Lack of STAT1 co-operative DNA binding protects against adverse cardiac remodelling in acute myocardial infarction

Figures

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  Figure 1. The STAT1-F77A mutation protects mice from adverse remodelling after myocardial infarction. (A,B) Kaplan-Meier curves showing the survival rates of female (A) and male (B) mice expressing homozygously either STAT1 WT (green) or the amino-terminal F77A mutation (orange) after experimental FIGURE 1 (Continued)MI due to LAD ligation. Log-rank test was used to test for a difference in survival between the two genotypes. (C–L) Echocardiographic assessment of haemodynamic parameters in WT (green) and STAT1F77A/F77A (orange) female mice before LAD ligation (n = 23 versus n = 22), 3 days (n = 12 versus n = 11), 1 week (n = 9 versus n = 8) and 4 weeks (n = 7 versus n = 8) following surgery. The box plots demonstrate data distribution between the two genotypes in left ventricular (C) end-systolic diameter, (D) end-diastolic diameter, (E) end-systolic area, (F) end-diastolic area, (G) end-systolic epicardial area, (H) fractional shortening, (I) fractional area shortening, (J) ejection fraction, (K) end-systolic volume, and (L) end-diastolic volume. Asterisks indicate statistically significant differences at a p-level of ≤0.05, as assessed by a two-tailed Student’s t-test.

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  Figure 2. Increased ratio of total STAT3 to STAT1 level in the myocardium of mice expressing homozygously the STAT1-F77A mutation on day 3 after MI. (A) Representative Western blots of proteins extracted from either infarcted ventricular tissue (+) or unaffected areas (−) from the ventricles of mice carrying the WT/WT, WT/F77A and F77A/F77A genotypes. Blots were probed with anti-phosphotyrosine STAT1 (p-STAT1), anti-pan STAT1 (STAT1), anti-phospho-STAT3 (p-STAT3) and anti-pan STAT3 antibodies, as well as a GAPDH-specific antibody (GAPDH) used for normalization (see also Supplementary Figure 3). (B–G) Quantification of immunoblots from three independent experiments, as shown in (A) and standardized to the WT non-infarcted value. Only the α-forms of STAT1 and STAT3 were evaluated. Asterisks in the box plots indicate statistically significant differences, as determined using t-test. All WT bands were normalized to 1.

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  Figure 3. A pronounced accumulation of infiltrating phospho-STAT1-expressing cells in the myocardium after MI, as assessed by means of histological analysis. (A–G) Immunohistochemical staining of p-STAT1, STAT1 and STAT3 in heart sections on day 3 after MI or sham operation. (A) Representative micrographs of the left ventricular wall of sham-(top panel) and MI-operated animals (bottom panel) in mice carrying the WT/WT and F77A/F77A genotype, respectively (scale bar: 50 μm). Arrows mark individual cells with positive immunohistochemical staining for the indicated antigen FIGURE 3 (Continued)(WT/WT male n = 4, female n = 3; F77A/F77A male n = 5, female n = 3). (B–F) Quantifications of the distribution of positively stained cells over the ventricular wall and septum (left panel), and of the density of positively stained cells within the infarcted area (right panel). Asterisks indicate statistically significant differences, as determined using t-test. (G–J) Haematoxylin and eosin (H&E) staining of paraffin-embedded cardiac tissue sections at day 3 after MI or sham operation. (G) Representative micrographs of hearts demonstrating the infiltration of immune cells in the infarct area. Strong…

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  Figure 4. Recruitment of different immune cells in WT and STAT1F77A/F77A mice. (A) Immunohistochemical staining of immune cell subsets in STAT1F77A/F77A mice with antibodies against CD3 (upper panel), CD68 (middle panel), and myeloperoxidase (MPO) (lower panel) as markers for T cells, monocytes and neutrophil granulocytes, respectively, on day 3 following MI or sham operation. Representative LV wall micrographs of sham-operated animals (top) and mice subjected to LAD ligation (bottom) from the two genotypes (scale bar: 50 μm, WT/WT male n = 4, female n = 3; F77A/F77A male n = 5, female n = 3). Arrows mark positively stained cells expressing the indicated antigen. Arbitrary quantification of the (B) distribution of CD3-positive cells and their (C) density over the ventricular wall and septum. Quantification of the distribution (D,F) and density (E,G) of CD68-positive (D,E) and MPO-positive cells (F,G) (WT/WT male n = 4, female n = 3; F77A/F77A male n = 5, female n = 3). (H–J) Analysis of the relative numbers of regulatory T cells (H,I) and inflammatory monocytes (J) in lymph nodes (H,J) and spleen (I) of 9-week-old females from the STAT1F77A/F77A mouse line as compared to WT controls.…

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  Figure 5. Higher number of differentially regulated genes in STAT1F77A/F77A mice following LAD ligation compared to WT mice. (A) Venn diagram showing the number of unique genes that are upregulated or downregulated in WT and transgenic mice (n = 12, for each group 3 mice) 24 h after LAD ligation. FIGURE 5 (Continued)A log2(fold change) >1 and a value of p < 0.05 were used. Green shows all the differentially expressed genes in WT and orange in STAT1F77A/F77A mice. (B) Hierarchical clustering of RPKM (reads per kilobase of transcript per million reads mapped) values in the WT and STAT1F77A/F77A mice in both sham- and LAD-operated mice. First heat map was generated using a list of genes that are differentially expressed in WT (MI vs. sham) comparison (n = 1,912 genes). Second heat map is generated using a list of genes that are differentially expressed in STAT1F77A/F77A (MI vs. sham) comparison (n = 4,055 genes). The R pheatmap package was used for visualization. (C) Volcano plot representation of differentially expressed genes in WT MI vs. sham-operated (top) and STAT1F77A/F77A MI vs. sham-operated (bottom) mice. Every point represents an individual transcript in each plot. Colored points…

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  Figure 6. Schematic presentation of the effects of disrupted co-operative DNA binding in a murine model of myocardial infarction (MI). Male mice with a homozygous F77A mutation show a higher survival rate following left anterior descending artery (LAD) ligation, while the haemodynamic function of the ischaemic heart is less affected in female mice. Changes in the STAT protein levels as well as differentially regulated KEGG cellular processes are depicted for female STAT1F77A/F77A mice. Upregulated pathways are displayed in red and downregulated pathways are in green. In the comparison between the transcriptomes from ischaemic and non-ischaemic myocardial tissues, prominent genotype-specific differences were observed for cardiac muscle contraction, fatty acid metabolism and oxidative phosphorylation.