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 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 mice treated with TGF-β (10 ng/ml) for the indicated times. Similar results were obtained from three independent experiments.

Fig 4: 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 analysis of ATF3 protein expression in cardiac fibroblasts transfected with Atf3 siRNA or control siRNA. j Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression levels in cardiac fibroblasts transfected with Kdm5b siRNA, Atf3 siRNA, control siRNA or cotransfected with Kdm5b and Atf3 siRNA followed by stimulation with TGF-β (10 ng/ml) for 24 h (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s t test (c, e, g) and ANOVA (b, j) were performed.

Fig 5: 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.