ERK signaling pathway in vivo and vitro

Accepted Manuscript Caffeic acid phenethyl ester attenuates pathological cardiac hypertrophy by regulation of MEK/ERK signaling pathway in vivo and vitro

Jie Ren, Nan Zhang, Haihan Liao, Si Chen, Ling Xu, Jing Li, Zheng Yang, Wei Deng, Qizhu Tang PII: DOI: Reference:

S0024-3205(17)30196-0 doi: 10.1016/j.lfs.2017.04.016 LFS 15188

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Received date: Revised date: Accepted date:

19 December 2016 20 April 2017 22 April 2017

Please cite this article as: Jie Ren, Nan Zhang, Haihan Liao, Si Chen, Ling Xu, Jing Li, Zheng Yang, Wei Deng, Qizhu Tang , Caffeic acid phenethyl ester attenuates pathological cardiac hypertrophy by regulation of MEK/ERK signaling pathway in vivo and vitro. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.04.016

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ACCEPTED MANUSCRIPT Caffeic acid phenethyl ester attenuates pathological cardiac hypertrophy by regulation of MEK/ERK signaling pathway in vivo and vitro Jie Ren1,2*, Nan Zhang1,2*, Haihan Liao1,2, Si Chen1,2, Ling Xu3, Jing Li1,2, Zheng Yang1,2, Wei Deng1,2, Qizhu Tang1,2#

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1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei

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Province, P. R. China,

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2 Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China.

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3 Department of Cardiology, The First Affiliated Hospital of Yangtze University,

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Jingzhou ,P.R .China.

Corresponding author: Qi-Zhu Tang, Department of Cardiology, Renmin Hospital of

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Wuhan University; Cardiovascular Research Institute,Wuhan University at Jiefang

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Road 238, Wuhan 430060, China. Tel.: +86 2788073385; Fax: +86 2788042292. E-mail: [email protected]

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Jie Ren and Nan Zhang contributed equally to this work.

Abstract

Aim: To explore the effects of caffeic acid phenethyl ester (CAPE) on cardiac hypertrophy induced by pressure overload. Main methods: Male wild-type C57 mice, aged 8-10 weeks, were used for aortic banding (AB) to induce cardiac hypertrophy. CAPE or (resveratrol) RS was administered from the 3rd day after AB surgery for 6 weeks. Echocardiography and

ACCEPTED MANUSCRIPT hemodynamic analysis were performed to estimate cardiac function. Mice hearts were collected for H&E and PSR staining. Western blot analysis and quantitative PCR were performed for to investigate molecular mechanism. We further confirmed our findings in H9c2 cardiac fibroblasts treated with PE or CAPE.

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Key findings: CAPE protected against cardiac hypertrophy induced by pressure

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overload, as evidenced by inhibition of cardiac hypertrophy and improvement in

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mouse cardiac function. The effect of CAPE on cardiac hypertrophy was mediated via inhibition of the MEK/ERK and TGFβ-Smad signaling pathways. We also

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demonstrated that CAPE protected H9c2 cells from PE-induced hypertrophy in vitro

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via a similar molecular mechanism as seen in the mouse heart Finally, CAPE seemed to be as effective as RS for treatment of pressure overload induced mouse cardiac

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hypertrophy.

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Significance: Our results suggest that CAPE may play an important role in the regulation of cardiac hypertrophy induced by pressure overload via negative

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regulation of the MEK/ERK and TGFβ/Smad signaling pathways. These results

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indicate that CAPE could potentially be used for treatment of cardiac hypertrophy.

Keywords: Caffeic Acid Phenethyl Ester; Cardiac hypertrophy; Cardiac fibrosis; MEK/ERK; TGFβ/Smad.

ACCEPTED MANUSCRIPT 1. Introduction Cardiac hypertrophy is a common pathway of pathological stimulus such as excessive pressure overload, sympathetic activation, and aberrant expression of myocardial contractile proteins caused by gene mutations1. It is a complex process involving the

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activation or inhibition of multiple signaling pathways. The maladjustment of these

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signaling pathways eventually promotes the progression of pathological cardiac

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hypertrophy evidenced by increased left ventricle volume, accumulation of proteins, and re-expression of fetal genes resulting in cardiac dysfunction and heart failure.

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Available reports have pointed out that the MAPK signaling pathway plays important

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roles in the regulation of cardiac hypertrophy2. ERK1/2, JNK1/2, and p38 can be activated by oxidative stress, pressure overload, and neurohumoral factors3. MAPK

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has been considered a therapeutic target for intervention in cardiac hypertrophy.

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Caffeic acid phenethyl ester (CAPE), a natural flavonoid-derivative, is an active phenolic part of bee propolis which has been used in folk medicine in Asia since

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ancient times. CAPE has been shown to have extensive biological effects, by

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modulating processes such as immune responses, cell proliferation, and apoptosis4, and there have been several reports on attempts to investigate the underlying mechanisms. Recent publications have illustrated that its anti-inflammatory effect was more apparent compared to other components of propolis, because it strongly modulates the arachidonic acid cascade5. Its anti-inflammatory function is also associated with the reduction of c-jun-N-terminal kinase and nuclear factor kappa-B (NF-κB), and the down-regulation of cyclooxygenase (COX)-2 expression. CAPE has

ACCEPTED MANUSCRIPT also been suggested to be involved in the regulation of the MAPK signaling pathway6, 7

. In human breast cancer MCF-7 cells, CAPE activates the phosphorylation of p38

and JNK leading to apoptosis through activation of Fas6, while in mouse intestinal epithelial cells, CAPE appears to be p38-independent for the regulation of

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inflammation mediated by TNF-α. In HepG2 cells, CAPE regulates oxidative stress

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associated with the post-translational phosphorylation of ERK8. These reports

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demonstrate that CAPE is closely linked to the regulation of the MAPK signaling pathway, but the roles of CAPE appear to be context-dependent.

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Epidemiological study has shown that regular honey intake is associated with a

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reduced risk of cardiovascular diseases9. It was also reported that CAPE shows cardioprotective effects in short-term myocardial ischemia in rats by reducing

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activities of xanthine oxidase (XO) and adenosine deaminase (ADA), and direct

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antioxidant effects10. The suggested mechanism was a reduction of cardiomyocyte apoptosis via CAPE-mediated inhibition of p38 MAPK activation and caspase-3

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activity, along with reduction of the proinflammatory cytokines (IL-1b and TNF-a) in

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cardiac tissues11. Therefore, CAPE-mediated protection of cardiac myocytes from I/R injury is possibly through suppression of both inflammatory signaling and cell death. However, the molecular mechanisms underlying cardiac hypertrophy are different from those of I/R injury. The purpose of the current study was to investigate the effects of CAPE on cardiac hypertrophy induced by pressure overload, and the underlying mechanisms.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Chemicals Caffeic acid phenethyl ester (CAPE) (>98% purity) was purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China).

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2.2. Animal models

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The animal protocol was approved by the Animal Care and Use Committee of

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Renmin Hospital of Wuhan University (protocol number: 00013274 Wuhan, China) and was in accordance with the Guide for the Care and Use of Laboratory Animals

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published by the US National Institutes of Health (NIH Publication No. 85-23,

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revised 1996). Forty male mice (C57 background) aged 8-10 weeks (weight, 23.5-27.5 g) were used in this study. The C57 male mice were purchased from the

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Institute of Laboratory Animal Science, CAMS & PUMC (Beijing, China). All

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animals were allowed to acclimatize to the laboratory environment for 1 week. Aortic banding (AB)12 was done to induce cardiac hypertrophy following reported

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methods13. Briefly, mice were anaesthetized and a horizontal skin incision was

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made at the 2-3 intercostal space. The descending aorta was isolated and a 24-gauge needle was placed next to the aorta, and then a 7-0 silk suture was tied around the needle and the aorta. Finally, the needle was quickly removed after ligation. The mice in sham groups underwent the same procedure without ligation. All animals were randomly assigned to four groups, 10 mice in each group: in the blank control group (CON), mice were treated with normal saline (NS) containing 0.5% carboxymethylcellulose; in the caffeic acid phenethyl ester treatment group (CAPE),

ACCEPTED MANUSCRIPT mice were treated with CAPE suspension at 100 mg/kg/day by oral gavage once a day; in the aortic banding group (AB), mice were treated with normal saline (NS) containing 0.5% carboxymethylcellulose; in the AB + CAPE group, mice were treated with CAPE suspension at 100 mg/kg/day by oral gavage once a day. All of

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these treatments began 3 days after AB surgery, for a period of 6 weeks. The dose of

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CAPE administered was based on the protocol described in a previous study14.

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CAPE suspension at a concentration of 10 mg/ml was prepared in 0.5% carboxymethylcellulose normal saline (NS) for the animal experiments.

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Suspensions were freshly prepared and administered at 100 mg/kg/day by oral

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gavage once a day. Six weeks after surgery, the mice were euthanized with 1.5% isoflurane for echocardiography and hemodynamics testing, and then the mice were

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sacrificed using cervical dislocation. Hearts, lungs, and tibiae of the sacrificed mice

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were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) ratio (in mg/g), the HW/tibial length (TL) ratio (in mg/mm) and the

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lung weight (LW)/BW (in mg/g) ratio in the different groups. All surgeries and

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analyses were performed in a blinded manner. To further validate the effects of CAPE in preventing the mouse heart from cardiac hypertrophy, we selected a well-known compound, Resveratrol (RS)15, for comparing the treatment effects between CAPE and RS. Briefly, the RS (100mg/kg.d) and CAPE was administration for mouse with or without AB surgery in a period of 4 weeks. And then, echocardiography was performed to evaluate the mouse function. The mouse hearts, LW and BW were used to assay the cardiac hypertrophy among different

ACCEPTED MANUSCRIPT group. This experiment was designed into 5 groups：CAPE treatment group (CAPE), RS treatment group (RS), AB surgery group (AB), AB+CAPE group and AB+RS group.

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2.3. Echocardiography and hemodynamics

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Echocardiography measurements were performed using a MyLab 30CV ultrasound

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(Biosound Esaote Inc.) with a 10-MHz linear array ultrasound transducer. The left ventricle (LV) dimensions were evaluated in the parasternal short-axis view.

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End-systole and end-diastole were identified as the phases in which the smallest and

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largest areas of the LV were obtained, respectively. The ejection fraction (EF) and fractional shortening (FS) were calculated based on the detected parameters.

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For hemodynamics determination, a micro-tip catheter transducer (SPR-839, Millar

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Instruments, Houston, Texas, USA) was inserted into the right carotid artery and advanced into the left ventricle. The pressure signals and heart rate were recorded

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continuously using an ARIA pressure-volume conductance system coupled to a

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Powerlab/4SP A/D converter. The signals were continuously recorded by a Millar Pressure Volume system, and the maximal rate of pressure development (dP/dtmax) and minimal rate of pressure decay (dP/dtmin) were processed using PVAN data analysis software16. 2.4. Histological analysis Hearts of mice were excised, arrested in diastole with 10% KCl to ensure that they were stopped in diastole, and placed in 10% formalin. After rehydration, heart tissue

ACCEPTED MANUSCRIPT sections (5 mm) were prepared and stained with hematoxylin-eosin (H&E) for histopathology, or Picrosirius red (PSR) for interstitial fibrosis, detected by light microscopy. A single myocyte was observed using an image quantitative digital analysis system (Image-Pro Plus 6.0). The outline of 150 myocytes in the left

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ventricle was traced for evaluation of cardiomyocyte hypertrophy in each group.

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2.5. Western blot

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LV tissues were lysed in RIPA lysis buffer. The lysates were placed on ice for 15 min, followed by centrifugation at 12000 g for 30 min at 4oC. The isolated proteins were

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quantified using a BCA Protein Assay Kit. Fifty micrograms of the extracted protein

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was used for SDS-PAGE gel electrophoresis. Subsequently, the protein blots were transferred to nitrocellulose membrane and were blocked with 5% (w/v) non-fat milk

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for 1 h at room temperature. Then, the protein blots were incubated with the specific

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primary antibodies overnight. The blots were then incubated with the secondary antibody for 1 h at room temperature in the dark. Finally, immunoblots were scanned

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using an Odyssey Imaging System. The expression levels of specific phosphorylated

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proteins were normalized to total protein or GAPDH on the same membrane. The following primary antibodies were used in this study: GAPDH (#2118), p-MRK (#9154S), T-MRK1/2 (#9122S), p-ERK1/2thr202/Tyr2041/2 (#4370p), T-ERK1/2(#4695), p-p38 (#4511), T-p38 (#9212), p-JNKT183/Y185 (#4668p), T-JNK (#9258), TGF-β (SC-9053), p-Smad1/5Ser463/465 (#9516), T-Smad1/5 (SC-6210), p-Smad3 (#8769), and T-Smad3 (SC-101154). 2.6. Quantitative real time RT-PCR

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ACCEPTED MANUSCRIPT Total RNA was isolated to detect the mRNA expression levels from the frozen heart tissue using TRIzol (Invitrogen). cDNA was synthesized from 2 µg RNA of each sample using the Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). The PCR amplifications were performed using a LightCycler 480

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SYBR Green 1 Master Mix (Roche, 04707516001). In order to examine the relative

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mRNA expression of Tollip, atrial natriuretic peptide (ANP), brain natriuretic peptide

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(BNP), β-MHC, α-MHC, TGF-β, Collagen Iα, and Collagen III, quantitative RT-PCR analysis was performed using the LightCycler 480 SYBR Green 1 Master Mix (Roche,

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04707516001) and the LightCycler 480 QPCR System (Roche). The target gene

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2.7. Cell culture and treatment

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mRNA expression levels were normalized to GAPDH. (table 1).

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H9c2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Chinese Academy of Sciences, Shanghai, China) with 1%

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penicillin and 1% streptomycin at a temperature of 37˚C. Cells were seeded into

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24-well or 100 mm cell culture dishes for subsequent experiments. H9c2 cardiomyocytes were seeded on chamber slides at a density of 1×105/ml and allowed to adhere for 24 h. The cells were then serum-starved for 12 h, and then incubated in medium with or without CAPE (20 µM) for another 12 h. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature and subsequently permeabilized with 0.3% Triton X-100 at 4oC for 5 min. After washing with PBS and blocking with 5% goat serum for 1 h at 37oC, the slides were incubated with rabbit polyclonal

ACCEPTED MANUSCRIPT anti-α-actin antibody (1:100) overnight. The slides were then washed with PBS and incubated with the secondary antibody conjugated to Alexa fluor 488 (1:200), and then with DAPI for 10 min before observation. The cell surface area was measured using an Eclipse E800 microscope (Nikon Nederland, Amsterdam, the Netherlands).

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The software IPP was used for calculation of cell areas.

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H9c2 cardiomyocytes were dissociated with 0.125% trypsin during the exponential

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phase and seeded in six-well culture plates at a density of 1x106/well, and incubated for 24 h. Cells were incubated in medium with or without CAPE (20 μM) for another

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30 min. Cellular proteins were extracted for detection of the MEK/ERK signaling

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pathway. In in vitro experiments, phenylephrine hydrochloride (PE, Sigma, PHR 1695) was dissolved in DMSO to obtain a stock concentration of 50 mM. A final

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concentration of 50 μM was used for inducing H9c2 hypertrophy in vitro. In the

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cell-based experiments, cells in the treatment groups were treated with PE after 2 h

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3. Results

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of treatment with CAPE.

3.1. Effects of CAPE against cardiac hypertrophy To investigate whether CAPE could effectively attenuate cardiac hypertrophy induced by pressure overload, mice were subjected to aortic banding (AB). HE staining indicated increased left ventricular and cardiomyocyte areas in the AB group, but decreased left ventricular and cardiomyocyte areas in the AB+CAPE group (Fig. 1). Six weeks after the AB surgery, mice allocated to the AB group showed significantly

ACCEPTED MANUSCRIPT increasing ratios of BW, HW, LW, HW/BW, HW/TL, and LW/BW, but treatment with CAPE significantly decreased these ratios (P<0.05) (Fig.2). Moreover, the mRNA expression of cardiac hypertrophy markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), α-myosin heavy chain (α-MHC), and β-myosin heavy

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chain (β-MHC) was also reduced in the AB+CAPE group compared to the AB group

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(P<0.05) (Fig.3). No differences in morphology and molecular markers were detected

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between the CON and CAPE groups.

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3.2. CAPE-mediated improvement in impaired cardiac function

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Echocardiography was performed to assay cardiac function of mice. Six weeks of AB led to decreased LV ejection fraction and LV fractional shortening, and increased

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LVDd, LVDs, LVPWD, and LVPWS resulting in damage to cardiac function in the

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mice. Administration of CAPE protected cardiac function in mice from damage, evidenced by increased LV ejection fraction (LVEF) and LV fractional shortening

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(LVFS), and decreased LVDd, LVDs, LVPWD, and LVPWS (P<0.05) (Fig.4). No

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statistically significant differences were detected between the control and CAPE groups (P>0.05). Hemodynamic assessment was used for further evaluation of cardiac function in mice. Treatment with CAPE markedly protected the hearts of mice from decompensation, evidenced by dP/dtmax, dP/dtmin, ESP and EDP, and shown in Fig. 5, while the HR showed nosatistically significant difference among different groups (P<0.05). (table 2.)

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3.3. CAPE-mediated attenuation of cardiac fibrosis Previous studies have demonstrated that cardiac fibrosis is a cumulative consequence of cardiac hypertrophy and heart failure. In this study, mice subjected to AB showed

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perivascular and interstitial fibrosis. As Figure 6 A shows, oral administration of

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CAPE could attenuate the fibrotic response and collagen deposition volume in hearts

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of mice (P<0.05). To further illustrate the effect of CAPE on cardiac fibrosis, we quantified the expression of fibrosis markers. Real-time PCR results showed that the

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mRNA expression of collagen I and collagen III was obviously decreased in the

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AB+CAPE group compared with the AB group (P<0.05) (Fig. 6B, 6C). LV collagen

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volume fraction also showed a similar result (Fig. 6D).

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3.4. CAPE-mediated regulation of the MAPK and TGF-β/Smad signaling pathways

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As mentioned earlier, the MAPK signaling pathway plays very important roles in the

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regulation of cardiac hypertrophy, and CAPE has been implicated in the regulation of the MAPK signaling pathway. We detected phosphorylation of MAPK family members (JNK, ERK, and p38). We found that all three members showed a high level of phosphorylation under conditions of pressure overload. However, treatment with CAPE significantly blunted the phosphorylation of ERK1/2. We further observed the inhibition of phosphorylation of MEK1/2 (P<0.05) (Fig.7), the upstream regulatory molecule.

ACCEPTED MANUSCRIPT The TGF-β/Smad signaling pathway has been demonstrated to be one of the key pathways for regulation of cardiac fibrosis. Consistent with the marked changes in fibrosis shown in figure 3, we also detected changes in the TGF-β/Smad signaling pathway. Our results showed that pressure overload stimulated the activation of the

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TGF-β/Smad signaling pathway, which was effectively inhibited by CAPE (P<0.05)

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(Fig. 8).

3.5. Effects of CAPE against PE-induced in vitro cardiomyocyte hypertrophy

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After 24 h of incubation with PE (50 µM), the surface of H9c2 cells was enlarged in

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the PE treatment group, but the surface enlargement in the PE+CAPE group was significantly restricted (Fig. 9 A and B).. Because the in vivo investigation

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demonstrated that CAPE inhibited hyperactivity of the MEK/ERK signaling pathway,

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we attempted to validate this in vitro as well. We found that PE significantly activated the MEK/ERK signaling pathway, and treatment with CAPE significantly blunted the

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activity of the PE-activated MEK/ERK signaling pathway (P<0.05) (Fig. 9 C and D).

3.6 Comparison of treatment effects between CAPE and RS RS have been demonstrated to be effective for treatment of cardiac hypertrophy and heart failure15, 17. We selected RS as an internal control to further validate the treating effects of CAPE in preventing cardiac hypertrophy. Interestingly, our data indicated that CAPE seemed to be as effective as RS in attenuating cardiac hypertrophy in mouse heart (Table 3 and 4). Both of CAPE and RS could protected the mouse heart

ACCEPTED MANUSCRIPT from pressure overload induced hypertrophy, but no statistically significant difference about cardiac hypertrophy and function were detected between the RS and CAPE groups. 4. Discussion

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In the present study, we aimed to investigate the role of CAPE in myocardial

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hypertrophy induced by pressure overload and the underling mechanism in vivo and in

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vitro. Our study demonstrated that CAPE protected hearts from pathological hypertrophy, fibrosis, and cardiac dysfunction, as shown by the decreased HW/BW

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ratio, HW/TL ratio, LVDd, LVDs, PWD, and collagen deposition, and increased EF

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and FS in animal models. We also found that CAPE exerted its anti-hypertrophic effect through down-regulation of the MEK/ERK signaling pathway. Finally, CAPE

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hypertrophic mouse.

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seemed to be as effective as RS in a period of 4 weeks' treatment in cardiac

CAPE, a bioactive compound of bee propolis extract, has been demonstrated to 18

, through which it exerts

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possess anti-inflammatory and antioxidant properties

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protective effects in many diseases. In a mouse obesity model induced by high-fat diet, CAPE ameliorated inflammatory damage through reduction of the expression of nuclear factor kappa B, c-jun-N-terminal kinase and cyclooxygenase (COX)-219. Oxidative stress has been implicated in cardiac remodeling in various pathological processes. CAPE administration decreased the levels of malondialdehyde (MDA) in aged rat heart20. Early treatment with CAPE also protected the rat hearts from radiation-induced damage owing to its anti-oxidative property, as evidenced by

ACCEPTED MANUSCRIPT down-regulation of MDA, oxidase (XO), and adenosine deaminase (ADA), and up-regulation of nitrate/nitrite and superoxide dismutase (SOD)21. The anti-oxidative stress mechanism might be mediated by the over-induction of heme oxygenase-1 (HO-1)22. Further, its function was established in other cardiac pathophysiological

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processes independent of its anti-oxidative property. In guinea-pig heart, CAPE

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showed anti-arrhythmic effects by the preferential inhibition of Ca (2+) inward and

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Na (+) inward currents23. Finally, CAPE treatment has been demonstrated to be involved in the regulation of cadmium-induced atrial and ventricular hypertrophy, in

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which the protective effects of CAPE might be at least partly associated with the

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inhibition of lipid peroxidation (LPO), MDA, and nitric oxide (NO)24．However, the benefits of CAPE treatment have not been reported in the regulation of pressure

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overload-induced cardiac hypertrophy.

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As is well-known, a comprehensive set of intracellular signaling pathways are involved in the regulation of pressure overload-induced cardiac hypertrophy25. The

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mitogen activated protein kinase (MAPK) signaling pathway, including p38, JNK,

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and ERK1/2 has been widely implicated in cardiac hypertrophy. In vitro studies on isolated myocytes revealed that activation of p38 promoted hypertrophy, and pharmacological inhibition or genetic blockade of p38 protected against cardiac hypertrophy26, 27. It was also reported that pressure overload-hypertrophy induced by AB resulted in activation of JNK and its downstream target transcription factors ATF-2 and c-Jun. Conversely, inhibition of JNK using DN-MKK4 prevented JNK activation in intact hearts during ET-1-induced hypertrophy28. As mentioned

ACCEPTED MANUSCRIPT previously, growth factors stimulate ERK1/2 signaling through RTKs and GPCRs to promote cardiac hypertrophy, while down-regulation of signaling molecules from the Ras-ERK pathway attenuated cardiac hypertrophy29. In accordance with previous findings, our study reveals that MEK/ERK serves as a

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pivotal regulator of cardiac hypertrophy development and plays an important role in

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the anti-hypertrophic effect of CAPE. We observed that the phosphorylation level of

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ERK1/2 was markedly inhibited in mice subjected to aortic banding. However, CAPE did not affect the phosphorylation of p38 and JNK1/2, which indicated that ERK1/2

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was the only downstream target of CAPE in cardiac remodeling. The ERK pathway is

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a critical regulator of cellular antioxidant activity, proliferation, stress responsiveness, and apoptosis30. Previous studies showed that CAPE may activate Nrf2 through

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activation of ERK, contributing to HO-1 induction and the resulting antioxidant

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activity in hepatic HepG2 cells8. However, in this study, we demonstrated that CAPE-mediated inhibition of cardiac hypertrophy in vivo and H9c2 hypertrophy in

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vitro depend on the regulation of the MEK/ERK signaling pathway. In this context,

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more research is essential to investigate the molecular mechanism of CAPE in different organs and pathological processes. Cardiac fibrosis involves a variety of cells including mainly fibroblasts, macrophages, and endothelial cells. Secreted cytokines stimulate the myofibroblast and induce the accumulation of extracellular matrix. CAPE has been tested in vivo to decrease pulmonary fibrosis in rats 31. Another report showed that the pro-fibrotic properties of transforming growth factor on human fibroblasts are counteracted by CAPE through

ACCEPTED MANUSCRIPT inhibition of myofibroblast formation and collagen synthesis32. Cardiac fibrosis, an important characteristic of cardiac hypertrophy, usually results from chronic stress damage to the myocardium together with the accumulation of extracellular matrix proteins33. The TGF-β/Smad signaling pathway is reported to participate in the

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progression of cardiac hypertrophy and cardiac fibrosis34. Overexpression of TGF-β

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in transgenic mice leads to cardiac hypertrophy and fibrosis, whereas blockade of

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TGF-β with neutralizing antibodies inhibits hypertrophic and fibrotic responses35. Our results are in agreement with previous research on the effect of CAPE in alleviating

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myocardial fibrosis. CAPE markedly decreased interstitial fibrosis in histological

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5. Conclusion

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collagen III expression in vivo.

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analysis and was associated with attenuated TGF-β, Smad1/5, Smad3, collagen I, and

In summary, the results of the present study demonstrated for the first time that CAPE

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protects against cardiac hypertrophy in vivo and in vitro by blocking the MEK/ERK

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signaling pathway. In addition, CAPE also inhibited fibrosis through modulation of TGFβ-Smad signal transduction. Our results provide experimental evidence for the application of CAPE in the treatment of cardiac hypertrophy and heart failure. Clinical studies are needed to address the potential clinical use of CAPE.

ACCEPTED MANUSCRIPT Acknowledgment Funding This work was supported by grants from the National Natural Science Foundation of China (81270303, 81470516, 81470402) and the Fundamental Research Funds for the

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Central Universities of China (No. 2015301020202).

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Boisvenue J, Soltys CL, Oudit GY, Dyck JR. Resveratrol treatment of mice

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Mansour HH, Tawfik SS. Early treatment of radiation-induced heart damage in rats by caffeic acid phenethyl ester. Eur J Pharmacol. 2012;692:46-51 Motawi TK, Darwish HA, Abd El Tawab AM. Effects of caffeic acid

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Effect of CAPE against cardiac hypertrophy. Gross hearts and hematoxylin and eosin (H&E) staining of heart sections from control and AB mice at 6 weeks after

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surgery.

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Figure 2. CAPE alleviates cardiac hypertrophy. (A) mean area of cardiomyocytes; (A)

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heart weight (HW) ratio; (B) lung weight (LW) ratio; (C) heart weight (HW)/lung weight (LW) ratio and (D) heart weight (HW)/tibia length (HW/TL) ratio. Control

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(CON), aortic banding (AB), *P<0.05 compared to Con group, #P<0.05 compared to

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AB group.

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Figure 3. CAPE inhibits the expression of mRNA of cardiac hypertrophy markers. (A)

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atrial natriuretic P (ANP), (B) B-type natriuretic peptide (BNP), (C) alpha-myosin heavy chain (α-MHC) and (D) beta-myosin heavy chain (β-MHC). Each of them was

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group.

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normalized to GAPDH; *P<0.05 compared to Con group, #P<0.05 compared to AB

Figure 4. CAPE improves cardiac function. (A) left ventricular (LV) end-diastolic diameters (LVDD), (B) LV posterior end-diastolic wall thickness (LVPWd); (C) LV end-systolic diameter (LVDS); (D) LV posterior end-systolic wall thickness (LVPWs) (C) ejection fraction (EF) and (D) shorting Fraction (FS); *P<0.05 compared to Con group, #P<0.05 compared to AB group.

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Figure 5. Hemodynamic analysis. (A) maximal rate of pressure development (dp/dt max), (B) minimal rate of pressure decay (dp/dt min), (C) end-systolic pressure (ESP), (D) end-diastolic pressure (EDP), (E) heart rate (HR), *P<0.05 compared to Con

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group, #P<0.05 compared to AB group.

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Figure 6. CAPE attenuates cardiac fibrosis. (A) Picrosirius red (PSR) staining of left ventricle, fibrosis is seen in red; (B) mRNA expression of collagen I; (C) mRNA

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expression of collagen III; (D) computed areas of PSR staining, *P<0.05 compared to

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Con group, #P<0.05 compared to AB group.

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Figure 7. CAPE inhibits the MER/ERK signaling pathway. (A) Immunoblots of

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p-MEK1/2, T-MEK1/2, p-ERK1/2, T-ERK1/2, p-JNK1/2, T-JNK1/2, p-P38, and T-P38 in the indicated groups; (B) computed expression of proteins in the different

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groups. *P<0.05 compared to Con group, #P<0.05 compared to AB group.

Figure 8. CAPE inhibits the TGF-β/Smad signaling pathway. (A) Immunoblots of TGF-β, p-Smad1/5, T-Smad1/5, p-Smad3, T-Smad3, and GAPDH in the indicated groups; (B) computed expression of proteins in the different groups. *P<0.05 compared to Con group, #P<0.05 compared to AB group.

Figure 9. CAPE inhibits H9c2 hypertrophy in vitro. (A) immunofluorescence showing

ACCEPTED MANUSCRIPT the surface area of H9c2s in the indicated groups; (B) estimation of cell areas; (C) Immunoblots of p-MEK1/2, T-MEK1/2, p-ERK1/2, and T-ERK1/2 in the indicated groups; (D) computed expression of proteins in the different groups. *P<0.05

transforming growth factor-β1(TGF-β1); α-myosin heavy

chain (α-MHC);β-myosin heavy chain (β-MHC).

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natriuretic peptide (BNP);

atrial natriuretic peptide (ANP); B-type

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Table 1. The primers used in this study .

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compared to Con group, #P<0.05 compared to PE group.

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Table2. Improvement of cardiac function by treatment of CAPE. body weight (BW) ; LV posterior end-diastolic

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left ventricular (LV) end-diastolic diameters (LVDd),

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wall thickness (LVPWs); ejection fraction (EF) and shorting Fraction (FS); *P<0.05

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compared to CON group , #P<0.05 compared to AB group. Table 3. Attenuated cardiac hypertrophy compared between CAPE and RS. body

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weight (BW); heart weight (HW); lung weight (LW). aortic banding (AB), caffeic

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acid phenethyl ester (CAPE), resveratrol (RS). *P<0.05 compared to CAPE or RS group, #P<0.05 compared to AB group. Table 4. Improvement of cardiac function compared between CAPE and RS. left ventricular (LV) end-diastolic diameters (LVDd),

LV posterior end-diastolic wall

thickness (LVPWd); LV end-systolic diameter (LVDs); LV posterior end-systolic wall thickness (LVPWs); ejection fraction (EF) and shorting Fraction (FS); *P<0.05 compared to CAPE or RS group, #P<0.05 compared to AB group.

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ACCEPTED MANUSCRIPT Table 1 Gene

The primers used in this study

Forward (5’-3’)

Reverse (5’-3’)

Name ANP

ACCTGCACCACCTGGAGGAG

CCTTGGCTGTTATCTTCGGTACC

GAGGTCACTCCTATCCTCTGG

GCCATTTCCTCCGACTTTTCTC

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TCAATCACTGTCTTGCCCCA

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Collage CCTCAAGGGCTCCAACGAG n Iα

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n III

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Collage ACGTAGATGAATTGGGATGCA GGGTTGGGGCAGTCTAGTC

AGGGTCTGCTGGAGAGGTTA

β-MHC CCGAGTCCCAGGTCAACAA

CTTCACGGGCACCCTTGGA

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α-MHC GTCCAAGTTCCGCAAGGT

ACCEPTED MANUSCRIPT Table 2

Improvement of cardiac function by

treatment of CAPE CAPE

AB

AB+CAPE

N

8

8

8

8

LVDd(mm)

3.31±0.12

3.58±0.14

5.00±0.13* 4.37±0.14#

LVDs(mm)

1.69±0.13

1.93±0.05

3.80±0.07* 3.07±0.14#

LVPWd(mm)

0.68±0.07

0.73±0.04

1.17±0.05* 0.816±0.02#

LVPWs(mm)

1.01±0.02

1.02±0.14

FS(%)

46.00±1.99

46.25±2.58 23.75±0.99* 30.00±0.72#

EF(%)

81.25±2.16

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1.33±0.03* 1.20±0.37#

78.87±1.95 47.87±2.62* 57.37±1.32#

ACCEPTED MANUSCRIPT Table 3 Attenuated cardiac hypertrophy

RS

AB

AB+CAPE

AB+RS

6

6

10

10

10

BW（g)

27.5±2.5

26.1±1.7

27.3±2.7

26.9±1.5

26.5±3.3

HW(mg)

113.7±5.5

115.3±4.7

187.9±15.6* 163.3±13.1#

155.4±8.3#

LW(mg)

123.3±11.2 121.4±9.9

167.7±22.4* 143.9±11.7#

140.9±7.9#

4.4±0.27

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6.9±0.31*

6.05±0.45#

5.7±0.29#

6.20±1.38*

5.41±1.57#

5.40±1.71#

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HW/BW(mg/g) 4.3±0.21

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LW/BW(mg/g) 4.47±.33

4.63±0.19

ACCEPTED MANUSCRIPT Table 4 Improvement of cardiac function compared between CAPE and RS RS

AB

AB+CAPE

AB+RS

N

6

6

10

10

10

LVDd(mm)

3.85±0.11

3.87±0.12

4.51±0.23*

4.13±0.14#

LVDs(mm)

2.23±0.09

2.19±0.10

3.13±0.19*

LVPWd(mm) 0.68±0.07

0.68±0.04

0.93±0.13*

LVPWs(mm) 0.93±0.05

0.92±0.04

4.07±0.10#

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1.41±0.11*

2.66±0.17#

2.51±0.15#

0.76±0.09#

0.74±0.08#

1.15±0.06#

1.11±0.11#

42.07±1.38 43.5±1.42

30.59±3.33* 36.61±2.11#

38.32±3.17#

EF(%)

73.65±3.07 76.58±2.56 59.44±4.39* 65.27±3.51#

69.84±2.77#

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FS(%)