miR-1929-3p Overexpression Alleviates Murine Cytomegalovirus-Induced Hypertensive Myocardial Remodeling by Suppressing Ednra/NLRP3 Inflammasome Activation

Abstract

MicroRNAs (miRNAs) play crucial roles in the development of essential hypertension (EH). Previously, we found that the expression of miR-1929-3p was decreased in C57BL/6 mice with hypertension induced by murine cytomegalovirus (MCMV). In this study, we explored the role of miR-1929-3p in hypertension myocardial remodeling in MCMV-infected mice. First, we measured MCMV DNA and host IgG and IgM after infection and determined the expression of miR-1929-3p and its target gene endothelin A receptor (Ednra) mRNA in the myocardium of mice. Then, we performed invasive blood pressure (BP) monitoring. Heart-to-body weight ratio (HW/BW%), along with mRNA levels of B-type natriuretic peptide (BNP) and beta myosin heavy chain (β-MHC), revealed myocardial remodeling. Hematoxylin/eosin and Masson's trichrome staining indicated morphological changes in the myocardium. Cardiac function was assessed via echocardiography. Moreover, MCMV-infected mice were injected with recombinant adeno-associated virus- (rAAV-) miR-1929-3p overexpression vector. Immunohistochemistry and western blotting showed the expression of Ednra and the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. And enzyme-linked immunosorbent assay (ELISA) revealed the concentrations of endothelin-1 (ET-1), interleukin-1β (IL-1β), and interleukin-18 (IL-18). In this study, we found that decreased expression of miR-1929-3p in MCMV-infected mice induced high BP and further development of myocardial remodeling cardiac function injury through increased expression of Ednra. Strikingly, overexpression of miR-1929-3p ameliorated these pathological changes of the heart. The positive effect was shown to be associated with inhibition of NLRP3 inflammasome activation and decreased expression of key proinflammatory cytokine IL-1β. Collectively, these results indicate that miR-1929-3p overexpression may effectively alleviate EH myocardial remodeling by suppressing Ednra/NLRP3 inflammasome activation in MCMV-infected mice.

Figure 1

MCMV infection raised BP and decreased the expression of miR-1929-3p in C57BL/6 mice. (a) Schematic of intervention here and in Figure 2 in the experimental group. (b) PCR analysis of the MCMV IE gene in the myocardium of C57BL/6 mice. (c) ELISA detection of IgG concentration in plasma. (d) ELISA detection of IgM concentration in plasma. (e) qRT-PCR analysis of miR-1929-3p expression in the myocardium. The value was normalized to the value observed with the control groups, which was set to 1: (f) SBP; (g) DBP; (h) MAP. The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05, the MCMV groups vs. the age-matched control groups. ^#P < 0.05, the MCMV 9-month groups vs. the control 7-month groups.

Figure 2

MCMV infection induced myocardial remodeling in mice. (a) Determination of HW/BW%. (b) Determination of LVW/BW%. (c) Representative images of H&E staining in the heart. (d) Quantitative analysis of the cell size (μm²) of cardiomyocytes in all groups. (e) Representative images of Masson's trichrome stain in the heart. (f) Quantitative analysis of fibrosis in all groups. The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05, the MCMV groups vs. the age-matched control groups. HW/BW%: heart-to-body weight ratio; LVW/BW%: left ventricle-to-body weight ratio. Images were magnified to ×400 power, scale bars = 20 μm.

Figure 3

miR-1929-3p overexpression relieved MCMV-induced myocardial remodeling in mice. (a) Schematic of intervention here and in Figures 4–7 in the experimental group. (b) The expression of miR-1929-3p in the myocardium. The value was normalized to the respective control groups, which was set to 1: (c) SBP; (d) DBP; (e) MAP; (f) HW/BW%; (g) LVW/BW%. (h) Representative images of H&E staining. (i) Quantitative analysis of the cell size (μm²) of cardiomyocytes in all groups. (j, k) qRT-PCR analysis of cardiac-specific fetal genes BNP and β-MHC. The value was normalized to the respective control groups, which was set to 1. (l) Representative images of Masson's trichrome stain. (m) Quantitative analysis of fibrosis in all groups. The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05 vs. the age-matched control groups. ^#P < 0.05, the MCMV+miR-1929-3p groups vs. the age-matched MCMV groups. Images were magnified to ×400 power; scale bars = 20 μm.

Figure 4

miR-1929-3p overexpression downregulated Ednra and alleviated MCMV-induced cardiac dysfunction. (a) Western blotting analysis of Ednra protein. (b) Densitometric analysis of (a). (c) qRT-PCR analysis of Ednra mRNA. The value was normalized to the control groups, which was set to 1. (d) Images of the M-mode of LV. (e–i) LVPWd, LVIDs, LVIDd, EF%, and FS%. The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05 vs. the age-matched control groups. ^#P < 0.05, the MCMV+miR-1929-3p groups vs. the MCMV groups. LVPWd: left ventricular end-diastolic posterior wall dimension; LVIDs: left ventricular end-systolic diameter; LVIDd: left ventricular end-diastolic diameter; EF%: ejection fraction; FS%: fractional shortening.

Figure 5

miR-1929-3p overexpression reduced MCMV-induced expression of the NLRP3 inflammasome of the myocardium in morphology. (a, c, e) Immunohistochemical analysis of NLRP3, ASC, and caspase-1. (b, d, f) Densitometric analysis of (a), (c), and (e). The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05 vs. the age-matched control groups. ^#P < 0.05, the MCMV+miR-1929-3p groups vs. the age-matched MCMV groups. Images were magnified to ×400 power; scale bars = 20 μm.

Figure 6

miR-1929-3p overexpression suppressed MCMV-induced expression of the NLRP3 inflammasome and secretion of IL-1β in the myocardium after MCMV infection. (a) Western blotting analysis of NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β, and IL-18 protein. (b–j) Densitometric analysis of (a). The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05 vs. the age-matched control groups. ^#P < 0.05, the MCMV+miR-1929-3p groups vs. the age-matched MCMV groups.

Figure 7

miR-1929-3p overexpression decreased ET-1 and IL-1β activation in MCMV-infected mice. (a, c, e) ELISA detection of ET-1, IL-1β, and IL-18 concentration in plasma. (b, d, f) ELISA detection of ET-1, IL-1β, and IL-18 concentration in cardiac tissues. The data are expressed as the means ± SEM (n = 5), ^∗P < 0.05 vs. the age-matched control groups. ^#P < 0.05, the MCMV+miR-1929-3p groups vs. the age-matched MCMV groups. (g) A schematic model showing the essential role of miR-1929-3p and Ednra in MCMV-induced hypertensive cardiac remodeling.