Gentisic acid attenuates pressure overload‐induced cardiac hypertrophy and fibrosis in mice through inhibition of the ERK1/2 pathway

Figures

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  Figure 1. Gentisic acid reverses cardiac hypertrophy in mice with TAC. Cardiac hypertrophy developed in mice over the 2 weeks following the TAC operation, after which, they were administered vehicle (n = 14), gentisic acid (100 mg/kg/d; n = 13), bisoprolol (5 mg/kg/d; n = 8), or diltiazem (10 mg/kg/d; n = 7) for 3 weeks. A, Representative images of H&E staining of cardiac tissue from the sham, TAC, TAC + gentisic acid, TAC + bisoprolol, and TAC + diltiazem groups. Scale bar = 50 μm. B, Quantified cross‐sectional areas of cardiomyocytes from the sham and gentisic acid, bisoprolol, or diltiazem‐treated TAC groups. C and D, HW/BW and HW/TL ratios in each group (n = 7–14 per group). Data are means ± SE. ***P < 0.001; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs the TAC group. TAC, transverse aortic constriction; HW/BW, heart weight to body weight; TL, tibia length; NS, not significant

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  Figure 2. Gentisic acid attenuates left ventricular hypertrophy in mice with transverse aortic constriction (TAC). Left ventricular hypertrophy was determined by echocardiography after 3 weeks of drug administration beginning 2 weeks after the TAC procedure. A, Representative B‐mode and M‐mode echocardiograms are shown. B, Interventricular septum thickness (IVSd); C, left ventricular posterior thickness (LVPWd); and D, fractional shortening (FS) (%)

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  Figure 3. Gentisic acid reduces expression of hypertrophic marker genes in mice with transverse aortic constriction (TAC). Total RNA was isolated from the hearts of mice in the sham, TAC, TAC + gentisic acid, TAC + bisoprolol, and TAC + diltiazem groups. Levels of Anp (A), Bnp (B), and skeletal α‐actin (C) transcripts were determined by RT‐qPCR and normalized to those of Gapdh. Data are means ± SE. ***P < 0.001; # P < 0.05 and ## P < 0.01 vs the TAC group; NS, not significant. D, Representative western blot images showing atrial natriuretic peptide (ANP) protein levels in the hearts of mice. GAPDH was used as a loading control. The images were cropped from the same blot. E, ANP protein expression was quantified using densitometry. ***P < 0.001; # P < 0.05 and ### P < 0.001 vs the TAC group

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  Figure 4. Gentisic acid reduces GATA4 and Sp1 expression in mice with transverse aortic constriction (TAC). Total RNA was isolated from the hearts of mice in the sham, TAC, TAC + gentisic acid, TAC + bisoprolol, and TAC + diltiazem groups. Levels of Sp1 (A) and Gata4 (B) transcripts were determined by RT‐qPCR and normalized to those of Gapdh. Data are means ± SE. C, Representative western blot images showing Sp1 and GATA4 protein levels in the hearts of mice. GAPDH was used as a loading control. The images were cropped from the same blot. D and E, Sp1 and GATA4 protein expression was quantified using densitometry. *P < 0.05, **P < 0.01, and ***P < 0.001; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs the TAC group

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  Figure 5. Gentisic acid attenuates ERK1/2 phosphorylation in mice with transverse aortic constriction (TAC). Representative immunoblots of phosphorylated (p‐)p38 MAPK (A), p‐ERK1/2 MAPK (C), and p‐JNK1/2 (E) levels in the sham, TAC, and drug‐treated TAC groups are shown. Levels of p‐p38 (B), p‐ERK1/2 (D), and p‐JNK1/2 (F) proteins were quantified using densitometry. Data are means ± SE. **P < 0.01; # P < 0.05 and ## P < 0.01 vs the TAC group; NS, not significant

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  Figure 6. Gentisic acid reduces interstitial and perivascular fibrosis in mice with transverse aortic constriction (TAC). Representative images of Masson's trichrome staining of the hearts of mice in the sham and TAC + vehicle, gentisic acid, bisoprolol, or diltiazem groups. Scale bar = 50 μm. A, Interstitial fibrosis and B, perivascular fibrosis are shown. Fibrotic areas are stained blue. C, Fibrotic areas in specimens from the sham, TAC, and drug‐treated TAC groups were quantified (n = 6 per group). Data are means ± SE. ***P < 0.001; ## P < 0.01 and ### P < 0.001 vs the TAC group

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  Figure 7. Gentisic acid attenuates expression of cardiac fibrosis marker genes in mice with TAC. Levels of collagen type I (A), collagen type III (B), fibronectin (C), and Ctgf (D) transcripts normalized to those of Gapdh. Data are means ± SE. ***P < 0.001; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs the TAC group. E, Representative immunoblots for collagen III, fibronectin, and connective tissue growth factor (CTGF)

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  Figure 8. Gentisic acid attenuates cardiac hypertrophy and fibrosis through downregulation of the ERK1/2 pathway. Pressure overload, such as transverse aortic constriction (TAC), activates the ERK1/2 signalling pathway in the heart. Given TAC stimulation, in addition to the increase in GATA4 or Sp1 expression, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression are also elevated. In the cardiac hypertrophy mouse model, gentisic acid administration ameliorates cardiac hypertrophy and fibrosis through suppressed activation of ERK1/2 and downregulation of Sp1 or GATA4