Role of GALNT4 in protecting against cardiac hypertrophy through ASK1 signaling pathway

Figures

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  Fig. 1. GALNT4 is upregulated in experimental models of hypertrophy. A Real-time PCR of GALNT4 expression in TAC group and sham group after 4 or 8 weeks. B, C The mRNA level of GALNT4 in NRCMs in the PE group and the Ang II group, respectively after 24 or 48 h. D–F Western blot of GALNT4 expression in every group. *p < 0.05 vs. sham or PBS, **p < 0.01 vs. sham or PBS.

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  Fig. 2. GALNT4 suppresses PE-induced cardiomyocytes hypertrophy. A The overexpression of GALNT4 was verified by western blotting of cardiomyocytes (n = 3 independent experiments). B Immunofluorescence staining images of NRCMS treated with AdshGALNT4 or AdshRNA after treatment with PE or PBS in each group (n = 30-40 cells). C Changes in cardiac hypertrophy markers were assessed after treatment with PE or PBS by real-time PCR (n = 3 independent experiments). D GALNT4 protein levels after transduction with AdGALNT4 and control (n = 3 independent experiments). E Immunofluorescence staining images of cells infected with AdGALNT4 or AdVector and treated with PE or PBS (n = 30-40 cells). F The level of cardiac hypertrophy markers in NCRMs was detected by real-time PCR (n = 3 independent experiments). **p < 0.01 vs. AdshRNA PBS or AdVector PBS, ##p < 0.01 vs. AdshRNA PE or AdVector PE.

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  Fig. 3. GALNT4 deficiency accelerates cardiac hypertrophy in vitro. A The levels of GALNT4 in GALNT4-KO mice and WT mice were analyzed by western blotting. B–D The ratio of HW/ BW, LW/BW, and HW/TL in each group (n = 10 mice per group). E–H LVEDd, LVESd, EF%, and FS% were analyzed in every group (n = 10 mice per group). I H&E staining images (n = 6 mice per group) and heart tissue CSA (n = 120 cells in each group) in the GALNT4-KO group and WT control. J Images of PSR staining (left, n = 6 mice per group) and percentage of LV collagen volume in the indicated group. L, M The expression of hypertrophic and fibrotic markers were analyzed by qPCR (n = 4 mice per group). *p < 0.05 vs. WT sham, **p < 0.01 vs. WT sham, #p < 0.05 vs. WT TAC, ##p < 0.01 vs. WT TAC.

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  Fig. 4. Cardiomyocyte-specific overexpression of GALNT4 attenuates the cardiac hypertrophy induced by TAC surgery. A The levels of GALNT4 in AAV9–GALNT4, AAV9-Vector mice were analyzed by western blotting. B–D The ratio of HW/ BW, LW/BW, and HW/TL in the AAV9-GALNT4 group and sham group (n = 10 mice per group). E–H LVEDd, LVESd, EF%, and FS% were analyzed in each group (n = 10 mice per group). I H&E staining images (n = 6 mice per group) and heart CSA (n = 120 cells in each group) in AAV9–GALNT4 and control mice. J Images of PSR staining (left, n = 6 mice per group) and percentage of LV collagen volume in the indicated group. L, M The levels of markers of hypertrophy and fibrosis were analyzed by qPCR (n = 4 mice per group). *p < 0.05 vs. AAV9-Vector sham, **p < 0.01 vs. AAV9-Vector sham, #p < 0.05 vs. AAV9-Vector TAC, ##p < 0.01 vs. AAV9-Vector TAC.

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  Fig. 5. GALNT4 inhibits the ASK1 signaling pathway. A, B The expression of phosphorylated and total ASK1 and its downstream molecules in cells treated with the indicated adenovirus. A, B **p < 0.01 vs. AdshRNA PBS or AdVector PBS, #p < 0.05 vs. AdshRNA PE or AdVector PE, ##p < 0.01 vs. AdshRNA PE or AdVector PE, n.s. indicates no significance. C, D Phosphorylated and total ASK1 and its downstream molecules in mouse hearts in each group subjected to TAC or sham surgery. C, D *p < 0.05 vs. WT sham or AAV9-Vector sham, **p < 0.01 vs. WT sham or AAV9-Vector sham, #p < 0.05 vs. WT TAC or AAV9-Vector TAC, ##p < 0.01 vs. WT TAC or AAV9-Vector TAC. n.s. indicates no significance.

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  Fig. 6. GALNT4 can regulate apoptosis and directly binds to ASK1 to inhibit its oligomerization. A Knockdown of GALNT4 increases the expression of apoptotic proteins in vivo. B The overexpression of GALNT4 inhibits cardiomyocytes apoptosis. A, B **p < 0.01 vs. AdshRNA PBS or AdVector PBS, #p < 0.05 vs. AdshRNA PE or AdVector PE, ##p < 0.01 vs. AdshRNA PE or AdVector PE. C, D The expression of Bax and C-caspase 3 in vitro. For C-D, *p < 0.05 vs. WT sham or AAV9-Vector sham, **p < 0.01 vs. WT sham or AAV9-Vector sham, #p < 0.05 vs. WT TAC or AAV9-Vector TAC, ##p < 0.01 vs. WT TAC or AAV9-Vector TAC. E, F The interaction between GALNT4 and ASK1 in 293T cells. G The interaction between GALNT4 and ASK1 in NRCMs. H, I A GST pull down assay was used to verify the direct combination between GALNT4 and ASK1. J The effect of GALNT4 on the dimerization of ASK1 (1–678) in 293T cells.

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  Fig. 7. Inhibiting ASK1 is the key step by which GALNT4 regulates hypertrophy. A The protein levels of GALNT4, pASK1, and ASK1 in NRCMS infected with the indicated adenovirus and treated with iASK1 or not after treatment with PE. B Images of NRCMS in each group and the CSA (n = 30-40 cells). C The mRNA expression levels of ANP and BNP were examined by RT qPCR. n = 3 independent experiments. **p < 0.01 vs. AdshRNA, ##p < 0.01 vs. AdshGALNT4.