Loss of KDM5B ameliorates pathological cardiac fibrosis and dysfunction by epigenetically enhancing ATF3 expression

Fig. 1. KDM5B expression is increased in cardiac fibroblasts after myocardial injury. a Q-PCR analysis of the mRNA expression levels of methylation modification enzymes in myocardial tissues from WT mice on Day 7 after MI or sham operation (n = 3 for sham group, n = 4 for MI group). b, c Q-PCR analysis of Kdm5b mRNA (n = 6 mice per group) or immunoblot analysis of KDM5B protein expression in myocardial tissues from WT mice on the indicated day after MI or sham operation. # indicates the serial numbers of the mice. d Q-PCR analysis of Kdm5b mRNA expression in myocardial tissues from WT mice on Day 28 following AngII or normal saline (NS) infusion (n = 6 mice per group). e Representative immunofluorescence staining of KDM5B (red) or α-SMA (green) in myocardial tissues from WT mice on Day 7 after MI or sham operation. Scale bar, 50 μm (magnified: 10 μm). f Representative immunofluorescence staining of KDM5B (red) or α-SMA (green) in myocardial tissues from WT mice on Day 28 after AngII or NS infusion. Scale bar, 50 μm (magnified: 10 μm). *p < 0.05, ***p < 0.001. Unpaired Student’s t test (a, d) or one-way ANOVA (b) was performed.

Fig. 2. KDM5B deficiency protects the heart from dysfunction and adverse cardiac remodeling after MI. a Echocardiographic measurement of the LVEF, LVFS, left ventricular end-diastolic internal dimension (LVIDd), and left ventricular end-systolic internal dimension (LVIDs) of KDM5B-KO or WT mice at baseline (Day 0) and on the indicated day after MI or sham operation (n = 6 mice per group). b–e Representative Masson’s trichrome staining images and quantitation of the scar size (b, c) or Sirius red staining images and quantitation of the fibrosis area (d, e) in myocardial tissues from KDM5B-KO or WT mice on Day 28 after MI. n = 6 mice per group. Scale bar, 1.6 mm (upper), 200 μm (bottom). f Q-PCR analysis of Col1a1 and Col3a1 mRNA levels in myocardial tissues from KDM5B-KO or WT mice on Day 14 after MI or sham operation (n = 6 mice per group). g Representative immunofluorescence staining of α-SMA (red) and Col III (green) in myocardial tissues from KDM5B-KO or WT mice on Day 14 after MI (n = 6 mice per group). Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s t test (c, e) or ANOVA (a, f) was performed.

Fig. 3. KDM5B deficiency prevents pressure overload-induced cardiac dysfunction and cardiac fibrosis. a Representative echocardiographic M-mode images of left ventricles from KDM5B-KO or littermate control WT mice on Day 28 after AngII or normal saline (NS) infusion. b, c Echocardiographic measurement of the LVEF (b) and LVFS (c) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). d, e The ratio of heart weight to body weight (HW/BW) (d) and the ratio of heart weight to tibia length (HW/TL) (e) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). f–i Representative Masson’s trichrome images and quantitation of perivascular (f, g) or interstitial (h, i) fibrosis in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). Scale bar, 200 μm (left), 100 μm (right). j Q-PCR analysis of Acta2, Col1a1, Col3a1 and Ctgf mRNA expression levels in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). k Immunoblot analysis of Col I and Col III protein expression in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS…

Fig. 4. KDM5B promotes fibrotic responses and the transition of cardiac fibroblasts. a, b Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (a) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (b) in KDM5B-deficient (KO) or littermate control WT cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. c, d Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (c) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (d) in Kdm5b-silenced or control siRNA-transfected cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. e, f Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (e) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (f) in cardiac fibroblasts treated with the KDM5B inhibitor GSK467 or DMSO followed by stimulation with TGF-β (10 ng/ml) for 24 h. g–i Representative immunofluorescence staining of α-SMA (red) in cardiac fibroblasts with KDM5B-deficiency (g), KDM5B knockdown (h) or GSK467 treatment (i) and the corresponding control cardiac fibroblasts (g–i) stimulated with TGF-β (10 ng/ml) for 24 h. Similar results were obtained from three…

Fig. 5. KDM5B facilitates SMAD-dependent or SMAD-independent TGF-β signaling activation. a Immunoblot analysis of phosphorylated (p-) or total levels of SMAD2 and SMAD3 proteins in the lysates of myocardial tissues from KDM5B-KO or littermate control WT mice after MI or sham operation. b Immunoblot analysis of phosphorylated or total levels of ERK, JNK, p38 and p65 proteins in the lysates of myocardial tissues from KDM5B-KO or WT mice after MI or sham operation. c Immunoblot analysis of phosphorylated or total levels of SMAD2 and SMAD3 proteins in the lysates of myocardial tissues from KDM5B-KO or WT mice after AngII or normal saline (NS) infusion. d Immunoblot analysis of phosphorylated or total levels of ERK, JNK, p38 and p65 proteins in the lysates of myocardial tissues from KDM5B-KO or WT mice after AngII or normal saline (NS) infusion. e Immunoblot analysis of phosphorylated or total levels of SMAD2 and SMAD3 proteins in the lysates of cardiac fibroblasts from KDM5B-KO or WT mice treated with TGF-β (10 ng/ml) for the indicated times. f Immunoblot analysis of phosphorylated or total levels of ERK, JNK, p38 and p65 proteins in the lysates of cardiac fibroblasts from KDM5B-KO or WT…

Fig. 6. KDM5B deficiency facilitates angiogenesis via the transition of fibroblasts to endothelial-like cells. a Q-PCR analysis of Vegfa, Cd31, Vegfr2, Nos3 and Vwf mRNA levels in myocardial tissues from KDM5B-KO or WT mice on Day 7 after MI surgery (n = 6 mice per group). b Gene Ontology enrichment analysis showing the top 15 differentially expressed genes in primary cardiac fibroblasts isolated from the heart tissues of KDM5B-KO and littermate control WT mice on Day 7 after MI surgery (BP biological process, CC cellular component, MF molecular function). c GSEA enrichment plot showing gene sets associated with the VEGF signaling pathway in primary cardiac fibroblasts as in b. d Heatmap showing differentially expressed genes relative to the positive and negative regulation of angiogenesis in primary cardiac fibroblasts as in b. e, f Representative immunofluorescence staining of VEGFR (green) (e), IB4 (green) (f) and Vimentin (red) (e, f) in myocardial tissues from KDM5B-KO or WT mice on Day 7 after MI surgery. Scale bar, 50 μm. Similar results were obtained from three independent experiments. g Q-PCR analysis of Vegfa, Cd31, Vegfr2, Nos3 and Vwf mRNA expression in KDM5B-deficient (KO)…

Fig. 7. KDM5B promotes cardiac fibrosis by inhibiting ATF3 expression. a Heatmap showing the top 20 upregulated differentially expressed genes in myocardial tissues from KDM5B-KO and littermate control WT mice on Day 7 after MI surgery. b Q-PCR analysis of Atf3 and Kdm5b mRNA expression in the myocardial tissues of WT mice on the indicated day after MI or sham operation (n = 6 per group). #p < 0.05 vs. Kdm5b mRNA levels in the sham operation group; *p < 0.05, **p < 0.01 vs. Atf3 mRNA levels in the sham operation group. c, d Q-PCR analysis of Atf3 mRNA (c) (n = 6 mice per group) or immunoblot analysis of ATF3 protein levels (d) in myocardial tissues from KDM5B-KO or WT mice on Day 7 after MI surgery. e, f Q-PCR analysis of Atf3 mRNA expression (e) (n = 6 per group) or immunoblot analysis of ATF3 protein expression (f) in Kdm5b-silenced or control siRNA-transfected cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. g, h Q-PCR analysis of Atf3 mRNA expression (g) (n = 6 per group) or immunoblot analysis of ATF3 protein expression (h) in cardiac fibroblasts treated with the KDM5B inhibitor GSK467 or DMSO followed by stimulation with TGF-β (10 ng/ml) for 24 h. i Immunoblot…

Fig. 8. KDM5B suppresses ATF3 expression through histone demethylation of the Atf3 promoter. a ChIP analysis of KDM5B enrichment levels at the Atf3 promoter in WT cardiac fibroblasts treated with TGF-β (10 ng/ml) or the control PBS (Ctrl) for 24 h. n = 6 per group. b–d Immunoblot analysis of H3K4me2 and H3K4me3 levels in cardiac fibroblasts with KDM5B deficiency (b), KDM5B knockdown (c) or GSK467 treatment (d) and the corresponding control cardiac fibroblasts (b–d) stimulated with TGF-β (10 ng/ml) for the indicated times. Similar results were obtained from three independent experiments. e, f ChIP analysis of H3K4me2 (e) or H3K4me3 (f) levels at the Atf3 promoter of WT cardiac fibroblasts treated with TGF-β (10 ng/ml) or control PBS (Ctrl) for 24 h. n = 6 per group. g, h ChIP analysis of H3K4me2 (g) or H3K4me3 (h) levels at the Atf3 promoter of KDM5B-deficient (KO) or WT cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. n = 6 per group. *p < 0.05, ***p < 0.001. Two-way ANOVA (a, e–h) was performed.