Secretory products from epicardial adipose tissue induce adverse myocardial remodeling after myocardial infarction by promoting reactive oxygen species accumulation

Figures

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  Fig. 1. Adverse myocardial remodeling in the rat model of myocardial infarction (MI). A Left: experimental protocols of control (n = 10), MI 1-week (n = 13), MI 2-week (n = 10), and MI 4-week (n = 15) groups. Right: wheat germ agglutinin (WGA) staining and Masson’s trichrome staining were carried out with the tissue at the location indicated by the dotted line. B Representative cardiac slices of rats with infarcts stained by 2,3,5-triphenyl tetrazolium chloride (TTC) in the control and MI 1-week groups. C Cardiomyocyte cross-sectional areas were examined using WGA staining (scale bar = 50 µm). D Myocardial fibrosis was examined using Masson’s trichrome staining (scale bar = 1 mm). **P < 0.01.

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  Fig. 2. The correlation between epicardial adipose tissue (EAT) and adverse myocardial remodeling. A Left: Representative images of hearts of rats in the control, MI 1-week, MI 2-week, and MI 4-week groups. Triangles: obvious EAT. Right: The ratio of hearts with obvious EAT in each group. The (B) cardiomyocyte cross-sectional areas and (C) myocardial fibrosis areas in the rats with (+) or without (−) EAT in MI 1-week group. *P < 0.05. D Pearson’s correlation coefficient (r) of EAT weights and cardiomyocyte size/myocardial fibrosis areas in rats in the MI 2-week and MI 4-week groups.

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  Fig. 3. Secretory products from EAT promoted cardiomyocyte hypertrophy and cardiac fibroblast activation by elevating ROS levels. Rat cardiomyocyte cell line H9C2 and primary rat cardiac fibroblasts were cultured in DMEM-F12 medium, EAT conditioned media (EAT-CM), or EAT-CM + N-acetyl-cysteine (NAC, 2 mM) for 72 h. A Immunofluorescent staining for α-actinin in H9C2 cells. B MitoSox fluorescence probe was used to observe the mitochondrial superoxide content in H9C2 cells. Cell nuclei were counterstained with DAPI. C Immunohistochemical staining for collagen III (CoI III) in primary rat cardiac fibroblasts (scale bar = 100 µm). D Immunofluorescent staining for alpha-smooth muscle actin (α-SMA) (green)/filamentous actin (F-actin) (red) in primary rat cardiac fibroblasts (scale bar = 100 µm). Cell nuclei were counterstained with DAPI. E The intracellular ROS level in primary rat cardiac fibroblasts was analyzed by flow cytometry using a DCFH-DA fluorescence probe. MFI: mean fluorescence intensity. F The protein levels of fibronection, smemb, and periostin in primary rat cardiac fibroblasts were measured by western blot. β-actin was used as an internal control. *P < 0.05, **P < 0.01 vs H9C2…

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  Fig. 4. The role of miR-134-5p in cardiomyocyte hypertrophy and cardiac fibroblast activation induced by secretory products from EAT. A Expression profiles of 35 candidate miRNAs in the left ventricular tissues of rats with (+) or without (−) obvious EAT in MI 1-week group. B Pearson’s correlation coefficient (r) of EAT weights and expression levels of 11 differentially expressed miRNAs determined in (A) in MI 4-week group. C The expression levels of miR-499-5p, miR-320-3p, miR-199a-3p, and miR-134-5p were measured by qRT-PCR in H9C2 cells and primary rat cardiac fibroblasts cultured in DMEM-F12 medium or EAT-CM. D, E H9C2 cells were divided into control, miR-134-5p inhibitor, EAT-CM, and EAT-CM + miR-134-5p inhibitor groups. D Immunofluorescent staining for α-actinin (scale bar = 20 µm). Cell nuclei were counterstained with DAPI. E MitoSox fluorescence probe was used to observe the mitochondrial superoxide content (scale bar = 20 µm). Cell nuclei were counterstained with DAPI. F–I Primary rat cardiac fibroblasts were divided into control, miR-134-5p inhibitor, EAT-CM, and EAT-CM + miR-134-5p inhibitor groups. F Immunofluorescent staining for (F) α-SMA (green)/F-actin (red) (scale bar =…

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  Fig. 5. KAT7 reversed the upregulation effect of miR-134-5p on intracellular ROS accumulation by promoting the transcriptions of MnSOD and catalase. A Bioinformatics databases TargetScan and miRDB forecasted there were 19 common target genes of miR-134-5p. The (B) mRNA and (C) protein levels of lysine acetyltransferase 7 (KAT7) in H9C2 cells and primary rat cardiac fibroblasts treated with EAT-CM or EAT-CM + miR-134-5p inhibitor were measured by qRT-PCR and western blot, respectively. β-actin was used as an internal control. D–F H9C2 cells and primary rat cardiac fibroblasts were divided into four groups: miR-134-5p inhibitor, the negative control of miR-134-5p inhibitor (inhibitor NC), miR-134-5p inhibitor + si- lysine acetyltransferase 7 (KAT7)-1, miR-134 -5p inhibitor + si-KAT7-2. All the cells were cultured in EAT-CM. D The intracellular ROS level was analyzed by flow cytometry using a DCFH-DA fluorescence probe. The (E) mRNA and F protein levels of manganese superoxide dismutase (MnSOD) and catalase. G H9C2 cells and primary rat cardiac fibroblasts were divided into three groups: EAT-CM, EAT-CM + miR-134-5p inhibitor, EAT-CM + miR-134-5p inhibitor + si-KAT7. Histone H3K14…

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  Fig. 6. miR-134 knockdown alleviated adverse myocardial remodeling in the rat model of MI. A The experimental protocols of sham (n = 10), MI + adeno-associated virus (AAV)-sh-miR-134-5p (n = 10), and MI + the negative control of AAV-sh-miR-134-5p (AAV-NC) (n = 10). B Myocardial fibrosis was examined using Masson’s trichrome staining. C Cardiomyocyte cross-sectional area was examined using WGA staining (scale bar = 50 µm). D The protein levels of KAT7, MnSOD, and catalase in the left ventricular tissues of rats in each group. E The miR-134-5p level and KAT7 mRNA level in the left ventricular tissues of rats in each group. B *P < 0.05 vs MI + AAV-NC. C–E *P < 0.05, **P < 0.01 vs sham; #P < 0.5 vs MI + AAV-NC.