The Selective Serotonin 2A Receptor Antagonist Sarpogrelate Prevents Cardiac Hypertrophy and Systolic Dysfunction via Inhibition of the ERK1/2–GATA4 Signaling Pathway

Figures

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  Figure 1. Sarpogrelate suppressed cardiomyocyte hypertrophy induced by a variety of hypertrophic stimuli. Primary cultured cardiomyocytes were treated with 0.3 or 1 μM sarpogrelate (SA) and then stimulated with 30 μM phenylephrine (PE), 0.1 µM angiotensin II (Ang II), or 0.1 µM endothelin 1 (ET-1) for 48 h. (a) Immunofluorescence staining was performed using anti-MHC antibodies and Alexa Fluor 555-conjugated anti-mouse IgG. Scale bar: 20 μm. (b) Cell surface area was measured using NHI ImageJ software. All data are presented as the mean ± SEM of four individual experiments. (c,d) Cardiomyocytes were harvested 48 h after stimuli and a luciferase reporter assay was performed for ANF (c) and ET-1 (d) promoters. Data are presented as the mean ± SEM of five individual experiments. (e,f) The mRNA levels of hypertrophy-related gene transcriptions of ANF (e) and BNP (f) at 48 h after stimulation were examined by quantitative PCR. Data are presented as the mean ± SEM of three individual experiments.

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  Figure 2. Sarpogrelate inhibited the ERK1/2–GATA4 signaling pathway in cardiomyocytes. (a) The mRNA levels of the 5-HT2A receptor were examined at 48 h after PE stimulation. Quantitative PCR data are presented as the mean ± SEM of three individual experiments. (b,c) WCL was extracted from cardiomyocytes at 48 h after PE stimulation. Representative Western blotting image (b) and quantified 5-HT2A receptor levels (c). Quantification is presented as the mean ± SEM of three individual experiments; n.s., no significance. (d,e) Quantitative PCR was performed for TPH1 (d), TPH2 (e), and 18S. Data are presented as the mean ± SEM of three individual experiments; n.s., no significance. (f) WCL was extracted from cardiomyocytes at 10 min after PE stimulation and then subjected to Western blotting using anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody and anti-p44/42 MAPK (ERK1/2) antibody. (g) Levels of phosphorylated ERK1/2 and total ERK1/2 were quantified. Data are presented as the mean ± SEM of three individual experiments. (h) The NE fraction was isolated from the cells at 1 h after PE stimulation and then subjected to Western blotting using anti-GATA4 (phospho S105) antibody and GATA4…

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  Figure 3. Sarpogrelate suppressed TAC-induced cardiac hypertrophy and systolic dysfunction in vivo. Eight weeks after TAC surgery, cardiac hypertrophy and cardiac function were assessed by echocardiography. Representative images of echocardiography from sarpogrelate- and vehicle-treated TAC and sham are shown.

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  Figure 4. Sarpogrelate suppressed TAC-induced cardiac hypertrophy and fibrosis. (a) Representative image of isolated heart from sham and TAC mice at 8 weeks after surgery. Scale bar: 5 mm. (b) Representative images of WGA-stained sections of LV myocardium from sham and TAC mice. Magnification: ×400. Scale bar: 20 μm. (c) Cardiomyocyte cross-sectional area was measured for 50 cells from five to seven mice from each group. (d) Representative photographs of the MT-stained perivascular fibrosis area of the LV myocardium of sham and TAC mice. Magnification: ×200. Scale bar: 50 μm. (e) The area of perivascular fibrosis in the LV was measured for at least three intramyocardial coronary arteries in each animal. Data are presented as the mean ± SEM of five to six individual experiments. (f,g) Quantitative PCR analyses were performed for ANF (f) and BNP (g). Data are presented as the mean ± SEM of five individual experiments.

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  Figure 5. Sarpogrelate suppressed TAC-induced phosphorylation of ERK and GATA4. (a,b) WCL from mouse heart was subjected to Western blotting to assess the levels of phosphorylated ERK1/2 and total ERK. Representative Western blotting images are shown in (a) and quantified levels of phosphorylated and total ERK1/2 in (b). (c,d) NE fractions from mouse heart were subjected to Western blotting to assess phosphorylated GATA4 and total GATA4. Representative images of Western blotting are shown in (c) and quantified phosphorylated and total GATA4 levels in (d). All data are presented as the mean ± SEM of four individual experiments.