Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Abstract Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.


| INTRODUC TI ON
Cardiac hypertrophy is usually considered to be a compensatory response to mechanical and neurohumoral stimuli. In the early stages, hypertrophy of cardiomyocytes and thickening of the myocardium contribute to improvements in contractile function. However, the continued presence of pathological stress often leads to myocardial interstitial fibrosis, contractile depression and ventricular dilatation, ultimately resulting in heart failure 1,2 and sudden death. 3,4 Therefore, cardiac hypertrophy is currently considered to be an independent risk factor for cardiovascular events.
Autophagy is a highly conserved lysosomal degradation pathway that helps to maintain cellular homoeostasis. 5 Recently, accumulating evidence 6,7 has suggested that autophagy plays an important role in the pathogenesis of cardiac remodelling and heart failure.
Although basal autophagy is adaptive and beneficial, excessive or persistent autophagy can be maladaptive and harmful. 8 Numerous studies 6,7,9 suggest that autophagy may be triggered in response to pathological stresses, such as pressure overload, and excess of such stresses may lead to cell death. 10 Thus, regulating autophagy could be important for treating pathological cardiac remodelling and heart failure.
Derived from the natural compound apigenin, sodium (±)-5bromo-2-(a-hydroxypentyl) benzoate (generic name: brozopine, BZP) is a potential cerebrovascular and cardiovascular drug that was approved for clinical trials for stroke treatment by the China Food and Drug Administration (CFDA). 11 BZP exhibits potent neuroprotective effects against ischaemic stroke, which may be related to its antioxidative effects 12 and inhibition of apoptosis. 13 As is well-known, autophagy can be induced by oxidative stress, 14,15 and antioxidants play a protective role in pressure overload-induced cardiac hypertrophy. [16][17][18] Besides, an increasing number of studies have revealed that crosstalk exists between autophagic and apoptotic machinery. 19,20 Therefore, it is reasonable to hypothesize that BZP may affect pressure overload-induced hypertrophy and autophagy.
In the present study, we investigated the effect of BZP on cardiac remodelling and autophagy regulation with regard to the hypertrophic response. We found that BZP could inhibit angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and excessive autophagy through AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signalling and could ameliorate pressure overload-induced cardiac hypertrophy and heart dysfunction.

| Experimental animals and transverse aortic constriction (TAC) surgery
Animal protocols were approved by the Ethics Committee of Zhengzhou University. Male C57/BL6 mice (8 weeks old, 17-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The animals were housed at room temperature under a 12 hours light/dark cycle and provided a normal diet and purified water ad libitum. Animals were acclimatized to the laboratory environment for at least 7 days before the experiments.
Mice were subjected to TAC or a sham operation under anaesthesia (isoflurane, inhalation) as previously described. 21 After anaesthesia and artificial ventilation were initiated, the transverse aorta was constricted by ligating the aorta with a 7-0 nylon suture around a blunted 27-gauge needle. The needle was removed immediately after the procedure. Mice in the sham group underwent all operation procedures except for the ligation. The effectiveness of aortic constriction was confirmed by performing echocardiography, and only mice with a pressure gradient over the aortic ligature ranging from 50 to 70 mm Hg were used in further experiments.
BZP bulk drug (purity 99.4%) was synthesized at the College of Chemistry and Molecular Engineering, Zhengzhou University (Zhengzhou, China). After the establishment of the TAC model, animals were treated with 20 mg/kg BZP or vehicle control by oral gavage once a day for 10 weeks.

| Echocardiography analysis
To measure global cardiac function, echocardiography was performed at baseline and at 2, 4 and 10 weeks after TAC. Echocardiography was performed with an ultrasonic echocardiographic system (Vevo2100, VisualSonics Inc, Toronto, Canada). Briefly, after the chests of mice were shaved, the mice were fixed and underwent two-dimensional echocardiography without anaesthesia. All parameters were evaluated by calculating the average of five cardiac cycles.
After treatment, the cells were harvested for analysis. LY294002, Compound C and metformin were purchased from MedChemExpress.

| Histological analysis
Following anaesthesia, the heart was excised and immediately placed in 4% paraformaldehyde at room temperature for 24 hours.
The myocardial specimens were embedded in paraffin and cut into 4 μm sections. Serial heart sections were stained with haematoxylin and eosin (H&E) or wheat germ agglutinin (WGA) to measure myocyte cross-sectional areas (CSAs). The degree of cardiac fibrosis was detected by Masson's trichrome staining. The fibrotic areas were stained blue, and normal tissues were stained red. Images were analysed using a quantitative digital image analysis system (Image-Pro Plus 6.0).

| Electron microscopy
Cardiac tissue was cut into 1 mm cubes immediately after the heart was excised. Tissue blocks or H9c2 cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C.
After fixation, the selections were immersed in 1% buffered osmium tetroxide for 2 hours. The specimens were then dehydrated through a graded ethanol series and embedded in epoxy resin. After that, the specimens were sliced into ultrathin sections (60-70 nm) with an ultramicrotome and post-stained with uranyl acetate and lead citrate.
Then, the sections were examined under an H-800 electron microscope (Hitachi, Tokyo, Japan).

| Quantitative real-time PCR (qRT-PCR) and Western blot analysis
Total RNA in NRCMs and tissues was extracted using TRIzol reagent (Invitrogen), and first-stand cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qRT-PCR was performed with FastStart Universal SYBR Green Master (Roche) to examine the relative mRNA levels of the indicated genes as previously described. 23 The sequences of the qRT-PCR primers are shown in Table 1.
For Western blot analysis, heart tissues and NRCMs were lysed in RIPA buffer (Solarbio). Proteins were isolated as previously described, 24 and the lysates (30 μg) were subjected to 10% SDS-PAGE and transferred to NC membranes. The membranes were then incubated with specific antibodies against different antigens overnight at 4°C. Then, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO) and visualized with X-ray film.

| Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. Student's t test was performed for analysis of two groups and one-way ANOVA followed by Newman-Keuls multiple comparison test for three or more groups. P < 0.05 was considered statistically significant.

| Ang II-induced hypertrophy and autophagy in cardiomyocytes
NRCMs were incubated with Ang II (1 μM) for 48 hours to induce cardiomyocyte hypertrophy. α-Sarcomeric actin staining and qRT-PCR were performed to validate the cardiomyocyte hypertrophic model. As shown in Figure 1A,1, Ang II stimulation significantly increased cell surface area. The average cell surface area in Ang II group was twice that in Control group. In addition, the qRT-PCR data ( Figure 1C) revealed that Ang II treatment clearly increased the mRNA expression levels of atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP), which are hypertrophic genes.
As shown in Figure 1D Figure 1H,I).
These data indicated that Ang II stimulation obviously induced cardiomyocyte hypertrophy and triggered autophagy.

| BZP attenuated Ang II-induced autophagy in cardiomyocytes
To investigate the effects of BZP on Ang II-induced autophagy, we performed Western blotting, immunofluorescence staining and electron microscopy. As shown in Figure 2A,2, exposure to BZP decreased the elevated LC3 II and Beclin-1 levels induced by Ang II in a dose-dependent manner in NRCMs. Moreover, a marked increase in p62 was also observed. Next, we used LY294002 as a positive control and examined the effect of BZP on Ang II-induced autophagy.
As expected, there was significantly lower protein expression of LC3 II, Beclin-1 and higher expression of p62 in the BZP treatment group and in the LY294002 treatment group than in the Ang II-only group ( Figure 2C,2).
Consistent with the results of the Western blotting assay, the immunofluorescence staining ( Figure 2E,2) and microscopic images ( Figure 2G,H) showed similar trends. These data indicated that Ang II-induced autophagy was significantly suppressed by BZP.

| BZP ameliorated Ang II-induced hypertrophy in NRCMs
To determine whether BZP could ameliorate cardiomyocyte hypertrophy induced by Ang II, we performed immunofluorescence staining and qRT-PCR. As shown in Figure 3A,3, cell surface area was dramatically increased by Ang II treatment, but BZP significantly prevented this increase. In addition, we used the autophagy inhibitor LY294002 as a positive control and found that LY294002 treatment also hindered the enlargement of cell size.
Next, the qRT-PCR data revealed that Ang II treatment clearly increased the mRNA expression levels of ANF and BNP, while BZP or LY294002 treatment reduced the Ang II-induced increases in the mRNA expression of these genes ( Figure 3C,D).

| BZP alleviated TAC-induced cardiac hypertrophy in mice
To investigate the in vivo effects of BZP on cardiac hypertrophy, we established an animal model by performing TAC surgery. Four weeks of TAC caused significant hypertrophy in the C57BL/6 mice.
At 4 weeks after TAC surgery, the heart weight/body weight (HW/ BW) and heart weight/tibia length (HW/TL) ratios were significantly lower in the BZP-treated group than in the TAC-only group. However, the lung weight/body weight (LW/BW) ratios did not vary among the three groups ( Figure 4C). In addition, the H&E staining and WGA staining results for myocyte CSAs also confirmed the protective effects of BZP against TAC-induced hypertrophy ( Figure 4A,D).
Subsequent analysis of the mRNA expression levels of hypertrophic genes showed similar trends ( Figure 4F).
Cardiac hypertrophy at 10 weeks after TAC surgery was more severe than that at 4 weeks after TAC surgery ( Figure 4A

| BZP ameliorated TAC-induced cardiac dysfunction in mice
To gain further insight into the effects of BZP, we evaluated cardiac function in the three groups of mice at different time points. A few representative short-axis views from each group are shown ( Figure 5A). According to echocardiographic evaluation ( Figure 5B) Figure 6C,6). These data indicate that BZP attenuated pressure overload-induced autophagy overactivation in vivo.

| BZP suppressed autophagy by suppressing the AMPK-mTOR signalling pathway
To elucidate the underlying molecular mechanism and associated signalling pathways of autophagy inhibition by BZP, components of the AMPK-mTOR signalling pathway were assayed by Western blotting. As shown in Figure 7A,7, significant inhibition of p-mTOR activity was observed in the Ang II group, indicating that suppression of mTOR contributes to the induction of autophagy by Ang II. In contrast, BZP and LY294002 reversed this process. In addition, BZP and LY294002 reduced the Ang II-induced phosphorylation of AMPKα, which is an upstream signal of mTORC1. Our results suggested that BZP suppressed Ang II-induced autophagy, which is mediated by the AMPK-mTOR signalling pathway.
We further investigated the role of metformin (a specific AMPK activator) and Compound C (a specific AMPK inhibitor) in the BZP-induced suppression of autophagy. Similar to Compound C, BZP acted as an AMPKα phosphorylation inhibitor and ultimately resulted in autophagy inhibition ( Figure 7C,7). Compared to the BZP + Ang II group, metformin reversed the BZP-induced suppression of autophagy, which was related to its strong activating effect on AMPK phosphorylation ( Figure 7C,7). These data suggested that BZP suppressed Ang II-induced autophagy by suppressing the AMPK-mTOR signalling pathway.

| D ISCUSS I ON
In this study, we established an in vitro cardiomyocyte hypertrophic model using Ang II treatment as well as a mouse model of myocardial hypertrophy using the TAC operation. Our in vitro data showed that Ang II-induced cardiomyocyte hypertrophy was accompanied by excessive autophagy (Figure 1). Furthermore, we confirmed that autophagic activity was increased in heart tissue due to the TAC operation ( Figure 6). We also demonstrated that BZP inhibited excessive autophagic activity both in vitro and in vivo (Figures 2 and 6), ameliorated cardiomyocyte hypertrophy ( Figure 3), alleviated TAC-induced cardiac hypertrophy and dysfunction (Figures 4 and 5), and suppressed autophagy via suppressing the AMPK-mTOR signalling pathway (Figure 7). Our results strongly suggest that BZP is a potential drug candidate for treatment of pressure overload-induced cardiac hypertrophy, and To explore the underlying mechanism by which BZP suppressed autophagy, we investigated intracellular signalling pathways. One of the important functions of autophagy is energy recycling. 33   . The data are expressed as the mean ± SEM. *P ＜ 0.05, compared to the Sham group; # P ＜ 0.05, compared to the TAC group inhibition of maladaptive cardiomyocyte autophagy through the AMPK-mTOR signalling pathway. Our findings also suggest that BZP may be a good candidate compound for treatment of pressure overload-induced cardiac remodeling and heart failure.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. F I G U R E 7 BZP suppressed autophagy by suppressing the AMPK-mTOR signalling pathway. (A-B) NRCMs were pre-treated with or without BZP (250 μM) or LY294002 (5 μM) for 2 h and exposed to Ang II (1 μM) for 48 h. p-AMPK/AMPK and p-mTOR/mTOR protein expressions are shown in Western blots and are presented in a bar graph (n = 3). (C-D) NRCMs were pre-treated with or without BZP (250 μM) or Compound C (5 μM) for 2 h and exposed to Ang II (1 μM) for 48 h. Metformin (10 mM) was added for 0.5 h before the addition of BZP. The expression of LC3 I/II, p62 and key factors in the AMPK/mTOR pathway are shown in Western blots and are presented in a bar graph (n = 3