The histone demthylase KDM3A protects the myocardium from ischemia/reperfusion injury via promotion of ETS1 expression

Figures

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  Fig. 1. KDM3A expression was downregulated after MIRI. a, b Levels of KDM3A protein (detected by Western blot assay) (a top panels are representative blots; bottom bar graphs show quantitative results) and mRNA (detected by qRT-PCR) (b) in rat heart at post I/R injury or a sham-operation (n = 6, *P < 0.05 vs. sham). c Representative dual immunofluorescence staining of KDM3A (green) and α-actinin (red) in heart sections with or without I/R injury (n = 4). The nuclei were stained with DAPI (blue). The upper panel shows heart sections from sham-operated animals; the lower panel shows heart sections from the I/R group. Scale bar = 20 μm. d, e Levels of KDM3A protein (d top panels are representative blots; bottom bar graphs display quantitative results) and mRNA (e) (n = 6, *P < 0.05 vs. control) in cultured NRCMs in the absence or presence of H/R injury. f Double immunofluorescence images of KDM3A (green) and α-actinin (red) in NRCMs treated with H/R or normoxic controls (n = 4). The top panels and bottom panels display cardiomyocytes subjected to normoxic control and H/R injury, respectively. Scale bar = 20 μm.

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  Fig. 2. KDM3A overexpression protects the myocardium against I/R injury. a Protein levels of KDM3A in rats intramyocardially transduced with AdKDM3A or AdGFP (n = 6, *P < 0.05 vs. AdGFP). Top: representative blots; bottom: quantitative results. b Representative section images (left panel) and averaged data of the ratio of IA to total left ventricular area (right panel) (detected by Evans blue+TTC staining) (n = 4, *P < 0.05 vs. I/R). The Evans blue-stained area (dark blue) represents the nonischemia/reperfusion area; TTC/Evans blue-negative regions (pale white) represent the infarcted myocardium. Scale bar = 1 cm. c, d Echocardiographic results of left ventricle ejection fraction (LVEF) and fractional shortening (LVFS) indicated cardiac function after KDM3A upregulation in response to MIRI or sham operation (n = 5, *P < 0.05 vs. sham; #P < 0.05 vs. I/R or I/R + AdGFP). e, f Myocardial death was estimated by the serum LDH/CK-MB concentrations with or without MIRI (n = 6, *P < 0.05 vs. sham; #P < 0.05 vs. I/R or I/R + AdGFP).

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  Fig. 3. KDM3A overexpression attenuates apoptosis, ROS and inflammation in MIRI. a, b Representative images (a) and averaged data of the percentage of TUNEL-positive cells (b) in rat hearts with or without viral transduction subjected to I/R injury (n = 4). TUNEL staining (green) indicates apoptotic nuclei; DAPI counterstaining (blue) indicates total nuclei. Scale bar = 20 μm. The apoptotic index (AI) is presented as the percentage of TUNEL-positive nuclei to the total number of nuclei. c Representative mitochondrial morphologies detected by TEM exhibiting the divergent status of swollen mitochondria, disrupted mitochondrial cristae and disorganized myofibrils with or without viral transduction either subjected to I/R injury or not (n = 3). Scale bar = 1 μm. d Mitochondrial function determined by myocardial ATP production (n = 6). e, f Cardiac protein levels of mitochondria-associated apoptotic mediators, including Bax (n = 6), Bcl-2 (n = 6), cleaved caspase-9/3 (n = 6), cytoplasmic cytochrome c (n = 4) and mitochondrial cytochrome c (n = 4), detected by western blot assay. Left panels show representative blots; right bar graphs show quantitative results (n = 6). g Representative images…

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  Fig. 4. KDM3A knockout exacerbates I/R-induced myocardial injury. a Representative western blots of KDM3A protein expression in heart tissue from KDM3A+/+ and KDM3A−/− (n = 6). b Representative section images (left panel) and averaged data of the ratio of IA to total left ventricular area (right panel) after sham or I/R surgery (n = 4). Scale bar = 1 cm. c, d Statistical analysis of echocardiographic parameters (LVEF and LVFS) indicating left ventricular function upon MIRI or sham operation in the indicated groups (n = 5). e Myocardial damage was estimated by LDH/CK-MB concentrations in the serum with or without MIRI (n = 6). *p < 0.05 vs. KDM3A −/−/sham; #p < 0.05 vs. KDM3A +/+/IR.

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  Fig. 5. KDM3A knockout exacerbates apoptosis, ROS and inflammation in MIRI. a Myocardial apoptosis determined by TUNEL staining. TUNEL staining (red) revealing apoptotic nuclei. DAPI counterstaining (blue) visualized total nuclei. Scale bar = 20 μm. b The apoptotic index (AI) was calculated using Image-Pro Plus software (n = 4). c, d Representative mitochondrial morphologies detected by TEM (n = 3, Scale bar = 1 μm) and mitochondrial function measured by ATP production (n = 6) in the presence or absence of MIRI in the indicated groups. e (n = 6) and f (n = 4), Cardiac protein levels of mitochondria-associated apoptotic mediators by western blot assay. Left panels show representative blots; right bar graphs show quantitative results. g Representative images (left panel) and mean fluorescence intensity (MFI) (right panel) of DHE staining on frozen myocardial tissue sections. n = 6. Scale bar = 50 μm. h, i Levels of pro-oxidative MDA and antioxidative SOD (n = 6). j, k The release of proinflammatory mediators (IL-6 and TNF-α) determined by ELISA kits (n = 6). *p < 0.05 vs. KDM3A −/−/sham; #p < 0.05 vs. KDM3A +/+/IR.

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  Fig. 6. The effects of KDM3A on H/R-exposed cardiomyocytes in vitro. Cell viability analysed by MTT (a, b) and culture medium LDH/CK-MB concentration (c, d). n = 6 per group, *P < 0.05 vs. AdGFP or AdshRNA/Control; #P < 0.05 vs. AdGFP or AdshRNA/H/R. e Representative images (upper panel) and quantitative results (lower panel) of apoptotic cardiomyocytes (detected by flow cytometry assay) infected with AdKDM3A or AdshKDM3A in response to H/R or control (n = 5). f ATP production in NRCMs infected with AdKDM3A or AdshKDM3A either exposed to H/R or not (n = 5). g–j Western blotting assay showing the levels of cytoplasmic cytochrome c, mitochondrial cytochrome c (g, h) Bax, Bcl-2 and cleaved caspase-9/3 (i, j) in primary cardiomyocytes infected with AdKDM3A or AdshKDM3A with or without H/R injury (n = 4). Upper panel: representative bots; lower panel: quantitative analysis. *P < 0.05 vs. AdGFP or AdshRNA/Control; #P < 0.05 vs. AdGFP or AdshRNA/H/R. k, l ROS accumulation detected by DHE staining and flow cytometry (upper panel) and quantified MFI (lower panel) in NRCMs (n = 5). m, n Levels of pro-oxidative MDA and antioxidative SOD in NRCMs (n = 4-5). o, p Quantification of proinflammatory…

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  Fig. 7. KDM3A is involved in MIRI by regulating ETS1 expression in vivo and in vitro. a Myocardial mRNA expression of the ETS1 gene detected by qRT–PCR with or without adenoviral transfection in response to MIRI or sham (n = 6, *P < 0.05 vs. sham; #P < 0.05 vs. I/R or I/R + AdGFP). b Representative western blots (upper panel) and quantitative results (lower panel) showing the protein expression of ETS1 and histone mark H3K9me2 with or without adenoviral transfection undergoing sham or MIRI (n = 4, *P < 0.05 vs. sham; #P < 0.05 vs. I/R or I/R + AdGFP). c mRNA levels of the ETS1 gene, as indicated by RT–PCR, in KDM3A+/+ and KDM3A−/− rats after sham or MIRI (n = 6, *p < 0.05 vs. KDM3A −/−/sham; #p < 0.05 vs. KDM3A +/+/IR). d Typical western blots (upper panel) and averaged data (lower panel) of ETS1 and H3K9me2 in KDM3A+/+ and KDM3A−/− rats with or without MIRI (n = 4, *p < 0.05 vs. KDM3A−/−/sham; #p < 0.05 vs. KDM3A+/+/IR). e mRNA levels of the ETS1 gene detected by qRT–PCR in NRCMs infected with AdKDM3A in response to control or H/R injury (n = 6, *P < 0.05 vs. AdGFP/Control; #P < 0.05 vs. AdGFP/H/R). f Representative western blots (upper panel) and quantitative data (lower panel) of the…

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  Fig. 8. ChIP-PCR assay demonstrated that KDM3A regulates the expression of ETS1 by modulating levels of H3K9me2 in its promoter region in the setting of H/R injury. a, b Primary NRCMs were transfected with AdKDM3A or AdGFP followed by H/R or control. ChIP followed by quantitative PCR (qPCR) with KDM3A and H3K9me2 antibodies. Data are expressed as the fold change compared to the corresponding control group (n = 4, *P < 0.05 vs. AdGFP/Control; #P < 0.05 vs. AdGFP/H/R). c, d NRCMs were infected with AdshKDM3A or AdshRNA with or without H/R. ChIP followed by quantitative PCR (qPCR) with KDM3A and H3K9me2 antibodies. Data are expressed as the fold change relative to the corresponding control group (n = 4, *P < 0.05 vs. AdshRNA/Control; #P < 0.05 vs. AdshRNA/H/R). e–h The enrichment of KDM3A and H3K9me2 on the GAPDH promoter were regarded as the control (n = 4). Data are expressed as the fold change relative to the corresponding control group (n = 4, NS: no significant difference).

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  Fig. 9. KDM3A ameliorates MIRI in an ETS1-dependent manner. a Representative western blots (upper panel) and quantitative data (lower panel) of the protein levels of three different ETS1 proteins in NRCMs (n = 5). *#P < 0.05 vs. vehicle. Cell viability was analysed using MTT reagent (b n = 6) and LDH/CK-MB concentration (c n = 4) in NRCMs infected with AdKDM3A followed by siETS1 transfection in response to H/R or control. ROS accumulation was detected by DHE staining and flow cytometry (d left panel), and the MFI (e right panel) was quantified in NRCMs (n = 5). Apoptosis was detected by flow cytometry using 7-AAD/AV-APC staining (f) and the apoptotic rate was quantified (g) (n = 5). h, i Quantitative results of IL-6/TNF-α in the culture medium of cardiomyocytes (n = 6). *P < 0.05 vs. H/R; #P < 0.05 vs. H/R/AdKDM3A.