JNK signaling-dependent regulation of histone acetylation are involved in anacardic acid alleviates cardiomyocyte hypertrophy induced by phenylephrine

Figures

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  Fig 1. Activity of HATs in hypertrophic cardiomyocytes induced by phenylephrine (PE). Different concentrations of PE (0, 25, 50, 100, and 150 μmol/L) were used to determine the optimal PE dose for the induction of cardiomyocyte hypertrophy in neonatal mice. (A) The effect of PE on cell viability was accessed using the Cell Counting Kit-8 assay in neonatal mouse cardiomyocytes. (B) Real-time PCR analysis of ANP mRNA expression after treatment with different concentrations of PE for 48 h. (C) The cardiomyocyte cytoplasm was stained with antibodies against α-actin, while the nuclei were stained with DAPI. The scale bars represent 20 μm. (D) Quantification of cell surface areas in each treatment group. (E) The activity of HATs were assayed by colorimetry, and PE (100 μmol/L) increased HATs activity. (F) The chemical structure of the HATs inhibitor anacardic acid (AA). (G) Effect of different concentrations of AA (0, 30, 40, 50, and 60 μmol/L) on cell viability of neonatal mouse cardiomyocytes by CCK-8 assays. (H) Different concentrations of AA (0, 30, 40, 50, and 60 μmol/L) were used to identify the optimal dose and 50 μmol/L AA was selected based on the level of H3K9ac. NS: normal saline.…

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  Fig 2. Effects of JNK/MAPK signaling and H3K9ac on mouse hypertrophic cardiomyocytes. (A) Effect of different concentrations of the JNK inhibitor SP600125 (0, 5, 10, 15, 20, and 25 μmol/L) on cell viability of neonatal mouse cardiomyocytes by CCK-8 assays, and optimal dose (20 μmol/L) of SP600125 was defined by taking into account myocardial cell viability. (B) Western blotting showing that p-JNK was significantly increased in myocardial cells exposed to phenylephrine (PE); the JNK inhibitor SP600125 decreased p-JNK overexpression in PE-induced hypertrophic cardiomyocytes but had not effect on T-JNK. (C) H3K9ac (green fluorescence) and α-actin (red fluorescence) combined with DAPI (blue fluorescence) staining in myocardial cells exposed to six different conditions. Scale bars, 20 μm. (D) The statistical analysis of average optical density of H3K9ac immunofluorescence in the six groups. All results are representative of at least three independent experiments. (E) The level of H3K9ac was significantly increased in PE-induced hypertrophic cardiomyocytes; the HATs inhibitor anacardic acid (AA) attenuated histone H3K9ac hyperacetylation in PE-induced hypertrophic cardiomyocytes. *P < 0.05…

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  Fig 3. p-JNK directly interacted with HATs and altered H3K9ac acetylation in primary mouse cardiomyocytes. (A and B) Co-immunoprecipitation (CoIP) of cell lysates from mouse myocardial cells exposed to six different experimental conditions with anti-p-JNK-protein G magnetic beads and immunoblot (IB) with an anti-P300-HAT, anti-PCAF-HAT, anti-H3K9ac, or anti-p-JNK antibody for the evaluation of protein expression. PE: phenylephrine; DMSO: dimethylsulfoxide; AA: anacardic acid; SP: SP600125 (JNK inhibitor); IP: immunoprecipitation; IB: immunoblotting; Input: positive control; IgG: negative control. Images are representative of three qualitatively similar independent experiments.

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  Fig 4. Effects of anacardiac acid (AA) and the JNK inhibitor SP600125 on the expression of HATs in phenylephrine (PE)-induced hypertrophic cardiomyocytes. (A) P300 (green fluorescence) and α-actin (red fluorescence) combined with DAPI (blue fluorescence) staining of myocardial cells exposed to six different conditions. Scale bars, 20 μm. (B) The statistical analysis of average optical density of P300 immunofluorescence in the six groups. (C) The levels of P300-HAT (histone acetyltransferase) significantly increased in the hypertrophic cardiomyocytes induced by PE; the HATs inhibitor AA and the JNK inhibitor SP600125 prevented this effect. (D) PCAF (green fluorescence) and α-actin (red fluorescence) combined with DAPI (blue fluorescence) staining of myocardial cells exposed to six different conditions. Scale bars, 20 μm. (E) The statistical analysis of average optical density of PCAF immunofluorescence in the six groups. (F) The level of PCAF-HAT was increased significantly in hypertrophic cardiomyocytes induced by PE compared to that in control cells. The HATs inhibitor AA or the JNK inhibitor SP600125 normalized the expression of PCAF-HAT in PE-induced hypertrophic cardiomyocytes. *P <…

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  Fig 5. Effects of anacarddic acid (AA) and the JNK inhibitor SP600125 on the expression of cardiac hypertrophy-related genes in phenylephrine (PE)-induced hypertrophic cardiomyocytes. (A) Chromatin immunoprecipitation (ChIP)-PCR results demonstrating the binding of P300 and PCAF to the MEF2A promoter. (−) negative control, amplification of DNA fragments after precipitation with normal mouse IgG; (+) positive control, amplification of DNA fragments after precipitation with anti-RNA polymerase II antibody. (B) qRT-PCR results showing that the mRNA expression of MEF2A was higher in PE-induced hypertrophic cardiomyocytes than in control cardiomyocytes, whereas histone acetyltransferases (HATs) and JNK inhibition with AA and SP600125, respectively, prevented MEF2A overexpression in these cells. (C) ChIP-PCR demonstrated that MEF2A could bind to the promoters of cardiac hypertrophy-related genes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC). (+): positive control,amplification of DNA fragments after precipitation with anti-RNA polymerase II antibody. (-): negative control, amplification of DNA fragments after precipitation with normal…

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  Fig 6. The HATs inhibitor anacardic acid (AA) and the JNK inhibitor SP600125 attenuates cardiomyocyte hypertrophy induced by phenylephrine (PE). (A) The cell surface area was measured by immunofluorescence staining to demonstrate hypertrophic responses in cardiomyocytes. Myocardial cell were stained using α-actin (red fluorescence) combined with DAPI (blue fluorescence). (B) Measurement of the cell surface area of myocardial cells for the quantification of cell size. (C) Determination of intracellular Ca2+ concentrations using the calcium fluorescent probe Flou-3/AM and laser scanning confocal microscopy. (D) Dynamic fluorescence curve of intracellular Ca2+ in myocardial cells. (E) Comparison of intracellular Ca2+ fluorescence intensity in myocardial cells among the six groups of PE-induced hypertrophic cardiomyocytes. *P < 0.05 vs. the control group, #P < 0.05 vs. the PE group (n = 6). Scale bars, 20 μm. PE: phenylephrine; DMSO: dimethylsulfoxide; AA: anacardic acid; SP: SP600125 (JNK inhibitor). Fluorescence quantification was done using ImageJ 1.8.0_112 software (https://imagej.nih.gov/ij/, 1997–2018). Figure created in IBM SPSS version 25…

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  Fig 7. Echocardiographic images of mice. Cardiac function of mice among the six groups were tested by echocardiography. M-mode images obtained from short-axis measurements were used to evaluate left ventricular anterior wall (LVAW) and left ventricular posterior wall (LVPW) thinkness of mice in left ventricular end diastole and left ventricular end systole. Red or green dotted lines represent the diameters of the left ventricle at diastolic or systolic stages. AA: anacardic acid, PE: phenylephrine, DMSO: dimethylsulfoxide, SP: SP600125 (JNK inhibitor).

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  Fig 8. Schematic representation of the possible mechanism through which anacardic acid (AA) attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy. AA could ameliorate cardiac hypertrophy by regulating JNK signaling-induced histone H3K9ac acetylation. AA enters the myocardial cell, and acts on P300-HAT and PCAF-HAT via p-JNK pathway, and downregulates P300-HAT and PCAF-HAT expression. Subsequently, the binding of P300-HAT and PCAF-HAT to the promoter of MEF2A is reduced, which leads to histone H3K9ac hypoacetylation in the promoter of MEF2A and further inhibits MEF2A transcription level, and finally downregulates cardiac hypertrophy-related genes ANP, BNP, and β-MHC overexpression induced by PE to prevent cardiac hypertrophy.