Runt‐related transcription factor 1 (Runx1) aggravates pathological cardiac hypertrophy by promoting p53 expression

Figures

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  FIGURE 1. Runx1 expression in cardiac hypertrophy. (A) Representative mRNA levels of Runx1 and Anp (atrial natriuretic peptide) in heart tissues from sham or transverse aortic constriction (TAC) mice. n = 6 mice per group. (B) Representative immunoblot images and statistics of the protein levels of Runx1 and Anp (atrial natriuretic peptide) in heart tissues from sham or transverse aortic constriction (TAC) mice. n = 6 mice per group. *P < .05, **P < .01

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  FIGURE 2. Knockdown of Runx1 alleviates cardiomyocyte hypertrophy induced by Ang II in NRCMs. (A) Left, representative images of NRCMs infected with the indicated adenoviruses and treated with saline or Ang II for 48 h. Cardiomyocytes were identified by a‐actinin staining (green), and nuclei were stained with DAPI (blue). (Scale bars, 50 μm). Right, quantitative results of the cell surface area of NRCMs (n ≥ 200 cells/group). (B) Representative mRNA levels of Anp and Myh7 in the indicated group. n = 4 samples per group. *P < .05, **P < .01

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  FIGURE 3. Cardiomyocyte‐specific Runx1 knockdown alleviates pressure overload–induced cardiac hypertrophy. (A) Statistical results of echocardiographic parameters in the indicated groups (n = 8‐10 mice per group). (B) Upper, representative images and of haematoxylin and eosin (HE)–stained heart sections from the indicated groups (scale bar, 50μm.), (n = 5‐7 mice per group). Low, statistical results of cardiomyocyte cross‐sectional area in the indicated groups, (n ≥ 50 fields per group). (C) Left, representative images of picro Sirius red (PSR)–stained heart sections from the indicated groups (scale bar, 100 μm.), (n = 5‐7 mice per group). Right, statistical results of left ventricle collagen volume determined by picro Sirius red staining. (D) The relative mRNA levels of atrial natriuretic peptide (Anp), B‐type natriuretic peptide (Bnp), and Myh7 in the left ventricle of mice from the indicated groups (n = 4 mice per group). (E) The relative mRNA levels of collagen I (Col I), collagen III (Col III) and connective tissue growth factor (CTGF) in the left ventricle of mice from the indicated groups (n = 4 mice per group). *P < .05, **P < .01

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  FIGURE 4. Runx1 contributes to cardiac hypertrophy by activating p53 signalling pathway. (A) Representative Western blots showing the protein levels of Runx1, p53, Noxa, Bax and Puma in mice heart tissues in the indicated groups, (n = 4 mice per group)

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  FIGURE 5. Analyses of transcriptional regulation of p53 by Runx1. (A) Representative mRNA level of Runx1 in neonatal rat cardiomyocytes (NRCMs) treatment with Ad Runx1 or Ad shRunx1 for 24 h (n = 3 samples per group). (B) Schematic images of PGL3‐p53‐Luc expression vectors containing the relevant mutations. (C) pcDNA‐Runx1 and PGL3‐p53‐Luc expression vectors containing the relevant mutations were co‐transfected into HEK293T cells. Cell lysates were collected at 24 h, and luciferase activity was measured using a luciferase reporter assay. (D) Schematic diagram of the primer locations of P1, P2, P3 and P4. (E) Quantitative PCR (qPCR) results of the chromatin immunoprecipitation (ChIP) assay results. (F) Relative values of the corresponding luciferase activities. *P < .05, **P < .01

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  FIGURE 6. p53 is required for Runx1‐induced cardiomyocyte hypertrophy in vitro. (A) Representative mRNA level of p53 was detected after treatment with the indicated adenovirus for 24 h (n = 3 per group). (B) Representative images of neonatal rat cardiomyocytes (NRCMs) infected with the indicated adenoviruses and treated with Saline or Ang II for 48h. Cardiomyocytes were identified by a‐actinin staining (green), and nuclei were stained with DAPI (blue) (scale bars, 50 μm). (C) Quantitative results of the cell surface area of NRCMs (n ≥ 200 cells/group). (D) Representative mRNA levels of atrial natriuretic peptide (Anp), B‐type natriuretic peptide (Bnp), and Myh7 in the indicated groups. n = 4 samples per group. *P < .05, **P < .01

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  FIGURE 7. Ro5‐3335 inhibits pathological cardiac hypertrophy. (A) Statistical results of echocardiographic parameters in the indicated groups (n = 7‐10 mice per group). (B) Upper, representative images of haematoxylin and eosin (HE)–stained heart sections from the indicated groups (scale bar, 50 μm) (n = 4‐6 mice per group). Low, statistical results of cardiomyocyte cross‐sectional area in the indicated groups (n ≥ 50 fields per group). (C) Left, representative images of picrosirius red (PSR)–stained heart sections from the indicated groups (scale bar, 100 μm.) (n = 4‐6 mice per group). Right, statistical results of left ventricle collagen volume determined by picrosirius red staining. (D) The relative mRNA levels of atrial natriuretic peptide (Anp) and Myh7 in the left ventricle of mice from the indicated groups (n = 4 mice per group). (E) The relative mRNA levels of collagen I (Col I), collagen III (Col III) and connective tissue growth factor (CTGF) in the left ventricle of mice from the indicated groups (n = 4 mice per group). *P < .05, **P < .01