Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis

Fig. 1. Gata4 and Gata6 show the highest expression among GATA family members in cardiac fibroblasts. a, b Quantification of Gata-factor RNA expression levels in isolated cardiac fibroblasts from RNA-sequencing [shown as counts per million reads (CPM, a)] and quantitative real-time-PCR (b) demonstrates that Gata4 and Gata6 are the highest expressed GATA family transcription factors in cardiac fibroblasts. RNA expression levels are normalized to Rpl7 (b). c Quantitative real-time-PCR of cell-type specific marker genes shows high purity of isolated cardiomyocytes (CM), fibroblasts (FB) and endothelial cells (EC) from adult mouse hearts. d Representative immunofluorescence images from transverse mouse heart sections show GATA-4 (upper images, green) and GATA-6 (lower images, green) in cardiac fibroblasts (stained with PDGFRα, red), nuclei are stained with DAPI (blue). Arrows indicate GATA-4 or GATA-6 positive cardiac fibroblasts, arrowheads indicate GATA-4 or GATA-6 positive cardiomyocytes. Scale bar 20 µm. e Quantitative real-time-PCR shows RNA expression level of Gata4 and Gata6 in FB compared to CM and EC. RNA expression levels are normalized to Gapdh (c, e). Data are shown as mean ± SD.…

Fig. 2. Combined deletion of Gata4 and Gata6 in activated cardiac fibroblasts aggravates heart failure after short- and long-term pressure overload. a, b Western blot of isolated cardiac fibroblasts from Gata4/6fl-Per-Cre and Gata4/6fl littermate control mice after pressure overload or sham (a) and quantification of relative protein levels normalized to GAPDH (b) showing efficient reduction of GATA-4 and GATA-6 protein levels after TAC in Gata4/6fl-Per-Cre mice. c–e Quantification of the ejection fraction (c), heart weight/tibia length ratio (HW/TL, d) and lung weight/tibia length ratio (LW/TL, e) in Gata4fl-Per-Cre and Gata4fl littermate control mice shows signs of compensated cardiac hypertrophy without significant differences in both groups after short-term pressure overload. f–h Quantification of the ejection fraction (f), HW/TL (g), and LW/TL ratio (h) in Gata6fl-Per-Cre and Gata6fl littermate control mice shows decreased cardiac function without significant differences in both groups after short-term pressure overload. i–n Quantification of the ejection fraction (i, l), HW/TL (j, m), and LW/TL ratio (k, n) in Gata4/6fl-Per-Cre mice and Gata4/6fl littermate controls shows that mice…

Fig. 3. Mice with fibroblast-specific deletion of Gata4 and Gata6 show a reduced capillary density after pressure overload. a–c Quantification of cardiac fibrosis from transverse mouse heart sections stained with Sirius-red after short (a) and long (b) pressure overload and representative pictures of Sirius-red stained hearts (c), scale bar 1 mm. d, e Quantification of the cardiomyocyte cross-sectional area after short (d) and long (e) pressure overload. f–h Quantification of the capillary density displayed as capillary per cardiomyocyte ratio after short (f) and long (g) pressure overload and representative pictures of WGA and IsolectinB4 stained heart sections (h), scale bar 50 µm. Data are shown as mean ± SD. Two-way ANOVA with Sidak’s multiple-comparisons test was used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4. Endothelial cells exert reduced migration and tube formation in co-culture with fibroblasts treated with siRNA to downregulate Gata4 and Gata6 compared to control-treated fibroblasts. a Schematic overview of co-culture experimental setup. Gata4 and Gata6 were downregulated in neonatal rat cardiac fibroblasts (NRC-Fib) by siRNA transfection 48 h before co-culture with HUVEC or HCMEC. b Relative mRNA level of Gata4 and Gata6 in rat cardiac fibroblasts 48 h after siRNA transfection. RNA expression levels are normalized to Gapdh. c, d Quantification of endothelial cell (HUVEC) migration (c) and representative images (d) from scratch assays. Pictures were taken directly after scratch (0 h) and 6 h later. e, f Quantification of tube formation (e) and representative pictures (f) of HUVEC/Fibroblast co-cultures on a 3D matrigel matrix. Images for quantification of tube formation were taken after 8 h of co-culture. Quantification of cardiac microvascular endothelial cell (HCMEC) migration (g) and representative images (h). Pictures were taken directly after scratch (0 h) and 10 h later. Scale bar (d, f, h) 200 µm. Data are shown as mean ± SD. One-way ANOVA with Dunnett’s…

Fig. 5. Transcriptome analysis of isolated cardiac fibroblasts showing an upregulation of anti-angiogenic genes in cells with Gata4/Gata6 deletion compared to control cells. a Heatmap of differentially regulated genes showing all analyzed groups (left panel) and direct comparison of Gata6fl-βGal (control) vs. Gata4/6fl-Cre (right panel) isolated cardiac fibroblast samples (n = 3 in each group). Gene ontology (GO, biological process) classification and selected example genes are demonstrated on the right. Bioinformatic analysis of RNA-Seq data is described in the “Materials and methods” section. b Venn diagram of significantly upregulated genes in fibroblasts with Gata4, Gata6, and Gata4/6 deletion compared to Gata6fl-βGal control samples. c RNA expression level of selected anti-angiogenic genes from RNA-sequencing. d Visualization of the top 20 GO BP clusters and relationships as a network plot. Each node represents an enriched term and is colored depending on its cluster ID, the cluster ID legend is shown on the right. Terms with a similarity > 0.3 are connected by edges. Data are shown as mean ± SD. Student’s t test was used for comparison of two groups. *p < 0.05, ***p < 0.001

Fig. 6. Proteomic profiling of the extracellular matrix (ECM) from whole hearts of Gata4/6fl and Gata4/6fl-Per-Cre mice shows significantly higher level of the anti-angiogenic glycoprotein CD36 in mice with fibroblast-specific downregulation of Gata4 and Gata6. a Schematic overview of sample preparation for proteomic analysis of extracellular matrix components. b PCA plot of detected extracellular matrix proteins from hearts of Gata4/6fl and Gata4/6fl-Per-Cre mice. c, d Heat map of all significantly different protein levels (c) and of important ECM components shown in color-coded clusters (d) in the cardiac ECM of Gata4/6fl and Gata4/6fl-Per-Cre mice. Detected proteins are indicated by encoding gene names. Normalized spectral counts are shown as log2 protein levels, blue color coding represents lower protein abundance, whereas red represents high protein levels. The anti-angiogenic glycoprotein CD36 was detected as the most significantly upregulated ECM protein in Gata4/6fl-Per-Cre mice compared to controls (highlighted with an orange box in c). e Normalized counts of CD36 from the ECM proteomic analysis. Data are shown as mean ± SD. Unpaired t test with Welch’s correction was used for…

Fig. 7. Anti-angiogenic effects of Gata4 and Gata6 downregulation in fibroblasts are mediated via CD36, thrombospondin-1 (THBS1) and angiopoietin-4 (ANGPT4). a, c Representative images of endothelial cell migration (a) and quantification (c) from scratch assays of HUVECs cultured in control medium (control) or in medium supplemented with recombinant THBS1 or ANGPT4 protein as indicated. Pictures were taken directly after scratch (0 h) and 10 h later. b, d Representative images showing the tube formation (b) and quantification of closed tubes (d) formed by HUVEC cultured on a 3D matrigel matrix. Images were taken after 8 h incubation in control medium (control) or medium supplemented with recombinant THBS1 or ANGPT4 protein. e Quantitative real-time-PCR of Angpt4, Thbs1, and Vegfa in fibroblasts after siRNA mediated downregulation of Gata4 and Gata6. RNA expression levels are normalized to Gapdh. f, h Representative images of endothelial cell migration (f) and quantification (h) from scratch assay of HUVECs in co-culture with fibroblasts. Gata4/6, Cd36 or Angpt4 were downregulated in fibroblasts by siRNA as indicated. Pictures were taken directly after scratch (0 h) and 6 h later. g, i…