MiR-200a promotes the survival of cardiac cells and improves cardiac injury in chronic heart failure rats

Abstract

Introduction: miRNAs play an important role in cardiovascular abnormalities such as heart failure. In the present work we evaluated the role of miR-200a in the condition of chronic heart failure and also the mechanism involved.

Material and methods: In the study 180 subjects, among whom 100 were reported for chronic heart failure and 80 as normal, were included. ELISA and qRT-PCR was done to evaluate levels of HMGB1 and miR-200a in subjects. The cardiac hemodynamics and functioning, oxidative stress and expression of mediators of inflammation were studied in rats with chronic heart failure induced after transfecting them with miR-200a or HMGB1. Luciferase activity was measured to establish any correlation between HMGB1 and miR-200a.

Results: The chronic heart failure patients included in the study showed suppressed levels of miR-200a and elevated HMGB1 compared to normal subjects. In chronic heart failure rats, the transfection of miR-200a attenuated the cardiac function and other hemodynamic parameters. In addition, improvement in oxidative stress as well as inflammatory mediators was observed. The outcomes also confirmed that HMGB1 was the potential target of miR-200a. It was also noted that upon transfection miR-200a resulted in suppression of protein as well as mRNA levels of HMGB1 in the cardiac tissue of chronic heart failure rats. Also overexpression of HMGB1 decreased the effects of miR-200a.

Conclusions: The outcomes indicate that miR-200a exerts a protective effect on cardiac cell injury via the HMGB1 pathway.

Keywords: HMGB1; chronic heart failure; miR-200a.

Figure 1

Serum profile of levels of HMGB1 and miR-200a in subjects reported for chronic heart failure. A – Results of ELISA analysis for serum levels of HMGB1 protein in chronic heart failure patients. B – qRT-PCR analysis for serum mRNA levels of HMGB1 in chronic heart failure subjects. C – qRT-PCR analysis of miR-200a levels in chronic heart failure subjects. D – Quantitative analysis showing inverse relation between levels of miR-200a and HMGB1 in chronic heart failure subjects evaluated by Spearman’s rank correlation. The results are mean ± SE. ***P < 0.001 compared to control

Figure 2

miR-200a attenuated the hemodynamic parameters in chronic heart failure rats. For the experiment the chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days. A – Evaluation of LVSP. B – Evaluation of LVEDP. C – Heart rate. D – –dp/dt. E – +dp/dt. All the parameters were compared among five treatment groups of animals. All the results are presented as mean ± SE. *P < 0.05, **P < 0.01 compared to normal control. ^#P < 0.05 compared to chronic heart failure rats

Figure 3

miR-200a attenuated the cardiac function in chronic heart failure rats. The chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days. The animals were evaluated by electrocardiogram for: LVESD (A), LVPWD (B), LVEDD (C), IVSD (D), FS (E), LVEF (F). The results were compared among five groups with defined treatments. The results are mean ± SE. *P < 0.05 compared to normal rats. ^#P < 0.05 compared to chronic heart failure rats

Figure 4

miR-200a enhanced the left and right ventricular mass index in chronic heart failure rats. A – LVMI, B – RVMI. The chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days. Both left and right ventricles were weighed and their ratio to body weight was compared among the treated groups. The data are presented as mean ± SE. *P < 0.05 compared to normal group. ^#P < 0.05 compared to chronic heart failure rats

Figure 5

miR-200a improves the status of inflammatory factors and oxidative stress in chronic heart failure rats. The chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days after which the serum levels of: BNP (A), CK-MB (B), SOD (C), MDA (D), IL-1 (E), and TNF-α (F) were evaluated by ELISA. The results are mean ± SE. *P < 0.05, **p < 0.01, and ***p < 0.001 compared with normal rats. ^#P < 0.05 and ^##p < 0.01 compared to chronic heart failure rats

Figure 6

miR-200a improved the status of oxidative stress and inflammatory factors in the cardiac tissues of chronic heart failure rats. The chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days; the levels of inflammatory mediators: TNF-α (A), IL-1 (B) and oxidative stress markers: MDA (C), and SOD (D) were evaluated by ELISA. Data are presented as mean ± SD. The results are mean ± SE. *P < 0.05, **p < 0.01, and ***p < 0.001 compared with normal rats. ^#P < 0.05 and ^##p < 0.01 compared to chronic heart failure rats

Figure 7

miR-200a suppressed the protein as well as mRNA levels of HMGB1 in cardiac tissues of chronic heart failure rats. The chronic heart failure rats were transfected with miR-200a mimics or negative control for 28 days and qRT-PCR was performed for the analysis of: miR-200a (A) and HMGB1 mRNA (B) levels in the cardiac tissues. C – The protein levels of HMGB1 in the cardiac tissues were assessed by western blot analysis. D – Quantitative analysis results f or protein levels of HMGB1. The results are mean ± SE. *P < 0.05, **p < 0.01, and ***p < 0.001 compared with normal rats. ^#P < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to chronic heart failure rats

Figure 8

miR-200a improved the status of oxidative stress and mediators of inflammation in rats with chronic heart failure via targeting HMGB1. A – Binding sequences showing potential binding sites of miR-200a on the 3’-UTR region of HMGB1 mRNA and the mutant. B – The wild type HMGB1 or the mutant HMGB1 was co-transfected with miR-200a mimics or respective negative control in the 293T cells. The luciferase activity was measured after 48 h. C – Relative HMGB1 mRNA levels were evaluated by qRT-PCR in chronic heart failure rats transfected with miR-200 mimics or miR-200a mimics + LV-HMGB1 for 28 days. D – Western blot analysis was done to evaluate the protein levels of HMGB1 in cardiac tissues. Quantitative results for protein levels of miR-200a. E–H – Results of ELISA for levels of TNF-α, IL-1, MDA and SOD in cardiac tissues. The results are mean ± SE. **P < 0.01 in comparison with miR-NC group. ^@P < 0.05 and ^@@@P < 0.001 compared to defined groups