione S‐transferase (GST) pull‐down assays. Blots were evaluated using anti‐His or anti‐GST antibodies, and visible His‐ADAMTS1 bands were detected in pull‐down lanes. Input: whole lysate. (F and G) Schematic visualization of the complexes ADAMTS1‐TFPI (F) and ADAMTS1‐TFPI2 (G). Three‐dimensional structure showing ADAMTS1 (grey)…

FIGURE 8. Graphical abstract. We conducted a single cell transcriptomic analysis to determine chamber‐specific characteristics of the atrial appendages. Potential mechanisms of pathological changes related to atrial fibrillation (AF), including atrial fibrosis through endothelial to mesenchymal transition (EndMT), macrophage polarization and imbalance of local anticoagulation in endocardial endothelial cells (EECs). Disturbed flow during AF‐reprogrammed EECs triggers high a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) expression and decreased tissue factor pathway inhibitor (TFPI) and tissue factor pathway inhibitor 2 (TFPI2), resulting in the prothrombotic state of left atrial appendage (LAA).