Fig 2: Knockdown of YB-1 exacerbates cardiac dilation and fibrosis. (A) Diagram of the experimental design. A single dose of 1 x 1011 vg of AAV9-shLuci or AAV-shYB-1 was injected into mice at postnatal day 8-12. Echocardiography was performed on 8-week-old mice post-injection. (B-C) Relative (B) Yb-1 expression and (C) immunoblotting of YB-1 on 8-week-old mice. Data represent means ± SEM, **P < 0.01, ***P < 0.001, Student's t-test. (D) Heart weight to body weight and heart weight to tibia length ratios on both AAV-shLuci and AAV-shYB-1 injected mice. Data represent means ± SEM, ***P < 0.001, Student's t-test. (E) Echocardiographic analysis of mice at 8 weeks post-injection. Data represent means ± SEM, **P < 0.01, Student's t-test. (F) Representative images of Trichrome staining of whole hearts, scale bar: 200 µm. (G-H) Relative (G) Col1a1 expression and (H) immunoblotting of COL1A1 after AAV injection. Data represent means ± SEM, *P < 0.05, **P < 0.01, Student's t-test. (I-J) Relative expression of (I) fibrosis markers Acta2, Postn and Vim, and (J) extracellular matrix components Col1a2, Col3a1 and Fn1. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test.

Fig 3: H19 directly interacts with YB-1 in cardiac fibroblasts under hypoxia. (A-B) Quantification of H19 in (A) neonatal cardiac fibroblasts (NCF) and neonatal cardiomyocytes (NCM), (B) adult cardiac fibroblasts (ACF) and adult cardiomyocytes (ACM). Data represent means ± SEM, *P < 0.05, **P < 0.01, Student's t-test. (C) Relative expression of H19 in different cell types including adult cardiac fibroblasts (ACF) and adult cardiomyocytes (ACM) in the peri-infarct and remote area post-MI day 7. Data represent means ± SEM, *P < 0.05, **P < 0.01, one-way ANOVA. (D-G) Relative expression of H19 in (D) neonatal cardiac fibroblasts (NCF), (E) adult cardiac fibroblasts (ACF), (F) human iPSC-derived cardiac fibroblasts (hiPSC-CF) and (G) NIH3T3 cells under normoxia and hypoxia conditions. Data represent means ± SEM, **P < 0.01, ***P < 0.001, Student's t-test. (H) Diagram showing chromatin isolation by RNA purification (ChIRP). LncRNA H19 was pulled down using biotinylated probes. (I) Mass spectrometry results showing proteins that interacted with H19. (J) Representative image of immunoblotting of YB-1 in ChIRP of H19, LacZ as control. (K) Detection of H19 after RNA immunoprecipitation of YB-1 by quantitative PCR in adult cardiac fibroblasts (ACF), human iPSC-derived cardiac fibroblasts (hiPSC-CF) and NIH3T3 cells under normoxia and hypoxia conditions. Data represent means ± SEM, ***P < 0.001, one-way ANOVA. (L) Representative image of electrophoretic mobility shift assay (EMSA) between H19 and YB-1. (M) Evaluation of YB-1 dissociation constant (Kd). (N) Representative images of immunostaining of YB-1 localization of NIH3T3 cells under hypoxia, YB-1 (green), nucleus (blue), scale bar: 50 µm. (O) Immunohistochemistry staining of YB-1 in mouse hearts at control (sham) and day 4 post-MI (MI4D), scale bar: 20 µm. (P) Representative immunofluorescent staining of YB-1 (red), Vim (green) and DAPI (blue), scale bar: 20 µm. Note YB-1 localization in the nucleus of cardiac fibroblasts from mouse hearts isolated on day 4 post-MI. (Q) Ratio of H19 distribution in the nucleus to cytoplasm under normoxic and hypoxic conditions measured by qPCR after subcellular fractionation. Data represent means ± SEM, *P < 0.05, one-way ANOVA. (R) Individual evaluation of H19-YB-1 interaction in the nucleus and cytoplasm after subcellular fractionation using RNA immunoprecipitation under hypoxia. H19 was detected using qPCR. Data represent means ± SEM, ***P < 0.001, one-way ANOVA.

Fig 4: Ablation of H19 attenuates cardiac dilation and fibrosis after injury. (A) Diagram for experimental design. Eight-week-old mice of both wild type (H19+/+) and H19 knockout (H19-/-) groups underwent LAD-ligation surgery and sham surgery (without ligation), respectively. Echocardiography was performed post-MI D1 and D4. (B) Echocardiography analysis, EDV (end diastolic volume), ESV (end systolic volume), EF (ejection fraction) and FS (fraction shortening) after MI. Data represent means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. (C) Cardiac catheterization on mice post-MI day 4. Peak rate of pressure rise (dP/dtmax), preload recruited stroke work (PRSW), end-systolic pressure-volume relation (ESPVR), peak rate of pressure decline (dP/dtmin), relaxation time constant (Tau) and end-diastolic PV relation slope (EDPVR) were evaluated. Data represent means ± SEM, *P < 0.05, Student's t-test. (D) Heart weight to body weight and heart weight to tibia length ratios in mice after MI. Data represent means ± SEM, *P < 0.05, ***P < 0.001, one-way ANOVA. (E) Representative photomicrographs of immunofluorescent staining of wheat germ agglutinin (WGA), and DAPI in myocardial tissue sections, scale bar: 50 µm. Data are expressed as mean ± SEM, *P < 0.05, Student's t-test. (F) Representative images of Trichrome staining of whole heart post-MI D4, scale bar: 500 µm. Data are shown as mean ± SEM, *P < 0.05, Student's t-test. (G) Evaluation of total collagen in the heart post-MI D4. Data represent means ± SEM, ***P < 0.001, one-way ANOVA. (H-J) Relative expression of (H) H19, (I) fibrosis markers, Acta2, Postn and Vim and (J) extracellular matrix components Col1a1, Col1a2, Col3a1 and Fn1 post-MI D4. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.

Fig 5: Ectopic overexpression of H19 exacerbates cardiac dilation and fibrosis after injury. (A) Dynamic expression of H19 in the peri-infarct and remote areas post-MI (n=3). Data represent means ± SEM, *P < 0.05, **P < 0.01, one-way ANOVA. (B) Diagram of experimental design. At postnatal day 8 to 12, mice were I.P. injected with a single dose of 1 x 1011 vg of AAV9-GFP (control) or AAV-H19OE (H19OE). LAD-ligation surgery and sham surgery (without ligation) were performed on 8-week-old mice in both the control and H19OE groups. A series of echocardiographies were performed post-MI to evaluate cardiac functions. (C) Echocardiography analysis, EDV (end diastolic volume), ESV (end systolic volume), EF (ejection fraction) and FS (fraction shortening), on control and H19OE mice at post-MI day 1 (D1) and day 4 (D4). Data represent means ± SEM, **P < 0.01, one-way ANOVA. (D) Evaluation of heart weight to body weight and heart weight to tibia length ratios in mice at post-MI D4. Data represent means ± SEM, **P < 0.01, ***P < 0.001, one-way ANOVA. (E) Representative photomicrographs of immunofluorescent staining of wheat germ agglutinin (WGA), and DAPI in myocardial tissue sections, scale bar: 50 µm. Data are expressed as mean ± SEM, Student's t-test. (F) Representative images of Masson's trichrome staining of the whole heart after MI, scale bar: 500 µm. Data are shown as mean ± SEM, *P < 0.05, Student's t-test. (G) Evaluation of total collagen in the heart at post-MID4. Data represent means ± SEM, **P < 0.01, one-way ANOVA. (H-J) Relative expression of (H) H19, (I) fibrosis markers Acta2, Postn and Vim and and (J) extracellular matrix components Col1a1, Col1a2, Col3a1 and Fn1 post-MID4. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.