MicroRNA-140 is elevated and mitofusin-1 is downregulated in the right ventricle of the Sugen5416/hypoxia/normoxia model of pulmonary arterial hypertension

Abstract

Heart failure, a major cause of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), is an outcome of complex biochemical processes. In this study, we determined changes in microRNAs (miRs) in the right and left ventricles of normal and PAH rats. Using an unbiased quantitative miR microarray analysis, we found 1) miR-21-5p, miR-31-5 and 3p, miR-140-5 and 3p, miR-208b-3p, miR-221-3p, miR-222-3p, miR-702-3p, and miR-1298 were upregulated (>2-fold; P < 0.05) in the right ventricle (RV) of PAH compared with normal rats; 2) miR-31-5 and 3p, and miR-208b-3p were upregulated (>2-fold; P < 0.05) in the left ventricle plus septum (LV+S) of PAH compared with normal rats; 3) miR-187-5p, miR-208a-3p, and miR-877 were downregulated (>2-fold; P < 0.05) in the RV of PAH compared with normal rats; and 4) no miRs were up- or downregulated with >2-fold in LV+S compared with RV of PAH and normal. Upregulation of miR-140 and miR-31 in the hypertrophic RV was further confirmed by quantitative PCR. Interestingly, compared with control rats, expression of mitofusin-1 (MFN1), a mitochondrial fusion protein that regulates apoptosis, and which is a direct target of miR-140, was reduced in the RV relative to LV+S of PAH rats. We found a correlation between increased miR-140 and decreased MFN1 expression in the hypertrophic RV. Our results also demonstrated that upregulation of miR-140 and downregulation of MFN1 correlated with increased RV systolic pressure and hypertrophy. These results suggest that miR-140 and MFN1 play a role in the pathogenesis of PAH-associated RV dysfunction.

Keywords: heart; heart failure; hypertension; hypertrophy; lungs; miR; pulmonary; rats.

Fig. 1.

Right ventricular systolic pressure and Fulton's index are increased in Su/Hx/Nx rats. A: a measure of right ventricle systolic pressure (RVSP) in normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. B: a measure of right ventricular (RV) hypertrophy (Fulton's index) determined by the RV to left ventricle plus septum (LV+S) ratio in normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. C and D: Pearson correlation between RVSP and RV hypertrophy (Fulton's index). *P < 0.05 vs. normoxic control, #P < 0.05 vs. 8-wk Su/Hx/Nx. Data are means ± SE (n = 4).

Fig. 2.

MiroRNA-140 is elevated in RV Su/Hx/Nx rats compared with RV normoxic rats. A: a subset of microRNA determined by unbiased microRNA microarrays that are differentially regulated more than 1 logFC between normal right ventricle (Nx RV) and hypertrophied RV at 8-wk Su/Hx/Nx PAH rats. B: qPCR validation of increase in miR-140 in the RV of the Su/Hx/Nx PAH rat. C: Pearson correlation between RVSP and miR-140 in the RV of 8-wk Su/Hx/Nx PAH rats. D: Pearson correlation between RV hypertrophy (Fulton's index) and miR-140 in the RV of 8-wk Su/Hx/Nx PAH rats. E: Pearson correlation between RVSP and miR-140 in the RV of 13-wk Su/Hx/Nx PAH rats. F: Pearson correlation between RV hypertrophy (Fulton's index) and miR-140 in the RV of 13-wk Su/Hx/Nx PAH rats. *P < 0.05 vs. normoxic control, #P < 0.05 vs. 8-wk Su/Hx/Nx. Data are means ± SE (n = 3–4).

Fig. 3.

MiroRNA-31 is elevated in RV Su/Hx/Nx rats compared with RV normoxic rats. A: qPCR validation of increase in miR-31 in the RV of the Su/Hx/Nx PAH rat. B: Pearson correlation between RVSP and miR-31 in the RV of 8-wk Su/Hx/Nx PAH rats. C: Pearson correlation between RV hypertrophy (Fulton's index) and miR-31 in the RV of 8-wk Su/Hx/Nx PAH rats. D: Pearson correlation between RVSP and miR-31 in the RV of 13-wk Su/Hx/Nx PAH rats. E: Pearson correlation between RV hypertrophy (Fulton's index) and miR-31 in the RV of 13-wk Su/Hx/Nx PAH rats. *P < 0.05 vs. normoxic control. Data are means ± SE (n = 4).

Fig. 4.

MiR-140 targets 3′UTR of MFN1 and decreases the expression of Mfn1 in cardiomyocytes. A: a predicted consequential pairing of target region (top) position 476–483 of MFN-1 3′UTR and miR-140 (bottom) obtained from targetscan.org. B: conserved target sequence (red box) for miR-140 in position 476–483 of MFN1 3′UTR among different species. C: representative Western blot showing the decreased levels of mitofusin-1 (MFN1) proteins in H9c2 cells after transfection of meridian mimic for miR-140-5p. D: quantitative determination of relative expression of mitofusin-1 (MFN1) in H9c2 cells after transfection of meridian mimic for miR-140-5p E: relative mitochondrial DNA copy number in H9c2 cells after transfection of meridian mimic for miR-140-5p. F: relative mitochondrial membrane potential (Δψm) expressed as a ratio of J-aggregates (Red) to monomers (Green), in H9c2 cells after transfection of meridian mimic for miR-140-5p or scramble control with and without enthodelin-1 (ET1) treatment. *P < 0.05 vs. control, #P < 0.05 vs. scramble control. Data are means ± SE (n = 4).

Fig. 5.

Expression of MFN1 is downregulated in RV Su/Hx/Nx rats compared with RV normoxic rats. A: representative Western blot of MFN1 from RV and LV of normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. Blots were extracted from different lanes of the same gel to compare side by side. B: quantitative determination of relative expression of MFN1 in RV normalized by LV-MFN1. C: Spearman correlation between RVSP and the relative expression levels of MFN1 in RV of 8-wk Su/Hx/Nx PAH rats. D: Spearman correlation between RV hypertrophy and the relative expression levels of MFN1 in RV of 8-wk Su/Hx/Nx PAH rats. E: Pearson correlation between miR-140 levels and the relative expression levels of MFN1 in RV of 8-wk Su/Hx/Nx PAH rats. F: Spearman correlation between RVSP and the relative expression levels of MFN1 in RV of 13-wk Su/Hx/Nx PAH rats. G: Spearman correlation between RV hypertrophy and the relative expression levels of MFN1 in RV of 13-wk Su/Hx/Nx PAH rats. H: Pearson correlation between miR-140 levels and the relative expression levels of MFN1 in RV of 13-wk Su/Hx/Nx PAH rats. *P < 0.05 vs. normoxic control, #P < 0.05 vs. 8-wk Su/Hx/Nx. Data are means ± SE (n = 3).

Fig. 6.

Expression of c-kit is upregulated in RV Su/Hx/Nx rats compared with RV normoxic rats. A: representative Western blot of c-kit from RV and LV of normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. Blots were extracted from different lanes of the same gel to compare side by side. B: quantitative determination of relative expression of c-kit in RV normalized by LV-c-kit. C: Spearman correlation between RVSP and the relative expression levels of c-kit in RV of 8-wk Su/Hx/Nx PAH rats. D: Spearman correlation between RV hypertrophy and the relative expression levels of c-kit in RV of 8-wk Su/Hx/Nx PAH rats. E: Pearson correlation between miR-31 levels and the relative expression levels of c-kit in RV of 8-wk Su/Hx/Nx PAH rats. F: Spearman correlation between RVSP and the relative expression levels of c-kit in RV of 13-wk Su/Hx/Nx PAH rats. G: Spearman correlation between RV hypertrophy and the relative expression levels of c-kit in RV of 13-wk Su/Hx/Nx PAH rats. H: Pearson correlation between miR-31 levels and the relative expression levels of c-kit in RV of 13-wk Su/Hx/Nx PAH rats. *P < 0.05 vs. normoxic control, #P < 0.05 vs. 8-wk Su/Hx/Nx. Data are means ± SE (n = 3).

Fig. 7.

Expression of TUBA1 is upregulated in RV Su/Hx/Nx rats compared with RV normoxic rats. A: representative Western blot of TUBA1 from RV and LV of normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. Blots were extracted from different lanes of the same gel to compare side by side. B: quantitative determination of relative expression of TUBA1 in RV normalized by LV- TUBA1. C: Pearson correlation between RVSP and the relative expression levels of TUBA1 in RV of 8-wk Su/Hx/Nx PAH rats. D: Pearson correlation between RV hypertrophy and the relative expression levels of TUBA1 in RV of 8-wk Su/Hx/Nx PAH rats. E: Pearson correlation between RVSP and the relative expression levels of TUBA1 in RV of 13-wk Su/Hx/Nx PAH rats. F: Pearson correlation between RV hypertrophy and the relative expression levels of TUBA1 in RV of 13-wk Su/Hx/Nx PAH rats. G: quantitative determination of relative expression of GAPDH in RV normalized by LV-GAPDH. H: representative Western blot of GAPDH from RV and LV of normoxic control, 8-wk, and 13-wk Su/Hx/Nx PAH rats. Blots were extracted from different lanes of the same gel to compare side by side. *P < 0.05 vs. normoxic control. Data are means ± SE (n = 3).

Fig. 8.

MicroRNA 208b is elevated in LV of Su/Hx/Nx rats compared with LV normoxic rats. A subset of microRNAs determined by unbiased microRNA microarrays that are differentially regulated more than 1 logFC between left ventricle (LV) of normoxic (Nx) and 8-wk Su/Hx/Nx PAH rats.