Inhibition of MEG3 ameliorates cardiomyocyte apoptosis and autophagy by regulating the expression of miRNA-129-5p in a mouse model of heart failure

Abstract

The long non-coding RNA, maternally expressed gene 3 (MEG3), are involved in myocardial fibrosis and compensatory hypertrophy, but its role on cardiomyocyte apoptosis and autophagy in heart failure (HF) remains unclear. The aim of this study was to investigate the effect of MEG3 on cardiomyocyte apoptosis and autophagy and the underlying mechanism. A mouse model of HF was established by subcutaneous injection of isoproterenol (ISO) for 14 days, and an in vitro oxidative stress injury model was replicated with H₂O₂ for 6 h. SiRNA-MEG3 was administered in mice and in vitro cardiomyocytes to knock down MEG3 expression. Our results showed that cardiac silencing of MEG3 can significantly ameliorate ISO-induced cardiac dysfunction, hypertrophy, oxidative stress, apoptosis, excessive autophagy and fibrosis induced by ISO. In addition, inhibition of MEG3 attenuated H₂O₂-induced cardiomyocyte oxidative stress, apoptosis and autophagy in vitro. Downregulation of MEG3 significantly inhibited excessive cardiomyocyte apoptosis and autophagy induced by ISO and H₂O₂ through miRNA-129-5p/ATG14/Akt signaling pathways, and reduced H₂O₂-induced cardiomyocyte apoptosis by inhibiting autophagy. In conclusion, inhibition of MEG3 ameliorates the maladaptive cardiac remodeling induced by ISO, probably by targeting the miRNA-129-5p/ATG14/Akt signaling pathway and may provide a tool for pharmaceutical intervention.

Keywords: ATG14; Heart failure; ISO; MEG3; apoptosis; autophagy; miRNA-129-5p; oxidative stress.

Graphical abstract

Figure 1.

SiRNA-MEG3 ameliorates the cardiac function and tissue structure of the LV in ISO-induced murine HF. (A) Representative M mode images of echocardiography in the mice and quantitative analysis of each cardiac function index. (B) Representative HE staining images of LV (scale bar: 100 μm; cell necrosis, nuclear lysis and inflammatory cell infiltration indicated by green arrows) and quantitative analysis of cross-sectional area (CSA) in the mice. Chromatin in the nucleus and the nucleic acid in the cytoplasm were stained purple-blue, and the components in the cytoplasm and extracellular matrix were stained red. (C) Representative Masson staining images (scale bar: 100 μm; myocardial fibrosis indicated by black arrows) and quantitative analysis of collagen volume of LV in the mice. Cardiomyocytes were stained red and collagenous fibers stained blue. (D) Representative images of picrosirius staining in mice (scale bar: 100 μm) and quantitative analysis of collagen I and III of LV in the mice. Collagen I was stained red strong orange or bright red, indicated by green arrow; collagen III was stained green, indicated by white arrow. HW/BW: heart weight/body weight; LVID: left ventricular internal dimension; LVVol: left ventricular volume; LVPW: left ventricular posterior wall; IVS: intraventricular septum; LVEF: left ventricular ejection fraction; FS: fractional shortening; d: diastole; s: systole; LV: left ventricular; CVF: collagen volume fraction. Data are expressed as mean ± SD. Mice, n = 12 (Figure.1 A)、n = 4 (Figure.1 B-D). **p < 0.01 versus control group; ^##p < 0.01versus ISO group.

Figure 2.

SiRNA-MEG3 reduced the expression of cardiac remodeling markers in the LV of mice with HF induced by ISO or oxidative damage of H9C2 cells induced by H₂O₂. (A) Representative western blotting bands of NPPA, NPPB and MYH7 and their quantitative analysis by densitometry based on immunoblot images in the LV of mice. (B) Representative Western blotting bands of NPPA, NPPB and MYH7 and their quantitative analysis in the H9C2 cells. Data are expressed as mean ± SD. Mice, n = 5; H9C2 cells = 2 batches of cells, repeat 2∼3 multiple wells for each batch of cells. *p < 0.05, **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01versus control group.

Figure 3.

SiRNA-MEG3 reduced myocardial apoptosis and ROS level of H₂O₂-stimulated H9C2 cells. (A-B) Representative western blotting bands of Bcl2 and Bax and their quantitative analysis in the mice and H9C2 cells. (C) Representative images of flow cytometry in H9C2 cells and quantitative analysis of apoptotic cells. (D-E) Representative images of DHE fluorescence staining and quantitative analysis of ROS level in H9C2 cells. The blue fluorescence represents the nucleus stained with DAPI, and the red fluorescence represents ROS (scale bar  = 50 µm). Data are expressed as mean ± SD. Mice, n = 5; H9C2 cells = 2 batches of cells, repeat 2∼3 multiple wells for each batch of cells. **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01versus control group.

Figure 4.

SiRNA-MEG3 inhibits autophagy in murine HF induced by ISO in vivo and H₂O₂-stimulated H9C2 cells in vitro. (A) Representative images of left ventricular autophagy in mice were obtained by TEM (scale bars: 1 μm). The typical tissue autophagosome is indicated by the red arrow. The higher the number of autophagosomes, the higher the autophagy. (B) Fluorescence representative image of H9C2 cells transfected with GFP-LC3B (scale bar: 50 μm). The red arrow represents free GFP-LC3B and autophagy vacuoles. The more the number, the stronger the autophagy. (C) Western blotting representation image of Beclin1, LC3II/LC3I and p62 proteins in mouse LV and quantitative analysis of Western blotting data. (D) Western blotting representation image of Beclin1, LC3II/LC3I and p62 proteins in H9C2 cells and quantitative analysis of Western blotting data. Data are expressed as mean ± SD. Mice, n = 5; H9C2 cells = 2 batches of cells, 2∼3 multiple wells repeated for each batch of cells. *p < 0.05, **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01versus control group.

Figure 5.

SiRNA-MEG3 regulates the expression of miRNA-129-5p and its downstream apoptotic proteins. (A-B) The levels of MEG3 and miR-129-5p gene were detected by RT-PCR in LV of mice and H9C2 cells. (C) Representative image of Western blotting of p-Akt, Akt, p-GSK3β and GSK3β in mice and their quantitative analysis. (D) Representative image of Western blotting of p-Akt, Akt, p-GSK3β and GSK3β in H9C2 cells and their quantitative analysis. Data are expressed as mean ± SD. Mice, n = 5; H9C2 cells = 2 batches of cells, repeat 2∼3 multiple wells for each batch of cells. *p < 0.05, **p < 0.01 versus control group; ^#P < 0.05,^##P < 0.01versus control group.

Figure 6.

SiRNA-MEG3 regulated the expression levels of ATG14 and p-mTORC1 protein. (A) Representative image of Western blotting of ATG14 and p-mTORC1 in mice and their quantitative analysis. (B) Representative image of Western blotting of ATG14 and p-mTORC1 in H9C2 cells and their quantitative analysis. Data are expressed as mean ± SD. Mice, n = 5, H9C2 cells = 2 batches of cells, repeat 2∼3 multiple wells for each batch of cells. *p < 0.05, **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01versus control group.

Figure 7.

SiRNA-MEG3 reduces H₂O₂-induced cardiomyocyte apoptosis and ROS levels by inhibiting autophagy. (A) Representative image of DHE immunofluorescence staining of H9C2 cells (scale bar  = 50 µm) and quantitative analysis of DHE fluorescence density in H9C2 cells. Blue fluorescence represents the nucleus stained with DAPI, and red fluorescence represents ROS. (B) Representative images of flow cytometry in H9C2 cells and quantitative analysis of apoptotic cells. Data are expressed as mean ± SD, H9C2 cells = 2 batches of cells, repeat 2∼3 multiple wells for each batch of cells. *p < 0.05, **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus H₂O₂ group; ^Δp < 0.05, ^ΔΔp < 0.01 H₂O₂+ siRNA-MEG3 + Rapa versus H₂O₂+ siRNA-MEG3.