Syringic acid mitigates isoproterenol‐induced cardiac hypertrophy and fibrosis by downregulating Ereg

Figures

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  FIGURE 1. Syringic acid pretreatment mitigates isoproterenol‐induced upregulation of cardiac hypertrophy and fibrosis marker genes in vitro. (A) Chemical structure of syringic acid. (B) The viability of H9c2 cells treated with three different concentrations of syringic acid for 24 h was evaluated (n = 6). NS, not significant. (C, D) H9c2 cells were serum‐starved overnight and pretreated with vehicle or syringic acid (10 μM) for 1 h, followed by co‐treatment with isoproterenol (ISO, 10 μM) for 24 h. Alexa 488 phalloidin staining was performed to determine cell size. Representative images of cells (C) and quantification of cell size (D) (n = 40). Scale bar = 50 μm. *** p < 0.001; ### p < 0.001. (E–J) H9c2 cells were pretreated with vehicle or syringic acid (10 μM) for 1 h before isoproterenol (10 μM for 9 h) stimulation. mRNA expression levels of Nppa (E), Nppb (F) and Col1a1 (G) were determined using quantitative real‐time polymerase chain reaction (n = 6). The protein expression levels (H–J) were quantified using Western blotting analysis. *** p < 0.001; ### p < 0.001; @@@ p < 0.001; NS, not significant. Data are presented as mean ± standard error of mean. Statistics: one‐way analysis of…

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  FIGURE 2. Syringic acid alleviates cardiac hypertrophy in isoproterenol‐treated mice. (A) Schematic representation of the experimental protocol. Syringic acid administration was initiated 7 days before isoproterenol infusion (5 days) in mice (sham + vehicle (dimethyl sulfoxide (DMSO), n = 8), sham + syringic acid (SA, 100 mg/kg bodyweight/day, n = 8), ISO + vehicle (DMSO, n = 8), ISO + SA (100 mg/kg bodyweight/day, n = 8)). (B) Representative images of whole hearts. (C) Heart weight to bodyweight (HW/BW) ratio (n = 8). *** p < 0.001; ### p < 0.001; NS, not significant. (D) Representative images of haematoxylin and eosin (H&E)‐stained sections. Scale bar = 50 μm. (E) Quantification of cross‐sectional area of the cardiac tissues. *** p < 0.001; ### p < 0.001; NS, not significant. (F, G) Nppa and Nppb mRNA levels were examined using quantitative real‐time polymerase chain reaction and normalized to those of Gapdh. * p < 0.05; *** p < 0.001; ### p < 0.001; NS, not significant. (H) Nppa and Nppb levels in the cardiac tissues. Actb was used as a loading control. (I, J) Quantification of Nppa and Nppb protein levels (n = 6). *** p < 0.001; ### p < 0.001

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  FIGURE 3. Syringic acid pretreatment mitigates isoproterenol‐induced pathological cardiac remodelling in mice. (A) Syringic acid administration was started 7 days before isoproterenol infusion (5 days) in mice (sham + vehicle (n = 8), sham + SA (n = 8), ISO + vehicle (n = 8) and ISO + SA (n = 8)). Representative M‐mode echocardiograms. (B, C) Quantification of the thickness of the interventricular septum (IVSd) and left ventricular posterior wall (LVPWd) (n = 8 per group). *** p < 0.001; ### p < 0.001; NS, not significant. (D, E) Quantification of left ventricular end‐systolic diameter (LVESd) and left ventricular end‐diastolic diameter (LVEDd) (n = 8 per group). *** p < 0.001; ### p < 0.001; NS, not significant. (F) Ejection fraction (%). ** p < 0.01; # p < 0.05; NS, not significant. Data are presented as mean ± standard error of mean. Statistics: one‐way analysis of variance, followed by Bonferroni post hoc tests. ISO, isoproterenol; SA, syringic acid

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  FIGURE 4. Syringic acid pretreatment mitigates isoproterenol‐induced cardiac fibrosis in mice. (A, B) Syringic acid administration was started 7 days before isoproterenol infusion (5 days) in mice (sham + vehicle (n = 8), sham + SA (n = 8), ISO + vehicle (n = 8) and ISO + SA (n = 8)). Representative images (A) of Picrosirius red‐stained cardiac tissues and quantification of staining intensity (B). Scale bar = 50 μm. *** p < 0.001; ### p < 0.001. mRNA levels of Col1a1 (C) and Fn1 (D) were determined using quantitative real‐time polymerase chain reaction and normalized to those of Gapdh. **p < 0.01 and *** p < 0.001; # p < 0.05 and ### p < 0.001. (E–G) Representative images of cardiac Col1a1 and Fn1 levels (n = 6 per group) and their quantification. Actb was used as a loading control. ISO and SA indicate isoproterenol and syringic acid, respectively. Data are presented as mean ± standard error of mean. Statistics: one‐way analysis of variance, followed by Bonferroni post hoc test

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  FIGURE 5. Syringic acid downregulates the expression levels of Ereg, Ngfr and Myc. (A) Heatmap of differentially expressed genes in the cardiac tissues of the sham + vehicle (n = 8), ISO + vehicle (n = 8) and ISO + SA (n = 8) groups. (B–D) RNA sequencing results of MAPK signalling‐related genes were validated using quantitative real‐time polymerase chain reaction. (E–G) Ereg, Ngfr and Myc mRNA levels in H9c2 cells transfected with si‐control or si‐Ereg. (H–L) Ereg, Ngfr, Myc, Nppb and Fn1 mRNA levels in H9c2 cells transfected with si‐control or si‐Ereg and treated with isoproterenol

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  FIGURE 6. Ereg knockdown mitigated isoproterenol‐induced cardiomyocyte hypertrophy and fibronectin (A) Representative images of phalloidin 488‐stained cells. Green and blue indicate phalloidin 488‐stained cells and nucleus, respectively. Scale bar = 50 μm. (B) Cell area of si‐Ereg‐transfected and isoproterenol‐stimulated H9c2 cells. *** p < 0.001; ### p < 0.001. (C) Representative Western blotting images of si‐Ereg‐transfected and isoproterenol‐treated cells. (D, E) Quantification of Nppb and Fn1 protein levels. *** p < 0.001; ## p < 0.01 and ### p < 0.001; @@ p < 0.01 and @@@ p < 0.001. (F) Schematic diagram of the mechanism of syringic acid involved in the suppression of cardiac hypertrophy and fibrosis. Isoproterenol upregulates Ereg mRNA levels in vivo and in vitro. Ereg knockdown mitigates the isoproterenol‐induced upregulation of Nppb, Fn1 and Ngfr. Syringic acid alleviates cardiac hypertrophy and fibrosis. Nppb, natriuretic peptide; Fn1, fibronectin; Ngfr, nerve growth factor receptor