The mechanism by which miR-494-3p regulates PGC1-α-mediated inhibition of mitophagy in cardiomyocytes and alleviation of myocardial ischemia-reperfusion injury.

OBJECTIVE: The purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-α and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R). METHODS: The expression of miR-494-3p was detected by RT‒qPCR, and the expression of PGC1-α, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (ΔΨm). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-α. RESULTS: The results showed that miR-494-3p and PGC1-α were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-α was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-α reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy. CONCLUSION: MiR-494-3p can target and negatively regulate the expression of PGC1-α to inhibit mitophagy in cardiomyocytes.

Upregulation of miR­423 improves autologous vein graft restenosis via targeting ADAMTS­7.

Coronary artery bypass graft (CABG) is one of the primary methods of treating coronary heart disease (CHD); however, vein graft restenosis is a major limiting factor of the effectiveness of CABG. Emerging evidence has indicated that miR­423 is associated with vascular diseases. Additionally, upregulation of a disintegrin and metalloproteinase with thrombospondin motifs­7 (ADAMTS­7) contributes to neointima formation by promoting the proliferation and migration of vascular smooth muscle cells and inhibiting the proliferation and migration of endothelial cells. The aim of the present study was to examine the effects of miR­423 target, ADAMTS­7, on regulating vein graft disease and identify novel biomarkers for use in therapy of vein graft failure (VGF). Aberrant expression of miR­423 in plasma of patients with CHD prior to and following CABG confirms that miR­423 may be a suitable target for preventing VGF. Furthermore, a dual­luciferase reporter gene assay indicated that miR­423 directly interacted with ADAMTS­7 and suppressed its expression. Ectopic expression of miR­423 suppressed ADAMTS­7, resulting in decreased proliferation and migration rates of human umbilical vein smooth muscle cells by targeting ADAMTS­7, but resulted in increased proliferation and migration of human umbilical vein endothelial cells in vitro. Overexpression of miR­423 also enhanced re­endothelialization and decreased neointimal formation in a rat vein graft model. In conclusion, the results of the present study demonstrated that the miR­423/ADAMTS­7 axis may possess potential clinical value for the prevention and treatment of restenosis in patients with CHD following CABG.

Resina draconis inhibits the endoplasmic-reticulum-induced apoptosis of myocardial cells via regulating miR-423-3p/ERK signaling pathway in a tree shrew myocardial ischemia- reperfusion model.

Ischemia-reperfusion (IR) is one of the significant medical problems in China. Triphenyltetrazolium chloride (TTC) staining is used to detect the status of the infarct size, and real-time PCR and western blotting are used to detect expressions of genes. TUNEL assay has been used to detect apoptosis. Using a tree shrew myocardial IR model, we found that in the reperfusion period, resina draconis (RD) treatment reduced the infarct size by TTC staining, and significantly enhanced the superoxide dismutase expression and down-regulated the malondialdehyde concentration in a dose-dependent manner. In hearts showing IR, Bax was increased and Bcl-2 was reduced, and RD treatment inhibited the IR-induced Bax expression and up-regulated the IR suppressed level of Bcl-2. TUNEL assay showed that IR induced the apoptosis of myocardial cells, and RD treatment suppressed the IR-induced apoptosis. CHOP and GRP78 were also upregulated in IR hearts, and RD treatment could significantly attenuate the CHOP and GRP78 levels compared with IR group. We further found that IR decreased the miR-423-3p expression and upregulated its target gene ERK both in mRNA and protein levels, and RD treatment upregulated miR-423-3p expression and downregulated ERK expression compared with the IR group. Importantly, miR-423-3p mimics inhibited IR increased ERK, CHOP and GRP78 expressions, and enhanced IR decreased Bcl-2 expression, and inhibited the IR-induced apoptosis of myocardial cells. The findings of this study suggest that RD treatment inhibited the endoplasmic reticulum induced apoptosis of myocardial cells via regulating miR-423-3p/ERK signaling pathway in a tree shrew myocardial IR model.

MALAT1/miR-15b-5p/MAPK1 mediates endothelial progenitor cells autophagy and affects coronary atherosclerotic heart disease via mTOR signaling pathway.

Objective: Present study focused on the influence of lncRNA MALAT1 on coronary atherosclerotic heart disease (CAD) by regulating miR-15b-5p/MAPK1 and mTOR signaling pathway.Method: Differentially expressed genes and activated pathway were investigated through bioinformatics analysis. QRT-PCR was conducted to verify expression of MALAT1, miR-15b-5p and MAPK1 in CAD blood samples and endothelial progenitor cells (EPCs). In addition, the interactions among MALAT1, miR-15b-5p and MAPK1 were revealed by Luciferase reporter assay. Cell autophagy of EPCs was examined by Cyto-ID Autophagy Detection Kit and transmission electron microscope. MTT assay and flow cytometry were carried out to assess cell viability and apoptosis in different interference conditions. Western blot was performed to testify the expression of pERK1/2 (MAPK1), phosphorylated mTOR, ATG1 and LC3-II. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by qRT-PCR. Finally, the effect of lncRNA MALAT1 on cell autophagy and atherogenesis was tested in vivo.Results:MALAT1 was overexpressed in CAD blood samples and EPCs. Knockdown of MALAT1 and MAPK1 promoted cell viability, autophagy and further suppressed the development of CAD. AntagoMALAT1 protects mice against atherosclerosis.Conclusion: LncRNA MALAT1 inhibited EPCs autophagy and increased cell viability while repressed apoptosis of CAD via activating mTOR signaling pathway.

Ginsenoside Rb1 inhibits autophagy through regulation of Rho/ROCK and PI3K/mTOR pathways in a pressure-overload heart failure rat model.

OBJECTIVE: This study was designed to explore the relationship between ginsenoside Rb1 (Grb1) and high-load heart failure (HF) in rats. METHODS: The parameters of cardiac systolic function (left ventricular posterior wall thickness (LVPWT), left ventricular internal diastolic diameter (LVID), fraction shortening (FS) and mitral valves (MVs)) of rat hearts in each group were inspected by echocardiogram. The expressions of rat myocardial contractile proteins, autophagy-related proteins and the activation of Rho/ROCK and PI3K/mTOR pathways were detected by Western blot. KEY FINDINGS: LVPWT, FS, MVs and the expression of myocardial contractile proteins α-MHC, apoptosis-related proteins Bcl-2 and signalling pathway involved proteins pAkt and mTOR were significantly reduced in the HF, HF+5 mg/kg Grb1 (HF+Grb1-5) and HF+Grb1+arachidonic acid (AA) groups with LVID, ß-MHC, cell apoptosis, cell autophagy and Rho/ROCK significantly increased compared with the control group, of which the tendency was contrary to the HF+20 mg/kg Grb1 (HF+Grb1-20) group compared with the HF group (P < 0.05). In the HF+Grb1+AA group, there was no significant change in the above indexes compared with the HF group. CONCLUSIONS: The results indicated that Grb1 can exert anti-HF function by inhibiting cardiomyocyte autophagy of rats through regulation of Rho/ROCK and PI3K/mTOR pathways.