Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Figure 1. miR-146b-5p is upregulated after myocardial infarction. (A) Levels of miR-146b-5p in the plasma of patients with chronic total occlusion (CTO) (n = 8 per group). (B) Representative images of infarcted mouse heart. Myocardium samples from different anatomic locations were harvested for the following analysis. (C and D) Expression levels of fibrotic genes Col1 (COL1A1) and αSMA (ACTA2) in myocardium samples (3 hearts, 5 samples per heart). (E) Expression levels of miR-146b-5p in mouse myocardium from different locations and time points, as measured by qPCR (n = 5 per group). Data are expressed as the mean ± SD. *P < 0.05 vs. control; **P < 0.01 vs. control. Data in panel (A), (C), and (D) were analyzed using Student’s unpaired t-test. Data in panel (E) was analyzed using one-way ANOVA followed by Tukey’s post-hoc analysis. Remo, remote zone; Infar, infarction zone.

Figure 2. Hypoxia-induced miR-146b-5p expression acts as a phenotypic modulator in fibroblasts, endothelial cells, and macrophages. (A) The expression profile of miR-146b-5p in primary cultured cardiac cells under hypoxia was studied using qPCR (n = 3 per group, 3 replicates). Multiple phenotypic studies were performed after 24 h of treatment with the miR-146b-5p mimic (miR-146b) or control (Ctrl) (B–H). (B) Representative images for EdU staining and EdU+ cell quantification of cardiac fibroblasts (n = 7 samples per group, 5 random fields per sample). Bar = 200 μm. (C and D), Representative migration images and quantification of data from Transwell and scratch assays in mouse fibroblasts (NIH3T3, n = 5 per group, 5 random fields per sample). Bar = 200 μm. (E) Representative images for immunostaining and quantification of expression of the fibroblast-to-myofibroblast transition genes Col1 (COL1A1) and αSMA (ACTA2) in cardiac fibroblasts (n = 3 per group, 7 fields per sample); bar = 5 μm. (F) Western blot analysis of the FMT genes in cardiac fibroblasts after treatment (n = 5). (G) Tube-formation assays of cardiac microvascular endothelial cells (CMVECs), with representative images on the…

Figure 3. Inhibition of miR-146b-5p consolidates the reparative phenotypes of fibroblasts, endothelial cells, and macrophages. Multiple phenotypic studies were performed after 24 h of treatment with miR-146b-5p inhibitor (Anti-146b) or control (Ctrl). (A) Representative images for EdU staining and EdU+ cell quantification of cardiac fibroblasts (n = 7 samples per group, 5 random fields per sample). (B and C), Representative migration images and quantification, according to Transwell and scratch assays of mouse fibroblasts (NIH3T3, n = 5 per group, 5 random fields per sample). (D and E) Representative western blot images and quantification of expression of the fibroblast-to-myofibroblast transition genes Col1 (COL1A1) and αSMA (ACTA2) in cardiac fibroblasts (n = 5 per group). (F) Tube-formation assays of cardiac microvascular endothelial cells (CMVECs), with representative images on the left and quantification data on the right (n = 3 samples per group, 5 random fields per samples, 3 replicates). (G) ELISA of macrophage-secreted inflammatory factors (n = 5 per group). Data are expressed as the mean ± SD for at least three independent assays unless otherwise noted. *P < 0.05 vs. control; **P…

Figure 4. Therapeutic modulation of miR-146b-5p restores cardiac function in the MI mouse. (A) Workflow for animal experiments to determine the effect of miR-146b-5p inhibition on infarcted hearts in vivo. The miR-146b-5p antagomir (Anti-146b) or control (Anti-Ctrl) was locally administered after MI surgery. The cardiac structure and function were studied for up to 4 weeks. (B) The ratio of heart weight to tibia length (n = 8 per group). (C) Representative images for hematoxylin and eosin (HE) and Masson’s staining and quantification of infarcted area (n = 8 samples per group, 10 random fields per sample). (D) Echocardiographic analyses of cardiac functions at 7, 14, 21, and 28 days post-MI and antagomir treatment (n = 7 per group). (E and F) Representative images for TUNEL staining and quantification of TUNEL+ apoptotic cells. Blue signals indicate cell nuclei. Brown signals represent TUNEL+ apoptotic cells (n = 7 per group, 10 random fields per sample); bar = 50 μm. (G and H) Representative images for CD31 staining and quantification of CD31+ vessels. Blue signals indicate cell nuclei. Brown signals indicate CD31+ vessels (n = 7 per group, 10 random fields per sample); bar = 50 μm. (I…

Figure 5. Therapeutic modulation of miR-146b-5p relieves cardiac fibrosis and remodeling in MI pig. (A) The LVEF of the experimental pigs was analyzed using echocardiography at different time points (sham group: n = 4, other groups: n = 8 each). (B) The survival analysis of the experimental groups. The survival curve was plotted using the Kaplan-Meier method. Pigs were sacrificed 4 weeks after MI. (C) Representative slices of the porcine myocardium. Infarction areas are indicated by the blue dashed line. Bar = 1 cm. (D) Infarction area analysis. Five whole heart sections per group were randomly selected, and the percentage of infarction area to total area in cross section was calculated. (E–G) The representative images for histological and immunohistochemical staining are shown (10 random fields per group, bar = 100 μm), indicating fibrosis, as per Masson’s staining (E); expression of collagen type 1 (Col1, F); and CD31+ capillaries (G). Cell apoptosis was analyzed by TUNEL staining (Fig. S5). Data are expressed as the mean ± SD. *P < 0.05; **P < 0.01. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc analysis. 1H, 1 h; 2W, 2 weeks; 4W, 4 weeks; Anti-146b, antagomir…

Figure 6. Therapeutic modulation of miR-146b-5p improves myocardial strains in MI pig. (A) The cardiac strains in different directions. (B and C) The definition of myocardial segmentation and the representative echocardiographic images of pig heart obtained 4 weeks post-MI (at end systole). (C) For speckle tracking, the left ventricle is divided into six anatomical zones in both the cross-sectional and longitudinal direction. (D) Representative segmental strain curves of the left ventricular (LV) mid-wall over one cardiac cycle at 4 weeks post-MI in the longitudinal, radial, circumferential, and rotation directions. (E–G) Bull’s eye diagrams of the absolute value of peak myocardial segmental strain in the radial (E), circumferential (F), and longitudinal (G) directions at 4 weeks post MI. BS, basal septum; MS, mid septum; AS, apical septal; AL, apical lateral; ML, mid lateral; BL, basal lateral; Ant-sept, anterior septum; Ant, anterior; Lat, lateral; Post, posterior; Inf, inferior; Sept, septum; Anti-146b, antagomir miR-146b-5p; Anti-Ctrl, antagomir control