High-Resolution Transcriptomic Profiling of the Heart During Chronic Stress Reveals Cellular Drivers of Cardiac Fibrosis and Hypertrophy

Figure 1.. Isolation and analysis of the cardiac cellulome by scRNA-seq. A, Schematic outline of the experimental procedure for the isolation and analysis of adult mouse cardiomyocytes and nonmyocytes by scRNA-seq. B, t-SNE projection of cardiac cells analyzed by scRNA-seq. Cells are colored by distinct cell populations as indicated. Note, the relative abundance of cell types displayed does not represent in vivo proportions (see Figure 1A and Expanded Methods in the Data Supplement). C, Heat map of relative expression of canonical cell markers in major cardiac cell populations. D, Top 5 distinct genes for each cell population, identified using an unsupervised approach. Dot color and size indicate the relative average expression level and the proportion of cells expressing the gene, respectively, within each cell population (also see Table I in the Data Supplement). DAPI indicates 4′,6-diamidino-2-phenylindole; DC, dendritic cells; ECs, endothelial cells; FACS, fluorescence-activated cell sorting; FSC-A, forward scatter area; lymph., lymphatic; NK, natural killer; and t-SNE, t-distributed stochastic neighbor embedding;

Figure 2.. Histological and cellular changes in the heart after induction of cardiac fibrosis. A, Experimental schema for AngII-mediated induction of cardiac fibrosis. Female and male mice were infused with AngII or saline (control) continuously for 2 weeks. B, Representative micrograph of trichrome-stained cardiac sections from mice infused with saline (Left) or AngII (Right). Example images shown are from female mice. C, Change in total cardiac area and cardiac fibrosis with and without induction of fibrosis in females and males (n=4–5 animals per group). Total cardiac area and fibrosis was quantified using high-resolution images of trichrome-stained midventricular sections as shown in B (also see Figure V in the Data Supplement). The entire cardiac section was used for measurements (see Expanded Methods in the Data Supplement). P values shown on plot are derived from the Wilcoxon rank sum test for differences between the groups. Whiskers of box and whisker plots represent highest and lowest value, except when a value is beyond the range of 1.5 interquartile. D, Fast Fourier transform–accelerated interpolation–based t-distributed stochastic neighbor (FIt-SNE) plot of cardiac cell clusters…

Figure 3.. Gene expression changes in cardiac cell populations. A, Lollipop plot summarizing number of up- and downregulated genes (uncorrected P<0.01) in AngII-treated mouse heart cells relative to control cohorts (see Table III in the Data Supplement). Note: We detected a statistically significant correlation between cell population size and number of differentially expressed genes discovered (Figure X in the Data Supplement). B, Dot plot summarizing the expression of genes identified as within the top 10 genes upregulated in response to AngII administration, for each cell type. Dot color and size are proportional to average expression within each cell cluster and fold increase in AngII cells relative to control cells, respectively. Black points at the centers of some dots indicate a significant difference in gene expression in response to AngII (uncorrected P<0.01). C, Sankey plot summarizing the top 3 GO terms for upregulated genes of each cell type (corrected P<0.05). Connections indicate GO terms associated with each cell type (see Table IV in the Data Supplement). Note: (1) many cell types upregulate genes that are associated with the same GO terms, consistent with a specific and…

Figure 4.. Distinguishing features of fibrosis-associated fibroblasts. A, GO terms enriched in a set of genes derived from comparing gene expression differences between Fibroblast-Cilp and Fibroblast-Thbs4 vs all other control cells. x axis indicates the number of genes mapped to each GO term, and color indicates the adjusted P value from GO enrichment analysis. Outline of FIt-SNE projection shown on Figure 2D, Left, indicates Fibroblast-Cilp and Fibroblast-Thbs4 population (red) being compared with other cells (black; see Tables V and VI in the Data Supplement). B, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp and Fibroblast-Thbs4 vs fibroblasts from control mice. C, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp and Fibroblast-Thbs4 vs fibroblasts from AngII-treated mice. Purple FIt-SNE outline indicates AngII-fibroblasts being compared with Fibroblast-Cilp and Fibroblast-Thbs4. D, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp to Fibroblast-Thbs4. E, Dot plot showing expression of fibroblast genes classified within the GO categories shown on y axis. Circle size represents…

Figure 5.. Shifts in fibroblast cell state in response to chronic stress imposed by AngII treatment. A, Stream plot depicting patterns of RNA velocity in fibroblasts from mice administered AngII. Velocities were calculated using scVelo.16 Clustering of fibroblasts and their placement in 2-dimensional space are identical to Figure 2D. RNA velocities are projected onto this Fourier transform–accelerated interpolation–based t-distributed stochastic neighbor embedding embedding for visualization purposes. Lines with arrows depict predominating changes in velocity and their associated directionality. B, Estimation of RNA velocity for Cilp in fibroblasts from AngII-treated mice. Relative velocities are colored according to scale on the right. C. As in B, but for Postn. AngII indicates angiotensin II.

Figure 6.. Intercellular connection network of the cardiac cellulome. A, Chord plot summarizing interconnections between different cardiac cell types from hearts of control mice. Lines represent potential interconnections between cell types, with line thickness proportional to the number of ligand-receptor pairs expressed in the connected cell types and line color reflecting the cell population producing transcript coding for the ligand. See Expanded Methods section “Ligand-receptor intercellular communication network analysis” in the Data Supplement for more details. B, Relative expression of a selection of essential growth factors across major cardiac cell types. Gene expression is normalized to the expression level of the cell type with the greatest mean expression. C, Spatial distribution of MRC1+ and MRC1– macrophages relative to the epicardium in Cx3cr1gfp/+ mouse hearts. Green fluorescent protein (white) labels both MRC1+ and MRC1– macrophages. Scale bar, 100 µm. Inset shows magnified view of the epicardium (indicated by white arrows). D, Number of receptor-ligand pairs potentially mediating cell–cell communication for each cell population with and without AngII treatment. Bar plot…

Figure 7.. Sexual dimorphic remodeling and cellular responses. A, Trichrome-stained sections from female and male hearts (Left, micrographs) and quantification of fibrosis (Right, boxplots). Plots summarize fibrosis in the left ventricles (LV), intraventricular septums (IVS), and right ventricles (RV) of male and female hearts (n=4–5 animals per group). Uncorrected P values determined using Wilcoxon rank sum test for differences between the groups. Whiskers of box-and-whisker plots represent highest and lowest value, except when a value is beyond the range of 1.5 interquartile. B, Illustration of the desegregation of female and male cells based on sex-specific gene expression. Cells are visualized as points in FIt-SNE space, identically to Figure 2D. As expected, female and male cells are each present within each cell cluster. C, Cellular abundances of Fibroblast-Cilp and Fibroblast-Thbs4 populations in females and males with and without fibrosis induction. Light gray lines indicate binomial proportion confidence intervals and are calculated using the Jeffreys interval. Individual female and male mice are indicated by red and blue dots, respectively. The y axis shows cell abundance and…