miR-210 overexpression increases pressure overload-induced cardiac fibrosis

Figures

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  Fig. 1. Tg-210 TAC mice develop eccentric hypertrophy. Wt and Tg-210 mice were treated with doxycycline starting from 7 days before TAC surgery and until the endpoint of the experiment. All the Wt and Tg-210 TAC mice underwent echocardiography at day 7 after surgery and then were randomly assigned to two different groups. The first one was sacrificed at day 7. The second one was analyzed and sacrificed at day 28. (A–H) M mode echocardiographic results at day 7. (I–R) M mode echocardiographic results at day 28. (A and I) The percentage of fractional shortening (FS%) ∗P < 0.02, ∗∗P < 0.001, ∗∗∗∗P < 0.0001; (B and L) the left ventricular mass (LV mass) ∗P < 0.04, ∗∗P < 0.009; (C and M) the left ventricular volume in diastole (LV vol d) ∗P < 0.05; (D and N) the left ventricular volume in systole (LV vol s) ∗P < 0.03 ∗∗P < 0.007; (E and O) the left ventricular internal diameter in diastole (LVID d) ∗P < 0.03; (F and P) the left ventricular internal diameter in systole (LVID s) ∗P < 0.02, ∗∗P < 0.002; (G and Q) the left ventricular posterior wall in diastole (LVPW d) ∗P < 0.01, ∗∗P < 0.004, ∗∗∗P < 0.0007; (H and R) the left ventricular posterior wall in systole (LVPW s) ∗∗P < 0.009. Anova…

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  Fig. 2. LV of Tg-210 TAC mice showed increased expression levels of hypertrophy markers. RNA was extracted from left ventricles of Wt and Tg-210 TAC mice and the expression levels of hypertrophy markers were measured by qRT-PCR at day 7 (A–B) or 28 post surgery (C–D). (A and C) Natriuretic peptide A (Nppa); (B and D) Natriuretic peptide B (Nppb). Scatter plots represent mean ± SD. Values are expressed using a log2 scale (-ΔΔCt). Test t ∗P < 0.05, ∗∗P < 0.009.

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  Fig. 3. LV of Tg-210 TAC mice showed increased expression levels of macrophages markers. RNA was extracted from left ventricles of Wt and Tg-210 TAC mice and the expression levels of macrophages markers were measured by qRT-PCR at 7 (A–D) or 28 days from surgery (E–H). (A and E) CD68; (B and F) Galectin-3 (Lgals3); (C and G) CD80; (D–H) Interleukin-1beta (Il-1β). Scatter plots represent mean ± SD. Values are expressed using a log2 scale (-ΔΔCt). Test t ∗P < 0.05, ∗∗P < 0.009, ∗∗∗P < 0.0005.

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  Fig. 4. miR-210 overexpression increased tissue fibrosis after pressure overload. (A–B and E-F) Representative Sirius red staining of Wt and Tg-210 TAC LVs at day 7 (A–B) and 28 (E–F). Calibration bar 2 mm. Quantification of the percentage of Sirius red positive areas in the septum (C and G) and in the free wall (D and H) at 7 (C and D) and 28 days (G and H). Scatter plots represent mean ± SD. Mann Whitney test ∗P < 0.03. (I–P) RNA was extracted from LVs of Wt and Tg-210 TAC mice and the expression levels of fibrosis markers were measured by qRT-PCR at 7 (I–M) and 28 days (N–P) after surgery. (I and N) expression levels of Col1A1; (L and O) expression levels of Col3a1; (M and P) expression levels of Myh10. Scatter plots represent mean ± SD. Values are expressed using a log2 scale (-ΔΔCt). Test t ∗P < 0.02, ∗∗P < 0.002.

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  Fig. 5. Characterization of α-SMA expression in Wt and Tg-210 TAC LVs at day 7 and 28. (A–B) RNA was extracted from LVs of Wt and Tg-210 TAC mice and the expression level of the myofibroblast marker actin alpha 2, smoot muscle (Acta2) was measured by qRT-PCR at day 7 and 28 post surgery. Scatter plots represent mean ± SD. Values are expressed using a log2 scale (-ΔΔCt). Test t ∗P < 0.02, ∗∗P < 0.005. (C–F) Representative immunofluorescence staining for alpha-smooth muscle actin (α-SMA) of Wt and Tg-210 TAC LVs. α-SMA positive cells were stained in green. Nuclei were stained by Hoechst (Blue). α-SMA stains both myofibroblasts and smooth muscle cells in the tunica media of the arteries. The insets show the enlargement of selected regions. White arrows indicate arteries, white asterisks indicate myofibroblasts. Calibration bar 500 μm. (G–L) Quantification of the percentage of the α-SMA positive areas in the septum (G and I) or in the free wall (H and L) of Wt and Tg-210 TAC mice. (G–H) Quantification at day 7. (I–L) Quantification at day 28. Scatter plots represent mean ± SD. Test t ∗P < 0.01.

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  Fig. 6. miR-210 overexpression enhance CFs adhesion. CFs were seeded on gelatin coated support and adhesion was measured at the indicated times; after fixation, the cells were stained by Hoechst 33342 and quantified by fluorescence measurement. miR-210 over-expressing Tg-210 CFs show increased adhesion compared to Wt CFs. Data are presented as scatter plots showing the mean ± SD (n = 6; ∗∗P < 0.01).

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  Fig. 7. Enhanced wound healing properties of Tg-210 CFs. Wt and Tg-210 CFs were seeded (2 × 10⁴ cells per incucyte imagelock plate 96 well) and allowed to form a confluent monolayer over 24 h. Reproducible wounds were created and phase-contrast images were acquired every 2 h for 40 h. (A) Time-lapse phase images show migration of Wt and Tg-210 CFs over 24 h, with segmentation masks showing initial scratch (yellow) and wound closure over time. (B) Relative wound density (expressed in %) was automatically measured in 2-h intervals for a 40-h time span following the initiation of the scratch assay. A mixed-effects model, incorporating cross-replicate variability as a random effects variable, was used to compare wound healing rates over time between Tg-210 (N = 36) and Wt (N = 35) CFs (P = 0.039). Post-hoc tests were performed to assess differences at individual time points. Blue and red lines and squares correspond to Tg-210 and Wt CFs respectively. Error-bars represent Standard Error of the Mean (SEM). Estimated marginal means post-hoc tests, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Fig. 8. The migratory capacity of Tg-210 CFs is increased compared to Wt. To test chemotaxis, 1 × 103 CFs (N = 36) were seeded into the IncuCyte Clearview 96 well insert in growth medium containing 0.5 % FBS and 1 ng/mL doxycycline. A chemoattractant (10 % FBS medium) or control (PBS) was added to the reservoir plate. The plates were equilibrated at 37 °C and imaged every hour for 20 h with a 10× objective under phase contrast to assess cell motility and migration (total cells area, μm2/well) was assessed making automated measurements. Mixed effects models were fitted, incorporating cross-replicate variability as a random effect, to compare migration rates over time between Tg-210 and Wt CFs in the presence (P = 3.6 × 10−10) or absence (P = 2 × 10−16) of FBS. Post-hoc testing enabled evaluation of differences at individual time points. Blue and red lines correspond to Tg-210 and Wt genotypes respectively, while solid and dashed lines correspond to FBS presence and absence respectively. Error-bars represent Standard Error of the Mean (SEM). Estimated marginal means post-hoc tests, ∗P < 0.05.

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  Fig. 9. HIF pathway activation in LV of Tg-210 TAC mice. (A–E) RNA was extracted from LVs of Wt and Tg-210 TAC mice and the expression levels of the HIF-1 responsive genes Pgk1 (A), Glut-1 (B), Eno1 (C), Ppar-γ (D) and Bnip3 (E) were measured by qRT-PCR at day 28 after TAC. Scatter plots represent mean ± SD. Values are expressed using a log2 scale (-ΔΔCt). Test t ∗P < 0.04, ∗∗P < 0.005.