Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

Figures

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  Figure 1. Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

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  Figure 2. Cardiomyocyte gene expression after TAC The cardiomyocyte gene expression was analyzed with RNA sequencing (A) A heatmap of differentially expressed genes in cardiomyocytes in sham vs. 1 week and vs. 8 weeks of TAC is shown. Eight different clusters were observed that each had a characteristic expression pattern. Example genes are indicated on the right. (B) Gene ontology terms (GO, biological process) related to the 8 clusters are shown. (C) Visualization of cluster gene regulation over time under the indicated conditions. Data are shown as mean ±SEM. (D–G) Relative mRNA levels of the indicated genes measured by qPCR in isolated cardiomyocytes from mice treated as indicated. Data are shown as mean ±SEM. ∗p<0.05, ∗∗p<0.01 as determined by two-tailed unpaired t-test, except for (F) in the right panel, where a two-tailed Mann-Whitney test was used. (H) Enrichment of top ten transcription factor binding motifs in the promoter region of regulated genes in the indicated clusters.

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  Figure 3. Endothelial cell gene expression after TAC The endothelial gene expression was analyzed with RNA sequencing. (A) A heatmap of differentially expressed genes in endothelial cells in sham vs. 1 week and vs. 8 weeks of TAC is shown. Eight different clusters were observed that each had a characteristic expression pattern. Example genes are indicated on the right. (B) Gene ontology terms (GO, biological process) related to the 8 clusters are shown. (C) Visualization of cluster gene regulation over time under the indicated conditions. Data are shown as mean ±SEM. (D) Immunofluorescence staining for the indicated markers from mouse heart sections after treatment as indicated. The arrows point at Ki-67 positive endothelial cells. Scale bar: 50μm. (E) Enrichment of top ten transcription factor binding motifs in the promoter region of regulated genes in the indicated clusters. (F) Immunofluorescence staining for the indicated markers from mouse heart sections after treatment as indicated. The arrows point at phospho(p)-SMAD3 positive endothelial cells. Scale bar: 50μm. (G) A quantitative analysis of pSMAD3 positive endothelial cells in hearts from mice treated as indicated. Data are shown…

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  Figure 4. Interaction of endothelial cell derived ligands in response to pressure overload (A) Number of interactions between upregulated (gain) or downregulated (loss) endothelial cell (EC) derived ligands after 1 week (A) and 8 weeks (B) of TAC with receptors expressed in EC, fibroblasts (FB) and cardiomyocytes (CM). Example interactions gained or lost after 1 week (C) or 8 weeks (D) of TAC are shown. (E and F) Immunofluorescence staining for the indicated markers from mouse heart sections after treatment as indicated. Scale bar: 50μm. (G) Isolated rate neonatal cardiomyocytes plated on different matrices as shown and stained for markers as indicated. Scale bar: 50μm. (H) Quantification from cardiomyocyte cell area from cells as shown in (G). Data are shown as mean ±SEM. (I) C166 endothelial cell proliferation plated on different matrices as indicated measured as BrDU incorporation by ELISA. Data are shown as mean ±SEM. (J and K) (J) Quantification and representative images (K) of endothelial cell migration after plating on different matrices as shown. Scale bar: 50μm. Data are shown as mean ±SEM. (L) Migration of primary cardiac fibroblasts plated on different matrices as indicated.…

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  Figure 5. Single cell sequencing of cardiac endothelial cells (A) UMAP plot from pooled single cell RNA sequencing data from isolated cardiac endothelial cells after sham, 1 and 8 weeks after TAC surgery (n = 2 samples/condition). 12 different subclusters were found. Main genes are shown on the right. (B) Gene ontology terms (GO, biological process) related to the clusters as indicated in (A). (C) Visualization of cluster gene regulation over time under the indicated conditions. (D) Quantification of the expression of the indicated genes in sham and 1 and 8 weeks after TAC in endothelial cells. Cdh5, Kdr, Fabp4 and Nrp1 are cardiac endothelial marker genes. N = 2 samples/condition.

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  Figure 6. Ribo-Tag Sequencing in endothelial cells after TAC (A) Scheme of the endothelial Ribo-Tag sequencing approach. (B) Venn diagrams to show the overlap between significantly upregulated genes and downregulated genes between endothelial RNA-sequencing (from previous Figures) and RiboTag sequencing. The numbers in the different sections indicate the number of genes (co-) regulated in the indicated sections of the diagram. (C) Heatmap of differentially translated genes in sham vs. 1 week TAC is shown (n = 4 sham, n = 5 TAC). Related gene ontology terms (GO, biological process) among the up- and downregulated genes with example genes is shown on the right. (D) Ligand-receptor analysis of gained new interactions between endothelial cells and cardiomyocytes is shown. The first two columns address endothelial cell derived ligands, the last two columns describe cardiomyocyte derived ligands. The ligand receptor pairs are shown on the right. (E) Venn diagrams to show the overlap of gained interaction between the indicated cell types computed from our RNA-seq versus the Ribo-Tag Seq results. The numbers in the different sections indicate the number of genes (co-) regulated in the indicated…

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  Figure 7. Collagen protein is produced by cardiac endothelial cells (A) Immunofluorescence staining of mouse heart sections for Isolectin B4 (IB4) to label endothelial cells and for Collagen 1 and 3 (Scale bar =50μm). (B and C) Immunostaining for the endothelial marker CDH5 and nuclear staining for DAPI in cultured confluent human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HCMECs). Scale bar= 50μm. (D) Col1 mRNA and Collagen 1 (Col1) protein from the indicated cells and their supernatant, respectively. Data are shown as mean ±SEM. (E) Collagen 1 protein from the supernatant of the indicated cells with and without TGF-β stimulation as indicated. (D) and (E), data are shown as mean ±SEM. The numbers indicated in the graph indicate the number of samples analyzed in that particular condition. ∗p<0.05, ∗∗p<0.01, ∗∗∗∗p<0.0001 as determined by two-tailed unpaired t-test (panel D) or by one-way ANOVA with Sidak’s multiple comparisons test.