RUNX1 promotes cardiac fibrosis and exacerbates myocardial infarction by transcriptionally activating PTN expression

Figures

• 

  Figure 1. RUNX1 expression increases following MI in mice. (A) Volcano plot showing 75 upregulated and 21 downregulated genes in infarcted versus sham hearts (|log2fold change| >1, P<0.05). (B) Heatmap displaying expression patterns of the top 20 upregulated genes. (C) KEGG enrichment analysis showing significant enrichment of ECM deposition and fibrosis pathways. *, P<0.05. (D) RUNX1 protein expression was significantly upregulated in infarcted myocardium (n=6). ***, P<0.001. Data are presented as mean ± SEM. CPM, counts per million; ECM, extracellular matrix; FC, fold change; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; MI, myocardial infarction; RUNX1, runt-related transcription factor 1; SEM, standard error of the mean.

• 

  Figure 2. RUNX1 knockdown improved cardiac function and cardiac fibrosis following MI in adult mice. (A) Western blot analysis of RUNX1 expression in cardiac fibroblasts of mice and quantification of the indicated proteins (n=6/group). (B,C) Immunofluorescence staining analysis showing RUNX1, GFP, and vimentin co-staining to confirm knockdown efficiency of the gene transfection system (n=6/group; scale bar =100 µm). (D) Cardiac function was assessed by echocardiography at pre-MI and 28 days after MI (n=6/group). (E) Representative images of H&E and Masson’s trichrome staining of mouse hearts (n=6/group; scale bar =1 mm). (F) Representative immunohistochemical staining of α-SMA and RUNX1 in transverse sections of hearts from the adult mouse MI model at 28 days after MI (n=6/group; scale bar =50 µm). (G) Western blot analysis of α-SMA, COL1A1, and COL3A1 protein levels in fibroblasts. The cells were isolated from the hearts of mice with MI (n=6/group). ***, P<0.001. Data are presented as mean ± SEM. α-SMA, α-smooth muscle actin; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; H&E, hematoxylin…

• 

  Figure 3. RUNX1 regulates cardiac fibroblast activation. (A) The mRNA levels of Col3A1, Col1A1, α-SMA, and Runx1 were measured by qPCR (n=3/group). (B) Western blot analysis of siRUNX1 transfection efficiency and the protein levels of COL3A1, COL1A1, and α-SMA after RUNX1 knockdown (n=3/group). (C) Representative images of wound-healing assays in fibroblasts transfected with siRunx1. Images of the wounds were captured at 0 and 24 hours. The area between the wound edges was measured and compared between the groups (scale bar =200 µm; n=3/group). (D) Cells were fixed with 4% paraformaldehyde for 30 min, followed by staining with crystal violet at room temperature for 1 h. Representative images of Transwell migration assay evaluating NRCF migration (scale bar =200 μm; n=3/group). *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM. α-SMA, α-smooth muscle actin; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; mRNA, messenger RNA; NRCF, neonatal rat cardiac fibroblast; qPCR, quantitative polymerase chain reaction; RUNX1, runt-related transcription factor 1; SEM, standard error of the mean; siRUNX1, small interfering RNA RUNX1; TGF-β,…

• 

  Figure 4. RNA-sequencing indicates that PTN is involved in ventricular remodeling regulation by RUNX1. (A) Volcano plot of differentially expressed genes in NRCFs after RUNX1 knockdown. (B) KEGG pathway analysis of differentially expressed genes. (C,D) Western blot analysis of PTN protein levels in myocardial tissue from MI mice (n=6; ***, P<0.001). (E,F) Western blot analysis of PTN protein levels in siRunx1-transfected NRCFs (n=3). (G,H) qPCR quantification of Runx1 and Ptn mRNA levels in siRunx1-transfected NRCFs (n=3). (I) Predicted RUNX1-binding sites in the PTN promoter identified using the JASPAR database. (J) ChIP-qPCR confirming RUNX1 occupancy at the Ptn promoter. (K) Dual-luciferase reporter assay demonstrating that RUNX1 positively regulates Ptn transcription. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. Data are presented as mean ± SEM. ChIP, chromatin immunoprecipitation; FC, fold change; IgG, immunoglobulin G; KEGG, Kyoto Encyclopedia of Genes and Genomes; MI, myocardial infarction; mRNA, messenger RNA; Mut, mutant; NRCF, neonatal rat cardiac fibroblast; oe, overexpression; PTN, pleiotrophin; qPCR, quantitative polymerase chain reaction; RUNX1, runt-related…

• 

  Figure 5. RUNX1 promotes cardiac fibroblast activation via PTN regulation. Western blot analysis of COL3A1, COL1A1, α-SMA, RUNX1, and PTN protein levels in NRCFs after RUNX1 overexpression alone or in combination with PTN-siRNA transfection (n=3). *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. Data are presented as mean ± SEM. α-SMA, α-smooth muscle actin; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; Con, control; NRCF, neonatal rat cardiac fibroblast; oe, overexpression; PTN, pleiotrophin; RUNX1, runt-related transcription factor 1; SEM, standard error of the mean; siRNA, small interfering RNA.