Arginyltransferase knockdown attenuates cardiac hypertrophy and fibrosis through TAK1-JNK1/2 pathway

Abstract

Myocardial hypertrophy, an inflammatory condition of cardiac muscles is a maladaptive response of the heart to biomechanical stress, hemodynamic or neurohormonal stimuli. Previous studies indicated that knockout of Arginyltransferase (ATE1) gene in mice and embryos leads to contractile dysfunction, defective cardiovascular development, and impaired angiogenesis. Here we found that in adult rat model, downregulation of ATE1 mitigates cardiac hypertrophic, cardiac fibrosis as well as apoptosis responses in the presence of cardiac stress i.e. renal artery ligation. On contrary, in wild type cells responding to renal artery ligation, there is an increase of cellular ATE1 protein level. Further, we have shown the cardioprotective role of ATE1 silencing is mediated by the interruption of TAK1 activity-dependent JNK1/2 signaling pathway. We propose that ATE1 knockdown in presence of cardiac stress performs a cardioprotective action and the inhibition of its activity may provide a novel approach for the treatment of cardiac hypertrophy.

Figure 1

Generation of cardiac hypertrophy and ATE1 knockdown in invitro and in invivo model Increase in mRNA levels of (A) ANP, (B) BNP, (C) β-MHC in Ang II treated H9C2 cells using Quantitative real-time PCR analysis (D) Graph showing significant reduction of ATE1 levels when knockdown by ATE1 siRNA compare to NS siRNA. Quantitative real-time PCR analysis of increased mRNA levels of (E) ANP, (F) BNP and (G) β-MHC in the heart samples of control (Sham) vs Renal artery ligated rat samples (Ligated). Experiment performed in triplicates and normalized to GAPDH content. Statistical analysis is carried out by Student’s two tailed unpaired T test. Data are represented as mean ± SE.

Figure 2

ATE1 expression is upregulated by hypertrophic stimuli. (A) Quantitative real-time PCR analysis of mRNA levels of ATE1 in Ang II treated H9C2 cells. (B) Transcriptional levels of ATE1 in heart samples from rat subjected to ligation of right renal artery (Ligated) and sham-operated control (Sham) rats. (C) Western blot analysis of ATE1 protein levels in heart samples from sham and renal ligated rats. Data were derived from experiments performed in triplicate and normalized to GAPDH content. Statistical analysis was carried out by student’s two-tailed t-test (*shows non-specific binding of antibody).

Figure 3

Knockdown of ATE1 attenuates cardiac hypertrophy. Real-time PCR analysis showing the mRNA levels of fetal gene (A) ANP in Ang II treated ATE1 knockdown H9C2 cells Decreased mRNA levels in (B) ANP (B1) Western blot analysis of ANP protein levels in heart samples from sham and renal ligated rats (B2) bar graph was generated by quantifying the blots and normalizing the intensities of bands to the untreated lane (C) BNP and (D) β-MHC in Ligated ATE1 siRNA when compared with Ligated NS siRNA samples. Statistical analysis was carried out by one-way ANOVA. Data are represented as mean ± SE.

Figure 4

Cardiac ATE1 deficiency restores cardiac dysfunction after right renal artery ligation. Measurements of echocardiographic parameters (A) LVDd and (B) % FS in the indicated groups.

Figure 5

ATE1 knockdown is involved in TAK1-JNK1/2 pathway. Representative western blots and their quantification showing the protein level of (A,A2) P-TAK1, (B,B2) TAK-1 (C,C2) P-JNK1/2 (D,D2) P- ERK1/2 (E,E2) ERK-1/2 in Ligated ATE1 Si RNA and ligated NS siRNA. Bar graph was generated by quantifying the blots and normalizing the intensities of bands to the untreated lane.

Figure 6

ATE1 knockdown impairs the TGF-β1 Smad signaling (A) TGF-β1 (B) Smad7 (C) Smad3 (D) Smad4 in (Ligated ATE1 siRNA) vs Ligated + NS siRNA (C1,D1) Western blot analysis of SMAD3 and SMAD4 protein levels in heart samples from (Ligated ATE1 siRNA) vs Ligated + NS siRNA (C2,D2) bar graph was generated by quantifying the blots and normalizing the intensities of bands to the untreated lane. Data derived from qRT-experiments performed in triplicate and normalized to GAPDH content. Data are represented as mean ± SE.

Figure 7

Quantitative real-time PCR analyses of fibrosis gene markers mRNA expression of (A) Collagen 3 (B) osteopontin (B1) Western blot analysis of osteopontin protein levels in heart samples from (Ligated ATE1 siRNA) vs Ligated + NS siRNA (B2) bar graph was generated by quantifying the blots and normalizing the intensities of bands to the untreated lane (C) CCN2 in the indicated groups. Data derived from independent experiments performed in triplicate and normalized to GAPDH content. Data are represented as mean ± SE.

Figure 8

Knockdown of ATE1 promoted cardiac apoptosis (A,A1) Western and quantitative analysis of protein levels of active caspase 3 in Ligated ATE1 siRNA and Ligated NS siRNA. GAPDH was used as loading control. Bar graph was generated by quantifying the blots and normalizing the intensities of bands to the untreated lane. (B) Graph representing the fluorescence values of cells treated with and without Ang II in the ATE1 knockdown H9C2 samples.