Sex-Specific Regulation of miR-29b in the Myocardium Under Pressure Overload is Associated with Differential Molecular, Structural and Functional Remodeling Patterns in Mice and Patients with Aortic Stenosis

Figures

• 

  Figure 1. Relative expression (RE vs. RNU6b) of miR-29b in the LV from male, female, orchiectomized (ORCH) male and ovariectomized (OVX) female mice subjected to a 4-week transverse aortic constriction (TAC) or sham (no-TAC) surgery. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA followed by Bonferroni post hoc test).

• 

  Figure 2. Relative expression changes in miR-29b (A) and Col1α1 (B) in cultured cardiac fibroblasts isolated from male and female myocardium, stimulated with TGF-β1 (10 ng/mL) for 24 h in serum-free medium. Relative changes in the expression of miR-29b in cardiac fibroblasts from male and female mice stimulated with 17β-estradiol (5 and 10 nM) for 24 h (C). Data are reported as mean ± SEM * p < 0.05, *** p < 0.001 (two way ANOVA and Bonferroni’s post-hoc test).

• 

  Figure 3. Morpho-functional echocardiographic changes induced by pressure overload in male (n = 19) and female (n = 29) mice subjected to 4-week transverse aortic constriction (TAC). LVMI: left ventricle (LV) mass indexed to body weight (A). PWTI: posterior wall thickness indexed to body weight (B). rPWT: relative posterior wall thickness (2xPWT/LVEDd) (C). LVEF: LV ejection fraction (D). MAPSE: mitral annular plane systolic excursion (E). E/e′: ratio of peak early transmitral flow velocity to peak early myocardial tissue velocity (e′) (F). Data are expressed as means ± SEM. ** p < 0.01; *** p < 0.001 (repeated-measures two-way ANOVA followed by Bonferroni test).

• 

  Figure 4. Relationship between LV miR-29b expression and morphological and functional echocardiographic parameters in mice. Linear regression and Pearson’s correlation analyses showing the relationship between LV miR-29b expression and morpho-functional echocardiographic parameters, in sham (n = 8 males; n = 11 females) and TAC (n = 17 males; n = 13 females) mice. IVS, interventricular septum (A,B); PWT, posterior wall thickness (C,D); rPWT, relative posterior wall thickness (E,F); LVM, LV mass (G,H); LVEF, LV ejection fraction (I,J); MAPSE, mitral annular plane systolic excursion (K,L); E/e′, the ratio of peak early transmitral flow velocity (E) to peak early myocardial tissue 1 velocity (e′) (M,N), r, Pearson’s correlation coefficient. * p < 0.05, ** p < 0.01, *** p < 0.001. $ Comparative analysis of female vs. male correlations with cocor.

• 

  Figure 5. Regulation by pressure overload of relevant LV remodeling related elements in mice. A and B: Fold change in mRNA levels of Col1α1 (A) and Col1α3 (B) in the LV from male and female mice subjected to sham surgery or transverse aortic constriction (TAC) for 4 weeks (sham males, n = 9; sham females, n = 11; TAC males, n = 15; TAC females, n = 12). (C) Representative images of LV sections showing myocardium stained with Masson’s trichrome. With this technique, muscle fibers are stained red, fibrosis is stained blue, the cytoplasm is stained light red or pink, and cell nuclei are stained dark brown to black. (D) Average fractional LV area of fibrosis in 4 sections from three to six mice per group. (E,F) Fold change in mRNA levels of β-MHC (E) and GSK-3β (F) in the LV from sham and TAC mice of both sexes. (G) Representative western blot images showing the protein levels of GSK-3β in the LV. (H) Average relative optical density (RD vs. GAPDH) quantified in three mice per group in two different experiments. Data are means ± SEM. * p < 0.05, ** p < 0.01; *** p < 0.001 (ANOVA followed by the Bonferroni post-hoc test).

• 

  Figure 6. Expression levels of miR-29b in the LV tissue and plasma in patients. (A) relative miR-29b expression in the LV (RE vs. RNU6b) from control individuals under 50 years (n = 6 men, n = 8 women) and over 50 years (n = 11 men, n = 15 women) of age, and (B) in plasma (RE vs. Cel-39) from control individuals under 50 years (n = 5 men, n = 8 women) and over 50 years (n = 10 men, n = 13 women) of age. (C) miR-29b expression in the LV from control patients (11 men, 15 women) and patients with AS (45 men, 50 women) over 50 years of age, and (D) in plasma from control patients (10 men, 13 women) and patients with AS (38 men, and 46 women) over 50 years of age. Data are means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal–Wallis followed by Dunn’s post hoc test).

• 

  Figure 7. Morphological and functional echocardiographic parameters in controls and men and women with AS who were older than 50 years of age. (A) LVMI, LV mass index; (B) LVEDdI, LV end-diastolic diameter index; (C) rPWT, relative posterior wall thickness; (D) LVEF, LV ejection fraction; (E) MAPSE, mitral annular plane systolic excursion; (F) E/e′, the ratio of peak early transmitral flow velocity to peak early myocardial tissue velocity (e′). Whiskers represent the 10th–90th percentiles. ** p < 0.01, *** p < 0.001 (two-way ANOVA followed by Bonferroni post hoc test). Control patients: men = 11, women = 15; AS patients: men = 45, women = 50. For MAPSE, control: men = 8, women = 10; AS patients: men = 42, women = 45. For E/e’, control: men = 5, women = 5; AS patients: men = 24 and women = 38.

• 

  Figure 8. Linear regression and Pearson’s correlation analyses showing the relationship between plasma miR-29b expression and morpho-functional parameters in patients older than 50 years. IVST, interventricular septum thickness in men (A) and women (B); rPWT, relative posterior wall thickness in men (C) and women (D); MAPSE, mitral annular plane systolic excursion in men (E) and women (F). r, Pearson’s correlation coefficient. * p < 0.05, ** p < 0.01. $Comparative analysis of women vs. men correlations with cocor.