Dual-specificity Phosphatase 9 protects against Cardiac Hypertrophy by targeting ASK1

Figures

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  Figure 1. DUSP9 expression was elevated in hypertrophic hearts and NRVM. (A) The mRNA level of DUSP9 in heart tissue after TAC or sham procedures at specified time point (n = 3 per group). (B) The mRNA level of DUSP9 in NRVM treated with PBS or angiotensin II (Ang II, 1 µM) for 48 h. (C) Western blotting assay and DUSP9 protein level quantification in the mice heart after four weeks of TAC or sham procedures (n = 3 per group). (D) Western blotting, and quantification of DUSP9 protein level in NRVM treated with PBS or angiotensin II (Ang II, 1 µM) for 48 h. Data are presented as the mean ±SD. *P<0.05, **P<0.01; NRVM refers to neonatal rat ventricular myocytes.

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  Figure 2. Cardiac DUSP9 deficiency exacerbated TAC-induced cardiac hypertrophy. (A) Representative western blot results to validate DUSP9 expression in αMHC-MCM, DUSP9-Flox and DUSP9-CKO mice (n = 6 per experimental group). (B-D) Statistical data of the ratios of Heart weight to body weight (HW/BW, B), lung weight to body weight (LW/BW, C) and heart weight to tibia length (HW/TL, D) in different genotypical mice (αMHC-MCM, DUSP9-Flox and DUSP9-CKO) at four weeks after sham or TAC procedures (n = 10 mice per group) (E, F) Histological analysis of gross morphology and left ventricular muscle of demonstrated groups four weeks after TAC surgery or sham procedures (n = 6 mice per group; scale bar, 50 µm). (G-J) Echocardiographic assessment of left ventricular end-diastolic diameter (LVEDd, G), left ventricular end-systolic diameter (LVESd, H), left ventricular fractional shortening (FS%, I) and left ventricular ejection fraction (EF%, J) in the demonstrated groups at four weeks after either sham or TAC procedures (n = 10 mice per group). (K, L) Representative imaging of PSR staining of perivascular and myocardial interstitial sections of the hearts from the demonstrated groups (n = 6 mice per…

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  Figure 3. Cardiac-specific DUSP9 overexpression attenuated TAC-induced cardiac hypertrophy. (A) Schematic graphic illustrating the establishment of cardiac-specific DUSP9 transgenic mouse strains. (B) Cardiac DUSP9 expression in transgenic mice and non-transgenic littermates (n = 4 per group). (C-E) Statistical data of the ratios of HW/BW (C), LW/BW (D), HW/TL (E), in non-transgenic and transgenic mice at four weeks after sham or TAC surgery (n = 10 mice per group) (F, G) Histological images of the gross morphology and left ventricular muscle of heart stained with HE from the demonstrated groups four weeks after TAC surgery or sham procedures (n = 6 mice per group; scale bar, 50 µm) (H-K) Parameters of the echocardiographic assessment for LVEDd (H), LVESd (I), left ventricular FS% (J), and left ventricular EF% (K), in the demonstrated groups (n = 10 mice per group). (L, M) Representative imaging of PSR staining of perivascular and myocardial interstitial sections of the hearts from the demonstrated groups (n = 6 mice per group; scale bar, 100 µm) (N, O) The mRNA levels of hypertrophic biomarkers (Anp, Bnp, Myh7) and fibrotic markers (collagen I, collagen III, Ctgf) in the demonstrated…

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  Figure 4. DUSP9 repressed Ang II-induced NRVM hypertrophy in vitro. (A) Respective western blots showing DUSP9 expression in NRVM infected with adenoviral vectors with shDUSP9 or controls (B, C) Representative immunofluorescence images of NRVM stained with α-actinin (green) and DAPI (blue) after infection by adenovirus of vectors with shDUSP9 or controls followed with a 48h-challenge of PBS or Ang II (1 µM). (D) The mRNA level of hypertrophic marker genes (Anp and Myh7) in NRVM after adenoviral vectors with shDUSP9 or controls infections undergoing Ang II (1 µM) or PBS treatment for 48 h (E) Respective western blots showing DUSP9 expression in NRVM infected with adenoviral vectors with Flag-DUSP9 or controls. (F, J) Representative immunofluorescence images of NRVM stained with α-actinin (green) and DAPI (blue) after the infections with adenoviral vectors with Flag-DUSP9 or controls followed by 48 h of PBS or Ang II (1 µM) challenge. (H) The mRNA levels of hypertrophic biomarkers (Anp and Myh7) in NRVM infected with adenoviral vectors with Flag-DUSP9 or controls undergoing Ang II (1 µM) or PBS for 48 h. Data are presented as the mean ±SD. *P<0.05, **P<0.01; NRVM refers to neonatal rat…

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  Figure 5. DUSP9 modulated cardiac hypertrophy via the ASK1-p38/JNK1/2 signaling axis. (A, B) Representative western blots and quantification of both phosphorylated and total protein levels of ASK1, p38, ERK1/2 and JNK in the hearts of control and DUSP9-CKO (A) or NTG and DUSP9-TG mice (B) four weeks after TAC treatment or sham (n = 4 mice per group) (C, D) Representative western blots and quantification of phosphorylated and total protein levels of ASK1, p38, ERK and JNK in NRVM with DUSP9 knockdown (C) or DUSP9 overexpression (D) after 48 h of PBS or Ang II (1 µM) challenge. Data are presented as the mean ±SD. *P<0.05, **P<0.01, n.s. means no significant difference; NRVM refers to neonatal rat ventricular myocytes.

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  Figure 6. DUSP9 regulated Ang II-induced cardiac hypertrophy by binding to ASK1. (A) Immunofluorescence staining of DUSP9 (red), ASK1 (green) and nuclei (blue) in HEK293T cells. (B) Co-immunoprecipitation assay was performed in HEK293T cells that co-transfected with Flag-DUSP9 and HA-ASK1 to measure the interaction between DUSP9 and ASK1. (C) Co-immunoprecipitation assay in NRVM infected with Flag-DUSP9 to exam the interaction between DUSP9 and endogenous ASK1. (D) Western blots of DUSP9 and phosphorylated ASK1 protein level in DUSP9 knockdown NRVM treated with DMSO or iASK1 along with Ang II (1 µM) insult for 48 h. (E, F) Representative fluorescent images and quantification of cell surface area of DUSP9 knockdown NRVM with DMSO or iASK1 with Ang II (1 µM) stimulation for 48 h (Scale bar = 20 µm) (G) mRNA levels of hypertrophic biomarkers (Anp and Myh7) in DUSP9 knockdown NRVM undergoing Ang II (1 µM) treatment for 48 h along with DMSO or iASK1. Data are presented as the mean ±SD. *P<0.05, **P<0.01; NRVM refers to neonatal rat ventricular myocytes.