Zinc-finger protein 418 overexpression protects against cardiac hypertrophy and fibrosis

Abstract

Background: This study aimed to investigated the effect and mechanism of zinc-finger protein 418 (ZNF418) on cardiac hypertrophy caused by aortic banding (AB), phenylephrine (PE) or angiotensin II (Ang II) in vivo and in vitro.

Methods: The expression of ZNF418 in hearts of patients with dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) and AB-induced cardiac hypertrophy mice, as well as in Ang II- or PE-induced hypertrophic primary cardiomyocytes was detected by western blotting. Then, the expression of ZNF418 was up-regulated or down-regulated in AB-induced cardiac hypertrophy mice and Ang II -induced hypertrophic primary cardiomyocytes. The hypertrophic responses and fibrosis were evaluated by echocardiography and histological analysis. The mRNA levels of hypertrophy markers and fibrotic markers were detected by RT-qPCR. Furthermore, the phosphorylation and total levels of c-Jun were measured by western blotting.

Results: ZNF418 was markedly down-regulated in hearts of cardiac hypertrophy and hypertrophic primary cardiomyocytes. Down-regulated ZNF418 exacerbated the myocyte size and fibrosis, moreover increased the mRNA levels of ANP, BNP, β-MHC, MCIP1.4, collagen 1a, collagen III, MMP-2 and fibronection in hearts of AB-treated ZNF418 knockout mice or Ang II-treated cardiomyocytes with AdshZNF418. Conversely, these hypertrophic responses were reduced in the ZNF418 transgenic (TG) mice treated by AB and the AdZNF418-transfected primary cardiomyocytes treated by Ang II. Additionally, the deficiency of ZNF418 enhanced the phosphorylation level of c-jun, and overexpression of ZNF418 suppressed the phosphorylation level of c-jun in vivo and in vitro.

Conclusion: ZNF418 maybe attenuate hypertrophic responses by inhibiting the activity of c-jun/AP-1.

Fig 1. ZNF418 expression is decreased in hearts of patients withdilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) as well as cardiac hypertrophy mice.

(A) Expression levels of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and Zinc-finger protein 418 (ZNF418) in donor hearts and patients hearts by western blotting analysis (n = 4 samples per group, *P < 0.05 vs. donor hearts); (B) The protein levels of ANP, β-MHC and ZNF418 in hearts of wild-type mice after sham or aortic binding (AB) surgery by western blotting analysis (n = 4 mice per group, *P < 0.05 vs. sham hearts); (C) Expression levels of ANP, β-MHC and ZNF418 in primary cardiomyocytes treated with angiotensin II (Ang II, 1 μM) or phenylephrine (PE, 100 μM), or phosphate buffered solution (PBS) for 48 h by western blotting analysis (n = 3 samples per group, *P < 0.05 vs. PBS).

Fig 2. ZNF418 overexpression suppresses angiotensin II (Ang II)–induced cardiomyocyte hypertrophy.

(A) Primary cardiomyocytes infected with AdZNF418, AdshZNF418, or their respective controls (AdGFP and AdshRNA) were analyzed by western blotting (n = 3 independent experiments, *P < 0.05 vs. AdGFP or shRNA); (B) Representative images of cardiomyocytes that have been infected with AdshZNF418 or AdZNF418 after treatment with Ang II (1 μM) for 48 h by the immunofluorescence analysis of α-actinin. Blue: nuclear; Green: α-actinin; Scale bar, 20 μm; (C) Cell surface area of cardiomyocytes that have been infected with AdshZNF418 (left) or AdZNF418 (right), as well as treated with Ang II for 48 h (n = 4 independent experiments, *P < 0.05 vs. AdGFP, AdZNF418, AdshRNA or AdshZNF418/PBS, ^#P < 0.05 vs. AdGFP or AdshRNA/Ang II); (D) The results of Real-time quantitative PCR showed the hypertrophy markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in ZNF418 overexpressed (above) and knockdown (below) cardiomyocytes after PBS or Ang II treatment (n = 4 independent experiments, *P < 0.05 vs. AdGFP, AdZNF418, AdshRNA or AdshZNF418/PBS, ^#P < 0.05 vs. AdGFP or AdshRNA/Ang II).

Fig 3. Loss of ZNF418 aggravates aortic banding (AB)-induced hypertrophy.

(A) The expression of ZNF418 in the hearts form wild-type (WT) and ZNF418 knockout (KO) mice by western blotting; (B) Statistical results for the percentage of heart weight/body weight (HW/BW), lung weight (LW)/BW and HW/tibial length (TL) (n = 13 for each group, *P < 0.05 vs. WT/sham; ^#P < 0.05 vs. WT/AB); (C) Statistical results for the parameters of the echocardiographic results, including left ventricle end-diastolic dimension (LVEDD), left ventricle end-systolic diameter (LVESD), ejection fraction (EF) and fraction shortening (FS) in WT and KO mice (n = 6–7 mice per experimental group); (D) Histological analyses of the cross sectional area of hearts form WT and ZNF418 KO mice after sham treatment or AB surgery for 4 weeks were stained with HE and WGA (n = 7 mice per group; scale bar, 100 μm for top HE staining, scale bar, 20 μm for middle HE staining and lower WGA staining); (E) Picrosirius red staining on histological sections from the left ventricles of WT and KO mice hearts after sham or AB surgery for 4 weeks (n = 7–8 mice per experimental group; Scale bars, 20 μm). The fibrotic areas of individual sections were quantified using Image-Pro Plus 6.0 software; (F) The mRNA expression levels of fibrotic markers-collagen 1a, collagen III, matrix metalloproteinase-2 (MMP-2), Fibronectin in the hearts of WT and KO mice were detected with real-time quantitative PCR after sham and AB surgery for 4 weeks (n = 5,*P < 0.05 vs. WT/sham or KO/sham, ^#P < 0.05 vs. WT/AB); (G) The results of real-time quantitative PCR showed the mRNA levels of hypertrophy markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC) and modulatory calcineurin-interacting protein1.4 (MCIP1.4), in ZNF418KO and WT mice hearts after AB or sham treatment for 4 weeks (n = 5–6 independent experiments, *P < 0.05 vs. WT/sham or KO/sham, ^#P < 0.05 vs. WT/AB).

Fig 4. Overexpression of ZNF418 protects against aortic banding (AB)-induced hypertrophy.

(A) The expression of ZNF418 in the hearts form wild-type (WT) and ZNF418 transgenic (TG) mice by western blotting; (B) Statistical results for the percentage of heart weight/body weight (HW/BW) and HW/tibial length (TL) (n = 13–15 for each group, *P < 0.05 vs. WT/sham; ^#P < 0.05 vs. WT/AB); (C) Statistical results for the parameters of the echocardiographic results, including left ventricle end-diastolic dimension (LVEDD), left ventricle end-systolic diameter (LVESD), ejection fraction (EF) and fraction shortening (FS) in WT and TG mice after sham or AB surgery for 4 weeks (n = 6 mice per experimental group); (D) Histological analyses of the cross sectional area of hearts form WT and ZNF418 TG mice after sham or AB surgery for 4 weeks were stained with HE and WGA (n = 9 mice per group; scale bar, 100 μm for top HE staining, scale bar, 20 μm for middle HE staining and lower WGA staining); (E) Picrosirius red staining on histological sections from the left ventricles of WT and TG mice hearts after sham or AB surgery for 4 weeks (n = 7–8 mice per experimental group; scale bars, 20 μm.). The fibrotic areas of individual sections were quantified using Image-Pro Plus 6.0 software; (F) The mRNA expression levels of fibrotic markers-collagen 1a, collagen III, matrix metalloproteinase-2 (MMP-2), Fibronectin in the hearts of WT and TG mice were detected with real-time quantitative PCR after sham and AB surgery for 4 weeks (n = 5–7, *P < 0.05 vs. WT/sham or TG/sham, ^#P < 0.05 vs. WT/AB); (G) The results of real-time quantitative PCR showed the mRNA levels of hypertrophy markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC) and modulatory calcineurin-interacting protein1.4 (MCIP1.4), in ZNF418 TG and WT mice hearts after AB or sham treatment for 4 weeks (n = 5 independent experiments, *P < 0.05 vs. WT/sham or TG/sham, ^#P < 0.05 vs. WT/AB).

Fig 5. ZNF418 regulates c-Jun/AP-1 pathway in the heart.

(A) The expressions of phosphorylated and total c-jun in samples of primary cardiomyocytes that were infected with AdshZNF418 or AdshRNA and then stimulated with angiotensin II (Ang II) for 48 h (n = 3, *P < 0.05 vs. AdshRNA; ^#P < 0.05 vs. AdshRNA/Ang II); (B) The expressions of phosphorylated and total c-jun in samples of primary cardiomyocytes that were infected with AdZNF418 or AdGFP and then stimulated with Ang II for 48 h (n = 3, *P < 0.05 vs. AdGFP/PBS; ^#P < 0.05 vs. AdGFP/Ang II); (C) The proteins levels of phosphorylated and total c-jun in samples from wild-type(WT) and ZNF418 KO mice after sham and AB treatment for 4 weeks (n = 3, *P < 0.05 vs. WT/sham; ^#P < 0.05 vs. WT/AB); (D) The proteins levels of phosphorylated and total c-jun in samples from WT and ZNF418 transgenic (TG) mice after sham and AB treatment for 4 weeks (n = 3, *P < 0.05 vs. WT/sham; ^#P < 0.05 vs. WT/AB).