Anacardic acid attenuates pressure‐overload cardiac hypertrophy through inhibiting histone acetylases

Abstract Cardiac hypertrophy has become a major cardiovascular problem wordwide and is considered the early stage of heart failure. Treatment and prevention strategies are needed due to the suboptimal efficacy of current treatment methods. Recently, many studies have demonstrated the important role of histone acetylation in myocardium remodelling along with cardiac hypertrophy. A Chinese herbal extract containing anacardic acid (AA) is known to possess strong histone acetylation inhibitory effects. In previous studies, we demonstrated that AA could reverse alcohol‐induced cardiac hypertrophy in an animal model at the foetal stage. Here, we investigated whether AA could attenuate cardiac hypertrophy through the modulation of histone acetylation and explored its potential mechanisms in the hearts of transverse aortic constriction (TAC) mice. This study showed that AA attenuated hyperacetylation of acetylated lysine 9 on histone H3 (H3K9ac) by inhibiting the expression of p300 and p300/CBP‐associated factor (PCAF) in TAC mice. Moreover, AA normalized the transcriptional activity of the heart nuclear transcription factor MEF2A. The high expression of cardiac hypertrophy‐linked genes (ANP, β‐MHC) was reversed through AA treatment in the hearts of TAC mice. Additionally, we found that AA improved cardiac function and survival rate in TAC mice. The current results further highlight the mechanism by which histone acetylation is controlled by AA treatment, which may help prevent and treat hypertrophic cardiomyopathy.

previous studies, we demonstrated that AA could reverse alcohol-induced cardiac hypertrophy in an animal model at the foetal stage. Here, we investigated whether AA could attenuate cardiac hypertrophy through the modulation of histone acetylation and explored its potential mechanisms in the hearts of transverse aortic constriction (TAC) mice. This study showed that AA attenuated hyperacetylation of acetylated lysine 9 on histone H3 (H3K9ac) by inhibiting the expression of p300 and p300/CBP-associated factor (PCAF) in TAC mice. Moreover, AA normalized the transcriptional activity of the heart nuclear transcription factor MEF2A. The high expression of cardiac hypertrophy-linked genes (ANP, β-MHC) was reversed through AA treatment in the hearts of TAC mice. Additionally, we found that AA improved cardiac function and survival rate in TAC mice. The current results further highlight the mechanism by which histone acetylation is controlled by AA treatment, which may help prevent and treat hypertrophic cardiomyopathy.

K E Y W O R D S
anacardic acid, cardiac hypertrophy, histone acetylation, thoracic aorta constriction development and progression of cardiac hypertrophy and that anacardic acid (AA) can decrease alcohol-mediated cardiac hypertrophy. 7 Unfortunately, the mechanism of AA underlying the Chinese herb extract-mediated attenuation of cardiac hypertrophy caused by transverse aortic constriction (TAC) remains unclear. Here, we aimed to assess the potential of AA as a pan-HAT inhibitor that corrects cardiac hypertrophy by suppressing HAT in an overload-mediated cardiac hypertrophy model. These studies were carried out to identify strategies to decrease pressure overload-induced cardiac hypertrophy. Mice were kept under fully controlled conditions (22 ± 1°C, 55% ± 5% humidity) and allowed food ad libitum along with equal light:dark cycles (12 h:12 h). Mice were exposed to pressure overload through thoracic aortic banding (TAB) (≈70% thoracic aortic diameter). TAC mice were given AA which is a pan-HAT inhibitor. Dimethylsulphoxide was used to dissolve AA for further preparations of 1 mg/ml and finally stored at 4°C. AA (3.75 ml/kg) was intraperitoneally injected into TAC mice every third day for up to 3 weeks after operation. Killing of mice was carried out by CO 2 narcosis, and finally, the heart tissues were collected for further analyses.

| Detection of HAT activity
The mouse myocardial tissues were homogenized to extract nucleoproteins using a Nuclear Extract Kit (Invent, Minnesota, USA) following the manufacturer's instructions. The HAT activity of the protein extracts was examined colourimetrically using a HAT assay kit (GenMed, Shanghai, China).

| Chromatin immunoprecipitation (ChIP)
The mouse heart tissue homogenates were placed in 1% formaldehyde for crosslinking of DNA-protein complexes. After crosslinking, the DNA was sheared through sonication followed by DNA-protein complex precipitation using monoclonal antibodies (anti-MEF2A, anti-p300, anti-PCAF and anti-GCN5) (ChIP grade, Abcam, Cambridge, England). A DNA purification kit (Merck Millipore, Darmstadt, Germany) was used to extract the pure DNA molecules.
All experiments had both positive control (precipitated by anti-RNA polymerase II antibody) and negative control (precipitated by normal mouse IgG) groups. Quantitative real-time PCR was performed after ChIP assays were conducted using a ChIP assay Kit (Merck Millipore, Darmstadt, Germany).

| Haematoxylin and Eosin (H&E) staining
Mouse heart tissues were collected and placed in 4% paraformaldehyde for 24 hours and were later transferred to 70% ethyl alcohol. Each heart tissue was placed into processing cassettes, passed through a gradient alcoholic series for dehydration and finally embedded in paraffin wax. Prior to immunostaining, heart tissue sections (5 μm) were dewaxed using xylene, rehydrated through reverse alcoholic gradient washed with PBS, and finally stained using haematoxylin and eosin.

| Echocardiography
Trans-thoracic echocardiograms of conscious-sedated mice were performed following the previously described methods 8,9 .

| Statistical analysis
The data obtained in this study were subjected to statistical analyses and are shown as the mean ± standard deviation (SD). One-way analysis of variance (1-way ANOVA) and LSD-t tests were applied to determine the significance of the results, where P < 0.05 was defined as statistically significant.

| Pressure overload-induced cardiac hypertrophy caused by TAB in mice
To investigate the TAC-induced myocardial hypertrophy in mouse hearts, we exposed the mice to pressure overload through TAB, and a histological examination was performed on the mouse heart tissues. The TAC mouse heart data obtained from stereoscopic analysis and haematoxylin and eosin staining showed apparent enlargement compared with those of the sham group. Myocardial hypertrophy was present, especially in the interventricular septum and left ventricle ( Figure 1A, 1). Moreover, haematoxylin and eosin staining revealed diffuse hypertrophy by myocytes, while nuclei in the myocardial cells were significantly inflated compared with those of the sham group ( Figure 1C). To assess the body-weight effect on heart size, we examined the cardiac mass index (CMI) and lung mass index (LMI) in this study. The results demonstrated that CMI was apparently increased in TAC mice compared to sham group mice, while LMI had no defined difference in the same mouse hearts (Table 1).
To further explore the changes in myocardial hypertrophy at the molecular and gene levels, we assessed biomarkers of myocardial F I G U R E 1 Anacardic acid (AA) blunts pressure overload-induced hypertrophy in mouse hearts. Stereoscope and haematoxylin and eosin staining analyses of the mouse hearts treated by thoracic aortic banding (TAB) showed that they were significantly inflated compared with those of the sham group, but myocardial hypertrophy, particularly in the left ventricular and interventricular septum, was observed in the transverse aortic constriction (TAC) mice. The HAT activity was increased significantly in the TAC mouse hearts compared to that of the sham group, but HAT activity was reduced in the hearts of mice treated with AA. *P < 0.05 vs the sham group, # P < 0.05 vs TAC (n = 6) TA B L E 1 The comparison of CMI and LMI in the hearts of mice (n = 6) hypertrophy in the TAC mouse hearts. The data showed that during hypertrophic growth in the hearts, expression of the β-MHC gene was significantly enhanced, while ANP was improved ( Figure 1D, 1).

| AA inhibits pressure overload-induced cardiac hypertrophy
To examine the effect of the HAT inhibitor AA on pressure overload-mediated cardiac hypertrophy, we exposed the mice to TAB, and the molecular structural formula of Chinese AA herbal extracts was defined ( Figure 1G). The optimal exposure dose of AA was first determined. TAC mice were injected intraperitoneally with various concentrations of AA (0, 1.25, 2.5, 3.75, 5.0 and 6.25 mg/kg) based on previous reports. 10 An optimum concentration (3.75 mg/ kg) was defined based on the ac-H3 level in the hearts of TAC mice ( Figure 1H). After operation, mice were randomly injected intraperitoneally with either AA or vehicle (Veh). Another group of mice was assigned as the sham operation group, which was injected with Veh every third day for up to 3 weeks. We observed pressure overloadinduced cardiac hypertrophy mediated by TAB in mouse hearts, and administration of AA (3.75 mg/kg) significantly suppressed hypertrophic growth measured as the heart mass normalized to the body mass ( Figure 1I), but the lung mass showed no change in the same samples ( Figure 1J).

| The HAT activity and echocardiography results in the hypertrophic hearts induced by TAC
Some evidence has suggested that the imbalance of histone acetylation modification is involved in cardiac hypertrophy induced by TAC.
To determine whether hyperacetylation of H3K9ac induced by HAT might be a vital factor in promoting myocardial hypertrophy, we tested the HAT activity in hypertrophic mouse hearts. Colorimetric assays revealed that HAT activity was significantly increased in TAC mice hearts ( Figure 1K). Meanwhile, the echocardiography results also demonstrated that AA could attenuate pressure overload cardiac hypertrophy induced by TAB in the mouse hearts (Figure 2A-C). These data suggested that AA attenuated pressure overload-induced cardiac hypertrophy. However, the potential mechanisms remain unknown. Next, we explored the potential mechanisms from an epigenetic perspective.

| The regulatory relationship between HAT and the transcription factor MEF2A
We further explored the effects of p300-HAT and PCAF-HAT, which control histone acetylation by regulating stress The ChIP-Q-PCR assays showed that AA significantly reduced the binding of p300-HAT and PCAF-HAT at the MEF2A promoter in TAC + AA mice compared with TAC + Veh mice ( Figure 3B).
Therefore, we have suggested that the H3K9ac level on the MEF2A promoter also decreased in TAC + AA mice compared to TAC + Veh mice. As expected, similar results were obtained when we examined H3K9ac level on the MEF2A promoter in the same samples ( Figure 3C). Meanwhile, the Western blot data showed that decreases in p300 and PCAF were observed in the TAC mice treated with AA compared to the TAC mice, but there was no significant change in the TAC + Veh mice ( Figure 3D,E). In previous experiments, we demonstrated that p300 and PCAF are involved in controlling the transcription of MEF2A using ChIP-PCR and that AA could inhibit the overexpression of p300-HAT and PCAF-HAT in the mouse heart. Thus, we have suggested that the level of histone acetylation was inhibited in the same samples. Western blots demonstrated that AA attenuated the hyperacetylation of H3K9ac at the translational level in TAC mice, as expected ( Figure 3F). In addition, ac-H4 was tested in the mouse heart, and immunoblot data showed that the ac-H4 level was increased in the hearts of mice treated with TAB compared to that of the sham group. Interestingly, AA significantly decreased the ac-H4 level in the hearts of TAC mice ( Figure 3G).

| AA decreases the transcriptional activity of MEF2A and normalizes the overexpression of downstream cardiac hypertrophic genes
MEF2A is a critical transcription factor that is involved in heart development, cardiac hypertrophy and many other cardiovascular diseases. Thus we first tested the mRNA expression of the MEF2A

| AA improves survival rate and cardiac function in the hearts of TAC mice
For clinical use of the HAT inhibitor AA, it is important to evaluate its long-term efficacy and tolerability. To explore this issue, we studied mice subjected to TAB or sham operation and treated them with AA (3.75 mg/kg, every 3 days) for 8 weeks, a period roughly corresponding to 6 to 8 years in humans. In this study, exposure to AA was well tolerated throughout the study (8 weeks (Table 2). Additionally, LVEF was observed in the mouse hearts, and the data showed that LVEF was increased significantly in TAC mice as time increased, but after 4 months of operation, LVEF was apparently decreased in TAC mice, perhaps due to heart failure. Importantly, LVEF in the TAC mice treated with AA had no apparent change compared with that of the sham group, and heart failure was not observed in the TAC mice treated with AA ( Figure 5B).

| D ISCUSS I ON
In this study, we demonstrated that pressure overload-mediated cardiac hypertrophy in TAC mice, in which histone acetylation played a significant role in myocardial hypertrophy. Additionally, we demonstrated that AA, a Chinese herbal extract, could attenuate cardiac hypertrophy by repressing HAT function, subsequently decreasing histone acetylation.
In fact, haemodynamic stress has been suggested to cause cardiac hypertrophy, resulting in cardiac malfunctioning and abrupt failure. [11][12][13] This process corresponds to the expression of genes produce stress responses. 23,24 A recent study confirmed that HAT p300 is required for cardiac myocyte gene expression. 6 Our results showed that the imbalance of histone acetylation (H3K9ac) induced by p300 and PCAF (isoforms of HATs) could be involved in promoting myocardial hypertrophy in the hearts of TAC mice. We also showed that MEF2A mRNA expression was significantly increased in the hypertrophic hearts of TAC mice. These results suggest that MEF2A is a vital transcription factor contributing to myocardial hypertrophy by pressure overload. However, AA administration could inhibit MEF2A transcription by decreasing H3K9ac hyperacetylation through repression of PCAF and p300 at the MEF2A promoters.
Interestingly, AA also normalized the overexpression of myocardial hypertrophy biomarker genes, for example, ANP and β-MHC, in the cardiac tissue of TAC mice. Our data suggest that H3K9 hyperacetylation is mediated by PCAF-HAT and p300-HAT and plays a significant role in pressure overload-induced cardiac hypertrophy. The findings are consistent with previous reports that the imbalance of histone H3K9ac modification induced by p300 and PCAF is involved to involve in pathological cardiac hypertrophy. 25,26 In our previous studies, the imbalance in acetylation of histones facilitated by HAT (PCAF, p300) was related to cardiac hypertrophy-mediated phenylephrine. 27