ANGPTL8 is a negative regulator in pathological cardiac hypertrophy

Figures

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  Fig. 1. Serum ANGPTL8 was increased in patients and animal models with cardiac hypertrophy. A Serum levels of ANGPTL8 in patients with hypertension (n = 30), patients with hypertension and myocardial hypertrophy (MH) (n = 100) and healthy people (n = 70). B, C The correlation between serum ANGPTL8 level and left atrium diastolic diameter (LAEDd) (n = 39) or left ventricular end diastolic diameter (LVEDd) (n = 54) in the subjects with hypertensive patients and myocardial hypertrophy. Serum ANGPTL8 levels in mice subjected to TAC for 4 weeks (D, n = 7 mice per group) and Ang II-infused mice (E, n = 5 mice per group) were detected by ELISA (***P < 0.001). F Representative image of immunofluorescence with an anti-ANGPTL8 antibody in slices from TAC and sham mice (n = 5 mice per group, scale bar, 100 μm). G Quantitative results of the relative fluorescence intensity of ANGPTL8 in the indicated groups (n = 5 mice per group, scale bar, 100 μm, **P < 0.01). H, I Representative image and quantitative results of immunofluorescence with an anti-ANGPTL8 antibody in the indicated groups (n = 5 mice per group, scale bar, 100 μm, **P < 0.01). J–M Representative immunofluorescence images and…

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  Fig. 2. ANGPTL8 deficiency accelerated cardiac hypertrophy induced by Ang II infusion. A Cardiac hypertrophy in mice was induced by Ang II infusion for 4 weeks. B Echocardiographic assessments of cardiac function in WT and ANGPTL8-KO mice at 4 weeks of Ang II infusion. PLAX = Long axis of the heart, M model (M type), Three-dimensional deformation ability (Strain 3D&strain rate), n = 4~6 per group. C–E Ejection fraction (EF%), fraction shortening (FS%) and left ventricular end-diastolic dimension (LVEDd) were assessed by echocardiography in WT and ANGPTL8-KO mice at 4 weeks after saline or Ang II infusion (n = 5, NS no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001). F Representative images of the gross cardiac morphology used for HW/BW and HW/HL calculation (scale bar, 2 mm) (n = 5). G Representative images of transverse sections stained with H&E (scale bar, 0.5 cm, n = 5). H, I Bar graphs showing quantitative data for HW/BW or HW/TL (n = 5 per group, NS no significant difference, *P < 0.05, ***P < 0.001).

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  Fig. 3. ANGPTL8KO enhanced hypertrophic and fibrotic marker expression after Ang II infusion for 4 weeks. Representative images of H&E staining (A), WGA staining (B), picrosirius red (PSR) staining (C) and collagen I staining (D) of slices from transverse sections of the left ventricles from the mouse hearts in the indicated groups (n = 5 mice per group, scale bars, 250 μm and 100 μm, respectively). Quantification of the average myocardial interstitial area (E) and myocyte cross-sectional areas (F) in WT and ANGPTL8-KO mice at 4 weeks (E n > 40 fields and F n = 100 cardiomyocytes from 5~6 mice in each group, NS no significant difference, **P < 0.01, ***P < 0.001). G Quantitative results of the collagen I-positive area from collagen I immunochemical staining in WT and ANGPTL8-KO mice (n = 5 mice in each group, **P < 0.01). H, I mRNA levels of ANF and BNP were analyzed by qRT–PCR from WT and ANGPTL8-KO mouse hearts (n = 5 mice in each group). J Representative Western blots of β-MHC and BNP in cardiac tissue from WT and ANGPTL8-KO mice (n = 3). K, L Quantitative statistical results of β-MHC and BNP protein levels (n = 3, NS no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 4. ANGPTL8 ameliorated cardiomyocyte hypertrophy induced by Ang II in vitro. Primary NRCM or H9c2 cells were treated with recombinant human full-length ANGPTL8 protein (rANGPTL8, 500 ng/ml) and Ang II (1 µM) for 48 h. A Representative microscopic images of NRCMs stained with rhodamine phalloidin in the indicated groups (scale bar, 50 μm and 25 μm, respectively). B Quantitative results of the cell surface area of cardiomyocytes in the indicated groups (n > 50 cells per group, 5 independent experiments, **P < 0.01, ***P < 0.001). C Representative microscopic images of H9c2 cells in the indicated groups (scale bar, 25 μm). D Quantitative results of the cell surface area of H9c2 cells in the indicated groups (n > 50 cells per group, 5 independent experiments, **P < 0.01, ***P < 0.001). E–H Quantification of the mRNA levels of ANF, BNP, collagen III and CTGF in NRCM in the indicated groups (n = 5 per group, **P < 0.01, ***P < 0.001). I–L Representative western blots and quantification of β-MHC in NRCM and H9c2 cells in the indicated groups (n = 3 per group, *P < 0.05, **P < 0.01). H9c2 cells were infected with the indicated lentivirus of ANGPTL8 for 24 h and then treated with Ang II (1…

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  Fig. 5. ANTPTL8 knockout aggravated pressure overload-induced cardiac hypertrophy. A pressure overload-induced cardiac hypertrophy model was established by transverse aorta constriction (TAC) for 4 weeks. A Schematic diagram depicting TAC. B Echocardiographic detection of cardiac function in mice with myocardial hypertrophy at 4 weeks after TAC surgery, long axis of the heart (PLAX), M model (M type), and three-dimensional deformation ability (Strain3D & strain rate). C–E, H, I Echocardiographic assessments of EF%, FS%, LVEDd, cardiac output (CO) and aortic diameter (AD) in WT and ANGPTL8-KO mice at 4 weeks after sham (n = 6 mice) or TAC surgery (n = 9~10) (NS no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). F Representative images of the gross cardiac morphology of WT and ANGPTL8-KO mice in the indicated groups. G Representative images of H&E staining from transverse sections of the left ventricles from the indicated groups (n = 5 mice for each group, scale bar, 0.5 cm).

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  Fig. 6. ANTPTL8 knockout enhanced hypertrophic and fibrotic features in the heart at 4 weeks after TAC. Representative images of H&E A (scale bar, 500 μm (up) and 75 μm (down), respectively) and WGA staining B (scale bar, 500 μm and 75 μm, respectively) in the indicated groups (n = 5 mice in each group). C Picrosirius red-stained transverse sections of the left ventricles from the indicated groups (scale bar, 500 μm, n = 6 mice in each group). D Representative immunohistochemical images for collagen I in mouse tissue with sham and TAC surgery in the indicated groups (scale bar, 500 μm and 75 μm, respectively, n = 6 mice in each group). Quantification of the myocyte-sectional area (E), myocardial interstitial fibrosis (F), myocardial vessel fibrosis (G) and collagen I-positive area (H) of slices from WT and ANGPTL8-KO mice in the indicated groups (n = 5 mice in each group). I, J qRT–PCR was performed to determine the mRNA levels of ANF and BNP in the indicated groups (n = 4 mice in each group). Representative Western blots (K) and quantification (L) of the protein levels of BNP in cardiac tissue from WT and ANGPTL8-KO mice in the indicated groups (n = 4 mice in each group), *P < 0.05, **P…

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  Fig. 7. Effects of ANGPTL8 on the Akt/GSK-3β signaling pathway. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the identified differentially expressed genes based on RNA-seq data set from the hearts of wild-type (WT) or ANGPTL8 KO mice at 4 weeks after Ang II infusion (n = 3 in each group). B Volcano plot of relative RNA expression from the hearts of WT mice infused with Ang II for 4 weeks compared to ANGPTL8 KO mice. Genes in the upper left and right quadrants are significantly differentially expressed (microarray, n = 2 per group). C–F Western blots and quantification of BNP and total and phosphorylated levels of GSK-3β and Akt proteins in cardiac tissues from WT and ANGPTL8-KO mice subjected to sham (n = 4) or TAC (n = 3). G–K The effect of rANGPTL8 on NRCM hypertrophy induced by Ang II and Western blotting was used to detect the hypertrophic markers β-MHC and BNP and the total and phosphorylated protein levels of GSK-3β and Akt. Representative western blots (G) and quantitative results (H–K) of these hypertrophic markers and signaling proteins (n = 3 in each group). L Microscopy images of NRCM hypertrophy treated with rANGPTL8 or the Akt activator SC79…

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  Fig. 8. ANGPTL8 attenuated cardiomyocyte hypertrophy by interacting with LILRB3. A Heatmap analysis of differentially expressed genes is presented. B, C The mRNA levels of PIRA2 and PIRB were determined by qRT–PCR in WT or ANGPTL8-KO mice at 4 weeks after sham or TAC surgery (n = 5–8 in each group). D, E The mRNA levels of PIRA2 and PIRB were detected by qPCR in WT or ANGPTL8-KO mice at 4 weeks after saline or Ang II infusion (n = 4–5 in each group). F, G Representative western blots and quantification of LILRB3 in NRCM-treated rANGPTL8 and/or Ang II cells (repeated n = 3 per group). H Models of the gene expression system based on chimeric receptors. I A luciferase reporter system detected the interaction between ANGPTL8 and PIRs (n = 4–5). J A schematic diagram of the experimental procedure was used to identify potential targets of ANGPTL8. K Representative Western blots performed with Flag or LILRB3 antibody after co-immunoprecipitation (IP) of ANGPTL8 or LILRB3 from HEK-T293 cell lysates using Flag antibody or LILRB3 antibody, respectively (n = 3). L, N Representative western blots and quantification of total and phosphorylated Akt and GSK-3β in the presence of rANGPTL8 and/or Ang II…