Myocardial fibrosis and calcification are attenuated by microRNA–129-5p targeting Asporin and Sox9 in cardiac fibroblasts

Figures

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  Figure 1. Bioinformatic analysis reveals downregulated miR–129-5p expression and upregulation of its targets SOX9 and ASPN in nonischemic HF LV. (A) Bioinformatic analysis pipeline. (B) Validation of miR–129-5p, Sox9, and Aspn transcript expression in LV of TAC versus sham mice by qPCR. (C) Validation of miR–129-5p, Sox9, and Aspn transcript expression by qPCR in LV of mice after chronic infusion of AngII or saline. (D) Representative image of miR–129-5p expression in CF in a healthy mouse heart as assessed by FISH with CF marker Ddr2. Arrows indicate miR–129-5p and Ddr2 colocalization. Scale bar: 10 μm. (E) Representative plot of flow cytometry–based assessment of miR–129-5p and Ddr2 expression in a healthy mouse heart. Repeated in hearts of 3 different mice. Data presented as mean ± SEM; n = 4–5 mice/group. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. AngII, Angiotensin II; Ddr2, Discoidin domain-containing receptor 2; DEG, differentially expressed gene; TAC, transverse aortic constriction.

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  Figure 2. miR–129-5p is downregulated and SOX9 and ASPN are upregulated in CF of patients with HF with LV fibrosis and calcification. (A) miR–129-5p (red) expression in CF assessed by FISH with CF marker Ddr2 (green). Scale bar: 20 μm. Arrows are miR-129-5p+ Ddr2+ cells. (B) Interstitial myocardial fibrosis (blue) assessed by Masson’s trichrome staining. Scale bar: 100 μm. (C) Calcification (black) in myocardial fibrotic areas assessed by von Kossa staining. Scale bar: 50 μm. Arrows are calcified fibrotic areas. (D) Nuclear expression of SOX9 (red) in COL1a1-expressing cells (green) — i.e., CF, assessed by immunofluorescence. Scale bar: 20 μm. Arrows are COL1a1+SOX9+ cells. (E) Intra- and extracellular ASPN (pink) expression in COL1a1-expressing cells (green) — i.e., CF, assessed by immunofluorescence. Scale bar: 20 μm. Arrows are COL1a1+ ASPN+ cells. (B–E) Correlations between the miR–129-5p/Ddr2 ratio and interstitial fibrosis, calcified interstitial fibrosis, percentage of SOX9-expressing COL1a1+ cells, and percentage of ASPN expressing COL1a1+ cells in human LV. Data are presented as mean ± SEM; n = 3–4 subjects/group. Student’s t test. Correlations tested by Pearson’s r. *P < 0.05,…

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  Figure 3. miR–129-5p inhibits both CF-to-MF and CF-to-OF transition. (A) miR–129-5p expression in CF upon AngII or TGF-β stimulation as assessed by qPCR. (B) miR–129-5p expression in CF upon miR–129-5p inhibition as assessed by qPCR. (C and D) AngII- and TGF-β–induced expression of Postn, Runx2, and Alp3 transcripts in CF upon miR–129-5p inhibition as assessed by qPCR. (E) miR–129-5p expression in CF upon miR–129-5p mimic overexpression as assessed by qPCR. (F) AngII- and TGF-β–induced CF proliferation upon miR–129-5p mimic overexpression as assessed by CCK8 assay. (G) CF migration upon miR–129-5p mimic overexpression as assessed by scratch wound assay. Original magnification ×10. (H) AngII- and TGF-β–induced CF collagen production upon miR–129-5p mimic overexpression as assessed by soluble Sirius red assay. (I) AngII- and TGF-β–induced αSMA expression in CF upon miR–129-5p mimic overexpression as assessed by immunofluorescence. Scale bar: 10 μm. Original magnification ×40. (J) Osteogenic growth medium–induced calcium nodules (dark red, arrows) in CF upon miR–129-5p mimic overexpression as assessed by alizarin red staining. Scale bar: 20 μm. Original magnification ×20. (K and L) AngII- and…

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  Figure 4. SOX9 and ASPN are direct targets of miR–129-5p. (A) AngII- and TGF-β–induced expression of Sox9 and Aspn transcripts in CF upon miR–129-5p inhibition as measured by qPCR. (B) AngII- and TGF-β-induced expression of Sox9 and Aspn transcripts in CF upon miR–129-5p mimic overexpression as measured by qPCR. (C) SOX9 and ASPN protein expression in CF upon miR–129-5p mimic overexpression with representative Western blots. (D) Luciferase activity upon NC or miR–129-5p mimic overexpression in HEK293T cells expressing Sox9 and Aspn constructs containing WT and mutant (MUT) miR–129-5p seed regions. (E) Expression of MF marker Postn and OF markers Runx2 and Alp3 in CF upon miR–129-5p inhibition in presence of control, Sox9, or Aspn siRNA as measured by qPCR. Data are presented as mean ± SEM; n = 3–4 independent experiments. Student’s t test (C) or ANOVA with Holm-Bonferroni post hoc correction.#P < 0.05 versus NC mimic ctrl stimulus, ##P < 0.01 versus NC mimic ctrl stimulus,;*P < 0.05, **P < 0.01, ***P < 0.001. AngII, Angiotensin II; NC, negative control.

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  Figure 5. SOX9 and ASPN promote both CF-to-MF and CF-to-OF transition. (A) AngII- and TGF-β–induced expression of Sox9 and Aspn transcripts in CF upon Sox9 or Aspn knockdown as assessed by qPCR. (B) SOX9 and ASPN protein expression in CF upon Sox9 or Aspn knockdown with representative Western blots. (C) AngII- and TGF-β–induced CF proliferation upon Sox9 or Aspn knockdown as assessed by CCK8 assay. (D) CF migration upon Sox9 or Aspn knockdown as assessed by scratch wound assay. (E) AngII- and TGF-β–induced CF collagen production upon Sox9 or Aspn knockdown as assessed by soluble Sirius red assay. (F) AngII- and TGF-β–induced αSMA expression in CF upon Sox9 or Aspn knockdown as assessed by immunofluorescence. Scale bar: 10 μm. (G) Osteogenic growth medium–induced calcium nodules (dark red, arrows) in CF upon Sox9 or Aspn knockdown as assessed by alizarin red staining. Scale bar: 20 μm. (H and I) AngII- and TGF-β–induced expression of Postn, Runx2, and Alp3 transcripts in CF upon Sox9 or Aspn knockdown as assessed by qPCR. (J) Expression of active β-catenin in CF upon Sox9 or Aspn knockdown as assessed by Western blot. Data presented as mean ± SEM; n = 3–4 independent experiments. Student’s…

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  Figure 6. miR–129-5p overexpression attenuates progression of AngII-induced fibrosis and calcification in mice and restores cardiac function. (A) Experimental setup for the chronic AngII infusion model. Arrows indicate tail-vein injections of NC mimic or miR–129-5p mimic. (B) LV miR–129-5p expression assessed by qPCR. (C) Cardiomyocyte hypertrophy assessed by wheat germ agglutinin staining. (D) Cardiac hypertrophy assessed by heart weight/tibia length (HW/TL) ratio. (E) Systolic function assessed by LV ejection fraction (EF) on serial echocardiography. (F) Diastolic function assessed by E’/A’ ratio on serial tissue Doppler imaging. (G and I) miR–129-5p (red) expression in LV CF assessed by FISH with CF marker ddr2 (green). Scale bar: 10 μm. (H and J) Interstitial myocardial fibrosis (blue) assessed by Masson’s trichrome staining. (K) LV expression of osteogenic markers Runx2 and Alp3 transcripts by qPCR. Data are presented as mean ± SEM; n = 6–8 mice/group. ANOVA with Holm-Bonferroni post hoc correction; *P < 0.05, **P < 0.01, ***P < 0.001. ddr2, discoidin domain-containing receptor 2; CSA, cross-sectional area; NC, negative control.

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  Figure 7. miR–129-5p rescues AngII-induced Sox9 and Aspn expression in CF in vivo. (A) Experimental setup for lineage tracing in the chronic AngII infusion model in mice. Arrows indicate tail-vein injections of NC mimic or miR–129-5p mimic. (B–D) SOX9 and ASPN expression in LV CF of the TCF21 lineage (eGFP+) upon chronic AngII infusion and NC or miR–129-5p mimic rescue injections, assessed by immunofluorescence. Scale bar: 10 μm. Data presented as mean ± SEM; n = 4–6 mice/group. ANOVA with Holm-Bonferroni post hoc correction. **P < 0.01. eGFP, enhanced green fluorescent protein; NC, negative control; Tcf21, transcription factor 21.