Polydatin prevents hypertrophy in phenylephrine induced neonatal mouse cardiomyocytes and pressure-overload mouse models

European Journal of Pharmacology 746 (2015) 186–197

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Polydatin prevents hypertrophy in phenylephrine induced neonatal mouse cardiomyocytes and pressure-overload mouse models Ming Dong a,1, Wenwen Ding b,1, Yansong Liao c, Ye Liu d, Dewen Yan e, Yi Zhang a, Rongming Wang a, Na Zheng a, Shuaiye Liu a, Jie Liu a,n a

Medical College, Shenzhen University, Shenzhen 518000, Guangdong, China Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China c Cardiology Division, Department of Medicine, The University of Hongkong, Hong Kong, China d Department of Anatomy, Hebei Medical University, Hebei 050017, China e Department of Endocrinology, The First Affiliated Hospital of Shenzhen University, Shenzhen 518060, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 16 October 2014 Received in revised form 7 November 2014 Accepted 11 November 2014 Available online 20 November 2014

Recent evidence suggests that polydatin (PD), a resveratrol glucoside, may have beneficial actions on the cardiac hypertrophy. Therefore, the current study focused on the underlying mechanism of the PD antihypertrophic effect in cultured cardiomyocytes and in progression from cardiac hypertrophy to heart failure in vivo. Experiments were performed on cultured neonatal rat, ventricular myocytes as well as adult mice subjected to transverse aortic constriction (TAC). Treatment of cardiomyocytes with phenylephrine for three days produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and atrial natriuretic peptide (ANP) protein expression. These effects were attenuated by PD in a concentration-dependent manner with a complete inhibition of hypertrophy at the concentration of 50 mM. Phenylephrine increased ROCK activity, as well as intracellular reactive oxygen species production and lipid peroxidation. The oxidizing agent DTDP similarly increased Rho kinase (ROCK) activity and induced hypertrophic remodeling. PD treatment inhibited phenylephrine-induced oxidative stress and consequently suppressed ROCK activation in cardiomyocytes. Hypertrophic remodeling and heart failure were demonstrated in mice subjected to 13 weeks of TAC. Upregulation of ROCK signaling pathway was also evident in TAC mice. PD treatment significantly attenuated the increased ROCK activity, associated with a markedly reduced hypertrophic response and improved cardiac function. Our results demonstrated a robust anti-hypertrophic remodeling effect of polydatin, which is mediated by inhibition of reactive oxygen species dependent ROCK activation. & 2014 Elsevier B.V. All rights reserved.

Keywords: Polydatin Hypertrophy Rho activity Reactive oxygen species

1. Introduction Cardiac hypertrophy is one of the leading causes of increased cardiac morbidity and mortality since it can result in myocardial ischemia, arrhythmia, sudden death and, eventually, heart failure (Levy et al., 1990). Many natural products have been used on heart failure therapy. Polydatin (PD) is a monocrystalline drug that can be isolated from a traditional Chinese herb (Polygonum cuspidatum). The molecular composition of PD is 3, 40 , 5-trihydroxystibene-3-monoglucoside, which is akin to the polyphenol resveratrol (Res) (3, 40 , 5-trihydroxystibene). PD has also been found to be one of the major stilbenoid compounds in red wine.

n

Corresponding author. Fax: þ86 75586671906. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejphar.2014.11.012 0014-2999/& 2014 Elsevier B.V. All rights reserved.

Similar to its analog resveratrol, PD has multiple biological activities in a cardiovascular and hematological system. The cardioprotection of PD may be related to activating cNOS, leading to an increase in NO production (Zhang et al., 2008) and decrease in apoptosis (Zhang et al., 2009). It can enhance heart function, improve microcirculatory perfusion in shock (Zhao et al., 2003) and survival rate in severe shock (Wang et al., 2013). It has shown that PD can cross the blood–brain barrier to protect brain tissues from ischemia–reperfusion injury or cerebral ischemia (Cheng et al., 2006; Su and Hsieh, 2011). Most of the studies thus far have focused on the beneficial effects of PD in the prevention of ischemia–reperfusion injuries and few concerns of its possible use as a therapeutic drug in hypertrophy. The only study of Gao et al. (2010) has firstly studied polydatin on pressure-overload rat models. However, the underlying mechanism of the beneficial effect is not completely clear.

M. Dong et al. / European Journal of Pharmacology 746 (2015) 186–197

Rho-kinase (ROCK), a target protein of the small GTP-binding protein Rho, has been shown to be involved in a number of models of cardiac hypertrophy (Hamid et al., 2007; Phrommintikul et al., 2008; Zeidan et al., 2006) and it can be activated by a variety of stimuli including angiotensin II (Aikawa et al., 2000), leptin (Zeidan et al., 2006), and stretch (Pan et al., 2005). Upon activation by hypertrophic stimuli, RhoA signals through downstream kinases (ROCK) to phosphorylate and inhibit the actin depolymerizing protein cofilin, thereby increasing filamentous actin. Of interest, pharmacological inhibition of RhoA (C3 exoenzyme), ROCK (Y-27632), or actin polymerization (latrunculin B) is sufficient to prevent cardiac hypertrophy induced by a variety of stimuli (Aikawa et al., 2000; Zeidan et al., 2006). Although the pro-hypertrophic mechanism of RhoA/ROCK pathway signaling remains unclear, it has been suggested that actin reorganization may signal via p38 MAPK and serum response factor to mediate hypertrophy (Kuwahara et al., 2007; Zeidan et al., 2008). Thus, these data suggest that the RhoA/ROCK signaling cascade may be a final common pathway to mediate the development of cardiac hypertrophy and heart failure and as such represents a logical target for therapeutic intervention. Evidences indicate that ROCK signaling pathway is activated by reactive oxygen species. Given that PD has prominent antioxidant activity, we speculated that PD may prevent the pathogenesis of hypertrophy by inhibiting ROCK activity via suppressing intracellular oxidative stress. The goal of the present study was to investigate the possible mechanisms of polydatin on ventricular remodeling induced by phenylephrine in neonatal rat cardiomyocytes and by transverse aortic constriction in mice. Here, we demonstrated a robust antihypertrophic and antiremodeling effect of polydatin, which is mediated by inhibition of reactive oxygen species dependent ROCK activation.

2. Materials and methods 2.1. Drugs and reagents Polydatin (with a purity of 98.87%) was supplied by Haiwang Co. (Shenzhen, Guangdong, China). The antioxidant MnTBAP (Manganese (III) Tetrakis (4-Benzoic Acid) Porphyrin) was from Biochemical Santa Cruz (MnTBAP chloride sc-221954A) and oxidizing agent DTDP (2,20 -dithiodipyridine) was from Sigma (Sigma 43791). L-NAME (NO inhibitor) was from Sigma. Because ROCK inactivates myosin phosphatase through the specific phosphorylation of myosin phosphatase target subunit 1 (MBS or MYPT1) atThr-696, which results in an increase in the phosphorylated content of the 20-kDa myosin light chain (MLC20) in this study, we used the ratio of pMBS to MBS to represent the ROCK activity which is the most widely used method to test ROCK activity. Primary monoclonal antibodies for western blots and immunostaining were anti-myosin phosphatase target subunit-1 (MYPT-1), phosphor T853-MYPT1 and atrial natriuretic peptide (ANP) from Santa Cruz Biotechnology (USA, all diluted: 1:1000) and β-actin from Sigma. Masson's trichrome kit was from Sigma HT15-1KT. RhoA activity assay kit (ab173237) was from USA. Briefly, the antiactive RhoA mouse monoclonal antibody was incubated with cell lysates containing RhoA-GTP. The bound active RhoA was pulled down by protein A/G agarose and the precipitated active RhoA was detected by immunoblot analysis using anti-RhoA rabbit polyclonal antibody. The ratio of active RhoA/total RhoA protein represented the activation of RhoA (Liu et al., 2007). 2.2. Cell culture All animal experiments were performed in accordance with ethical standards as formulated in the Guide for the Care and Use of

187

Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of Shenzhen University. Neonatal cardiomyocytes were isolated and cultured using previously described methods (Liao et al., 2012). The ventricular heart from 1-day-old neonatal rats was removed, cut into small chunks and washed with Hanks' balanced salt solution (HBSS). Then, the tissue was incubated in 4 ml trypsin/EDTA solution (GIBCO, Carlsbad, CA) at 4 1C for 30 min with rotation. The digestion was stopped by addition of 6 ml DMEM containing 10% fetal calf serum (FCS, GIBCO, Carlsbad, CA). After centrifugation at 200g for 5 min, the supernatant was removed, and the tissues were incubated in 4 ml Liberase TH (0.1 U/ml in HBSS, Roche Diagnostics GmbH, Mannheim, Germany) at 37 1C for 15 min. The supernatant containing the released cells to DMEM-10% FCS was removed, and fresh Liberase TH was added to the undigested tissues, which were then incubated for a further 15 min. This digestion procedure was repeated until most of the cells had been released from ventricular tissue and the obtained cells were resuspended in DMEM. All collected cells were seeded into fibronectin-coated 12-well tissue culture plates (Costar; Corning, NY). After 1.5 h of incubation with 5% CO2 at 37 1C, the attached fibroblasts were discarded and cardiomyocytes in the supernatant were enriched and seeded into fibronectin-coated tissue culture plates after cell concentration was adjusted. Cardiomyocytes were used in experiments when they had formed a confluent monolayer and beat in synchrony at 72 h. To initiate hypertrophy, myocytes were then treated with10 mM adrenoceptor agonist phenylephrine and then in the absence or presence of polydatin (30, 40, or 50 mM). 2.3. Measurement of cell surface area Myocytes were visualized using a Leica DMIL inverted microscope (Leica, Wetzlar, Germany) equipped with an Infinity 1 camera. At least 10 random photographs were taken from each dish, and the cell surface area of a minimum of 30 cells from each treatment was measured using SigmaScan Software (Systat, Richmond, CA). 2.4. Western blotting Total protein was measured with a bicinchoninic acid assay kit (Pierce) after sonication of the harvested cells. Various protein components were separated in 7.5% polyacrylamide gels, transferred onto nitrocellulose paper, and stained with primary and secondary antibodies. Reactive bands were visualized by the Supersignal ECL Western blotting detection kit (Pierce), and densitometry was obtained in ImageJ software. 2.5. Fluorescence of intracellular reactive oxygen species and measurement of malondialdehyde (MDA) In neonatal rat cardiomyocytes a fluorescentprobe,5-(and-6)chloro-methyldichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes) were used for the assessment of intracellular reactive oxygen species formation as described previously (Jiang et al., 2013). Briefly, cells were loaded with10 μM DCFDA for 30 min at room temperature followed by 15 min for de-esterification. Frame fluorescence images (excitation at 488 nm and emission at 505– 530 nm, laser intensity 4%, 6.6 s/frame) were acquired with a Zeiss510 inverted confocal microscope with 20  lens. Each experiment was performed within 5 min after15 min for de-esterification of DCFDA. The MDA levels were determined using colorimetric assays with commercial kits following the manufacturer's instruction (Nanjing Jiancheng Biotechnology Institute, China). L-NAME was treated as 100 mM for 30 min before MDA testing.

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Fig. 1. Effect of different concentrations of polydatin (30–50 mM) on cell surface area and ANP protein expression in myocytes treated with phenylephrine (20 mM) for 3 days. (A) Representative micrographs of cardiomyocytes (20  ). (B) Cell surface area in control, PD, phenylephrine and different phenylephrine treatment groups. (C) Expression of ANP protein in different PD (50 mM) treatment days. (D) Expression of ANP protein in different PD concentrations (30, 40 and 50 mM) for three days. Data are shown as mean 7S.E.M., nP o0.05 vs control; #P o0.05 vs phenylephrine group, þ P o0.05 vs phenylephrineþ PD1, PD; n¼ 6 and 7. PD, polydatin; PD1, PD2 and PD3 means 30 mM, 40 mM and 50 mM respectively.

2.6. Animals and treatments 10-week-old C57BL/6 male mice 20–22 g supplied by the animal center of Shenzhen Medical University were anesthetized and transverse aortic constriction (TAC) or sham operation was carried out as previously described (Sano et al., 2007). The transverse aorta was constricted with a 7-0 nylon suture by ligating the aorta together with a blunted 26-gauge needle, which was removed later. For post-operative analgesia, the mouse is injected with buprenorphine (0.1 mg/kg) intraperitoneally. Mice

were randomly divided into 3 groups (20 in each group): sham (control), TAC group, TAC plus polydatin). One week after TAC, mice were fed by gavage with 50 mg/kg polydatin daily. The feed time was till to the eighth week (Fig. 1). 2.7. Cardiac weight indexes and morphological examinations After 13 weeks, the heart was taken out. Heart weight (HW) and body weight (BW) were measured, and HW/BW ratio was calculated. Then, the left ventricular tissue was divided into two parts after

M. Dong et al. / European Journal of Pharmacology 746 (2015) 186–197

separating the atria, aorta and adipose tissue. One halfpart was rapidly frozen in liquid nitrogen and then stored at a  70 1C freezer for western blotting. Another halfpart was immersed in formalin (10% formaldehyde) and was dehydrated and embedded in paraffin, and then cut into 5-μm thick slices and heated overnight in a 60 1C incubator. The sections were stained with hematoxylin and eosin (H&E) for measurement. Each sample slice was photographed (5  magnification) under the microscope (Olympus BX51, Olympus, Tokyo, Japan). Myocyte hypertrophy was quantitated by measuring the diameter of 30 randomly selected longitudinal sectioned myocytes at the nuclear level from the histological sections.

189

2.8. Echocardiographic and hemodynamic analysis of cardiac function Mice were anesthetized with 2% isoflurane and placed in a supine position on a heated platform. The chest and abdomen were shaved and the extremities were fixed to electrodes on the platform surface by use of tape and a highly conductive electrode gel. Echocardiography evaluations were performed using a Vevo 2100 high-resolution in vivo microimaging system equipped with a real-time microvisualization scan head of 17.5 MHz (VisualSonics, Toronto, ON, Canada). M-mode 2-dimensional echocardiography images were obtained from the parasternal short axis. Images were analyzed using the Vevo 2100 Protocol-Based Measurements software. Left ventricular (LV) posterior wall thickness in diastolic or systolic (LVPWD, LVPWS), end-diastolic LV internal diameter (LVIDD), and end-systolic LV internal diameter (LVIDS) were measured. All measurements were done from leading edge to leading edge according to the American Society of Echocardiography guidelines (Lang et al., 2005). The percentage of LV fractional shortening (FS) was calculated as [(LVIDD  LVIDS)/ LVIDD]  100. Echocardiographic acquisition and analysis were performed by a technician who was blinded to treatment groups. 2.9. Statistical analysis All values were expressed as mean 7standard error. Differences were evaluated using unpaired Student's t test between two groups and one-way ANOVA for multiple comparisons, followed by a post-hoc Student–Newmann–Keuls test when necessary. All analyses were done using SPSS 15.0 (SPSS, Chicago, IL), and statistical significance was set at P o0.05.

pMBS

tMBS actin

3. Results 3.1. Effect of polydatin on phenylephrine-induced cardiomyocyte hypertrophy Myocytes treated with phenylephrine for three days demonstrated a significant increase in cell surface area from 789791 mm2 to 12017140 mm2 (Fig. 1A and B; Po0.05); whereas administration of 40–50 mmol/l polydatin reduced the enlarged cell surface area to 9027101 mm2 and 842780 mm2 (Fig. 1B; both Po0.05 vs phenylephrine group). No obvious decrease was observed in 30 mmol/l polydatin treatment when compared with phenylephrine group (Fig. 1B). Phenylephrine increased protein expression of ANP by around 2-fold (Fig. 1C; Po0.05) compared with controls. Polydatin treatment inhibited phenylephrine-induced increase of ANP protein level in a dose and time-dependent manner. The increase of ANP protein was almost completely prevented by 50 mM polydatin treatment (Fig. 1C and D; Po0.05). In addition, PD had no direct effect on either parameter in the absence of phenylephrine (Fig. 1B– D). So in this study we chose 50 mmol/l polydatin as the treatment dose. 3.2. Effect of polydatin on phenylephrine-induced RhoA and ROCK activation

Fig. 2. Effect of polydatin in different treatment days (1–3 days) on RhoA activity and the quantification of ROCK activity expression. (A) The fold increase of RhoA activity in control, phenylephrine and different PD treatment groups. (B) The upper is western blotting of pMBS and t(total)MBS in control, phenylephrine and different PD treatment groups. The lower bar chart figure represents the of ROCK activity in different groups. Phosphorylation levels of MBS (pMBS) and total MBS (tMBS) were determined by western blotting and the ratio represents the ROCK activity. Data are shown as mean7S.E.M., nPo0.05 vs control; #Po0.05 vs phenylephrine group; n¼ 5–9. PD, polydatin; PD1, PD2 and PD3 means 30 mM, 40 mM and 50 mM respectively.

Since we and others have shown an important role for the RhoA/ROCK signaling pathway in cardiac hypertrophy (Dong et al., 2012), we sought to determine whether the anti-hypertrophic effects of polydatin were related to inhibition of the ROCK signaling cascade. Fig. 2 shows that phenylephrine induced more than 2-fold and around 1.7-fold increases of RhoA (Fig. 2A) and ROCK activity (Fig. 2B) (all Po 0.05 vs control), which was consistent with the increase of ANP (Fig. 1D) in response to

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Control

phenylephrin

e

DTDP

nt

Co

pMBS tMBS ANP

phenylephrine +PD

phenylephrine +MnTBAP

DTDP+PD

rol

actin

ne hri p e yl DP en ph DT

e ne rin hri ph e D lep l P y y A +P en en ph D ph nTB TDP D +P +M

M. Dong et al. / European Journal of Pharmacology 746 (2015) 186–197

LP A+ Y2 7

ANP

LP A

tro l

63

2

PD e+ e ir n in h hr p p e e yl yl 2 en en 763 h h p p Y2 +

Co n

l

tro

n Co

e in hr p e yl A en h LP p

191

ANP

pMBS tMBS

pMBS tMBS actin

actin

Fig. 4. Effect of RhoA agonist LPA and ROCK inhibitor Y27632 on ROCK activity and reactive oxygen species production in hypertrophic cardiomyocytes induced by phenylephrine. (A) Western blotting of ANP, pMBS and t(total)MBS in control, phenylephrine, LPA, phenylephrine plus PD and phenylephrine plus Y27632. (B) Statistics of MDA contents in various groups. (C) Western blotting of ANP, pMBS and t(total)MBS in control, LPA, LPA plus Y27632. LPA: lysophosphatidic acid, PD: polydatin. nPo 0.05 vs control; #Po 0.05 vs phenylephrine group.

phenylephrine stimulation. The increased RhoA and ROCK activities were both inhibited by the treatment of polydatin, again in a dose-dependent manner. A significant inhibition of RhoA and ROCK activities was observed in 40 and 50 mM of polydatin treatment (P o0.05 vs phenylephrine treatment) but not 30 mM of polydatin treatment. The data suggested that the antihypertrophic effects of polydatin were mediated through inhibition of RhoA/ROCK pathway. 3.3. Effect of polydatin on phenylephrine-induced reactive oxygen species increase It has been suggested that reactive oxygen species involves inactivation of ROCK signaling pathway. To investigate whether PD suppresses ROCK by reducing intracellular reactive oxygen species level, we examined the effects of polydatin on phenylephrineinduced reactive oxygen species production. Imaging DCFDA fluorescence reflecting intracellular reactive oxygen species level in myocytes showed significant increase in intracellular reactive oxygen species levels in phenylephrine treated cardiomyocytes

(3.14 fold vs control, P o0.05) (Fig. 3A). Consistently, lipid peroxidation as indicated by MDA levels was increased by phenylephrine stimulation (1.65 70.09 nmol/mg protein, P o0.05 vs control) (Fig. 3C). The effects of phenylephrine on intracellular reactive oxygen species production were reproduced by the oxidizing agent DTDP (10 μM) (Fig. 3A–C). As anticipated, PD treatment remarkably reduced the increase of intracellular reactive oxygen species level (P o0.05 vs phenylephrine group, Fig. 3A–C), similar to the effects of the antioxidant MnTBAP (100 μM). Moreover, DTDP increased hypertrophic protein ANP and ROCK activity as phenylephrine did, which was attenuated by polydatin, and MnTBAP attenuated the hypertrophic remodeling induced by phenylephrine (Fig. 3D–F). The results collectively indicate the important role of reactive oxygen species–ROCK signaling cascade in phenylephrine induced cardiomyocyte hypertrophy and suppression of this signaling pathway contributes largely to the antihypertrophic effect of PD. In this study, L-NAME was used to find out how PD inhibits reactive oxygen species. A significant increase of MDA was found the in phenylephrine and phenylephrineþ LNAME groups (Fig. 3G). Polydatin could suppress the MDA partly

Fig. 3. Polydatin decreased reactive oxygen species production, ANP protein level and ROCK activity in hypertrophic cardiomyocytes. (A) Representative images of reactive oxygen species-sensitive indicator DCFDA-loaded cells from control, phenylephrine, phenylephrine plus PD, DTDP, phenylephrine plus MnTBAP and DTDP plus PD groups. (B) Averages of DCFDA fluorescence in various groups compared to control. (C) Statistics of MDA contents in various groups. (D) Representative western blots in different groups. (E) Quantification of ANP protein level and (F) ROCK activity in the cells in different groups. (G) Statistics of MDA contents in control, L-NAME, phenylephrine, phenylephrineþ L-NAME, PD, PD þ L-NAME, phenylephrineþPD and phenylephrine þ PD þ L-NAME. Data are expressed as the mean7 S.E.M., nPo 0.05 vs control; #P o 0.05 vs phenylephrine and DTDP. (G) Suppressive effects of L-NAME on nitric oxide production and PD markedly inducing nitric oxide production. nPo 0.05 vs control; #Po 0.05 vs PD. (H) PD significantly suppressing the increase of reactive oxygen species production induced by phenylephrine. nPo 0.05 vs control; #Po 0.05 vs phenylephrine group.

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0

1 wk

TAC

13 wk

8 wk

Tissue preparation

PD

Sham

TAC

TAC+PD

Fig. 5. Body weights and indices of cardiac hypertrophy in animals subjected to either TAC or sham operation, with or without daily polydatin treatment (50 mg/kg) for 13 weeks. (A) Schematic diagram of study timeline. One week after TAC, mice were fed by gavage with 50 mg/kg polydatin daily. The feed time was till to the eighth week. (B) Representative global heart photographs in different groups. Gravimetric data for heart weight (HW), body weight (BW) and HW/BW ratio are shown in (C), (D), and (E), respectively. Data are shown as data are expressed as the mean 7S.E.M. nPo 0.05 vs sham and PD; #Po 0.05 vs phenylephrine; þ P o 0.05 vs PD. PD, polydatin; TAC, transverse aortic constriction.

in phenylephrine þ L-NAME group when compared with phenylephrine group, which means polydatin inhibits reactive oxygen species production partly from NO-mediated pathway (Fig. 3G). In order to investigate that phenylephrine-induced cardiac hypertrophy is indeed due to increase reactive oxygen species induced ROCK activation, we used RhoA agonist lysophosphatidic acid (LPA) and ROCK inhibitor Y27632 in cell culture (Fig. 4A). LPA significantly increased reactive oxygen species production. Furthermore, Y27632 obviously decreased ROCK activity. However, reactive oxygen species production just slightly decreased in phenylephrine plus Y27632 treatment group when compared with only phenylephrine treatment (Fig. 4B). Furthermore, PD prevented ROCK activation and ANP production by reactive oxygen species agonist LPA (Fig. 4C). The present data combined with our previous finding that reactive oxygen species scavenger can inhibit phenylephrine induced reactive oxygen species production and subsequently inhibited ROCK activation and ANP production, collectively indicate that phenylephrine-induced cardiac hypertrophy is indeed due to increase reactive oxygen species induced ROCK activation.

3.4. Effect of polydatin on TAC-induced cardiac hypertrophy and left ventricular dysfunction We investigated the effect of polydatin on cardiac hypertrophy at 13 weeks after TAC. All the experimental mice survived 13 weeks after TAC, and significant cardiac hypertrophy developed by TAC procedure (Fig. 5B). The heart weight and the ratio of heart weight to body weight were consistently increased, while the BW was not significantly changed (Fig. 5C–E). Histology cross sections showed increase of LV wall thickness and expansion of the heart chamber in the TAC group (Fig. 6A). Enlarged size of myocytes and bizarre nuclei was observed in TAC group (8907142 mm2 vs normal group

6347172 mm2, Po0.05, Fig. 6B). Masson-trichrome staining clearly showed increase fibrin accumulation in TAC compared with the normal group (Fig. 6A). Quantitative analysis of fibrotic areas confirmed that PD could reduce the increased fibrosis in the TAC group (Fig. 6C). In TAC groups, in the myocardium multifocal infiltrates associated with myocyte necrosis were clearly seen (Fig. 6A). At the same time, we found increased leukocyte number and infiltration in TAC group and this increase could be suppressed by polydatin (Fig. 6A). The in vivo cardiac structure and function were evaluated in real-time by echocardiography during the 13 weeks of TAC. Cardiac structure assessment involved measurement of left ventricular posterior wall (LVPW) and left ventricular internal dimensions (LVID). Both systolic (LVPWS) and diastolic LVPW (LVPWD) were increased dramatically after 2 weeks of TAC. The LVPWS was increased to a peak 5 weeks of TAC, and then decreased gradually (Table 1). Despite this, the LVPWS 13 weeks after TAC was still significantly larger than that in sham (36.2%, Po0.05 Fig. 7B). The LVPWD increased 48.7% at 13 weeks of TAC relative to sham (Fig. 7B). The LVIDS and LVIDD decreased significantly 5 weeks of TAC (data not shown) and then increased gradually which were significantly larger than control after 13 weeks of TAC (30.8%, 20.0%, Fig. 7B), suggesting that eccentric hypertrophy developed following the concentric hypertrophy. PD treatment significantly attenuated TAC-induced hypertrophic remodeling. LVPW and LVID at both systole and diastole in PD-treated TAC mice were not significantly different from those in sham. The cardiac systolic function indicated by left ventricular fraction shortening (FS) and LV ejection fraction (LVEF) was significantly increased 5 weeks of TAC, but returned to normal 8 weeks after TAC (data not shown) and significantly lower than normal 13 weeks of TAC (Fig. 7B). There was no significant difference of FS and EF between sham and TAC with PD treatment but a significant increase in TAC without PD treatment was observed (Fig. 7B).

M. Dong et al. / European Journal of Pharmacology 746 (2015) 186–197

TAC

Sham

193

TAC+PD

→ ← MT

←

HE

→

←

Fig. 6. (A) The first line pictures are the cross sections of the hearts in sham, TAC and TAC þPD. The middle and the third lines represent the Masson trichrome and hematoxylin-eosin staining, respectively. Fibrosis and infiltrating cells were observed in TAC group clearly (arrows). (B) Note enlarged size of myocyte, bizarre nuclei, myofiber disarray, and abnormal vacuolization in TAC from Masson-trichrome staining. However, no obvious difference was observed between normal and TAC þPD treatment groups. (C) Quantitative analysis of fibrotic area (Masson trichrome-stained area in light blue normalized to total myocardial area; magnification,  400). Data are shown as data are expressed as the mean 7 S.E.M. nPo 0.05 vs sham and PD; #Po 0.05 vs phenylephrine; þ Po 0.05 vs PD. PD, polydatin; TAC, transverse aortic constriction.

3.5. Effect of polydatin on TAC-induced RhoA, ROCK activation, ROCK1, ROCK2 and ANP increase Consistent with in vitro finding, we found that mice subjected to TAC had significantly increased RhoA and ROCK activity (Fig. 8A–C), which was markedly reduced by polydatin (all Po0.05). However, no significant difference of ROCK1 and ROCK2 was found in these four groups (Fig. 8B). The indicator of hypertrophy, ANP protein levels, was significantly increased in hearts subjected to TAC, although this response was largely abrogated by polydatin (Fig. 8D, all Po0.05).

4. Discussion In this study, we studied the therapeutic effects of polydatin on the development of hypertrophy in vitro and in vivo. The major findings in

this study are that polydatin has a robust anti-hypertrophic effect against pressure overload-induced cardiac hypertrophy and heart failure. Meanwhile, PD prevents phenylephrine-induced cardiomyocyte hypertrophy, indicating that one of the possible underlying mechanisms is due to directly targeting on cardiomyocytes to prevent the development of cardiac hypertrophy in vivo. Furthermore, we revealed that PD prevents cardiac hypertrophy might largely through inhibition of the reactive oxygen species–RhoA–ROCK signaling pathway. Our group previously demonstrated that ROCK activity increased in hypertrophic heart failure patients and it might be a complementary biomarker for predicting long-term outcomes and mortality (Dong et al., 2012; Phrommintikul et al., 2008). The RhoA/ROCK signaling cascade has emerged as a potential convergence point for a variety of hypertrophic stimuli suggesting that it may represent a logical target for therapy (Aikawa et al., 2000; Brown et al., 2006; Zeidan et al., 2006). Further support for the role of ROCK in cardiac

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Table 1 Comparison of echocardiographic cardiac dimensions of different sham, TAC, PD and TAC plus PD groups at different timepoints.

LVPWs (mm)

LVPWd (mm)

LVIDs (mm)

LVIDD (mm)

LVFS (%)

EF (%)

Groups

2w

5w

8w

13w

Sham TAC PD TAC þ PD Sham TAC PD TAC þ PD Sham TAC PD TAC þ PD Sham TAC PD TAC þ PD Sham TAC PD TAC þ PD Sham TAC PD TAC þ PD

1.047 0.05 1.36 7 0.05a 0.98 7 0.06 0.977 0.06b 0.677 0.05 0.95 7 0.04a 0.63 7 0.02 0.65 7 0.04b 2.46 7 0.02 1.99 7 0.10a 2.36 7 0.05 2.26 7 0.08b 3.63 7 0.15 3.187 0.07a 3.517 0.04 3.477 0.05 0.337 0.02 0.38 7 0.02a 0.337 0.03 0.357 0.02 0.46 7 0.05 0.54 7 0.07a 0.42 7 0.05 0.52 7 0.05

1.147 0.08 1.647 0.03a 1.077 0.06 1.28 7 0.03b 0.717 0.06 1.02 7 0.06a 0.69 7 0.03 0.79 7 0.04b 2.26 7 0.18 1.53 7 0.12a 2.50 7 0.06 1.86 7 0.13 3.497 0.14 2.65 7 0.14a 3.717 0.04 3.137 0.09 0.32 7 0.03 0.497 0.02 0.357 0.02 0.40 7 0.01 0.52 7 0.02 0.767 0.02a 0.55 7 0.03 0.617 0.01

1.11 70.02 1.51 70.02a 1.11 70.03 1.2470.03b 0.72 70.04 1.01 70.04a 0.7070.02 0.82 70.04b 2.57 70.06 2.48 70.04 2.36 70.02 2.20 70.04b 3.63 70.17 3.61 70.10 3.73 70.04 3.48 70.14b 0.3370.02 0.31 70.02a 0.34 70.02 0.42 70.02b 0.54 70.02 0.53 70.02 0.57 70.03 0.64 70.02b

1.12 70.04 1.38 70.06a 1.0770.05 1.21 70.08b 0.76 70.02 0.99 70.03a 0.73 70.03 0.85 70.03b 2.45 70.15 3.21 70.09a 2.36 70.07 2.32 70.11b 3.73 70.24 4.31 70.07a 3.61 70.04 3.58 70.09b 0.3570.02 0.21 70.02a 0.36 70.02 0.38 70.03b 0.50 70.03 0.38 70.03a 0.55 70.03 0.58 70.02b

PD, polydatin; TAC, transverse aortic constriction. LV, left ventricular; LVPWD, left ventricular posterior wall thickness in diastolic; LVPWS, left ventricular posterior wall thickness in systolic; LVIDD, left ventricular internal diameter in diastolic; LVIDS, left ventricular internal diameter in systolic; FS, fractional shortening; EF, ejection fraction. All parameters are expressed as mean 7 standard division (n¼ 10–20). a b

P o0.05 vs sham and PD treatment group. Po 0.05 vs phenylephrine treatment group.

hypertrophy comes from data indicating that Rho activates fetal genes, such as ANP, and is associated with cardiomyocyte growth (Sah et al., 1996). Thus, the ability of polydatin to suppress ROCK activity in phenylephrine treatment could explain the main mechanism for its anti-hypertrophy action. Many studies have focused on the correlation of ROCK activity and reactive oxygen species. Jernigan et al. (2008) demonstrated that RhoA/ROCK signaling in response to ET-1 is reactive oxygen species mediated. Knock et al. (2009) found that ROCK activation in rat pulmonary arteries could be initiated by LY83583, a superoxide anion generator. There is controversy over the species of reactive oxygen species involved in activating ROCK. For example, Knock et al. showed RhoA is activated selectively by superoxide but not by H2O2. However Chi et al. (2010) demonstrated that treatment with H2O2 can elicit a significant increase of RhoA activity. The discrepancy may be accounted for by different experimental conditions. Nevertheless, there is a consensus that reactive oxygen species have a direct effect on activating ROCK. Multiple studies in human and various animal models of myocardial hypertrophy and failure have demonstrated that the level of oxidative stress is elevated in myocardial hypertrophy and failure because of increased production of reactive oxygen species (Sawyer et al., 2002). In this study, we found that phenylephrine stimulation increased reactive oxygen species production, indicated by increase of intracellular DCFDA fluorescence and MDA concentration in the supernatant of phenylephrine-stimulated cardiomyocytes. Meanwhile, we found that suppressing intracellular reactive oxygen species production inhibited phenylephrine-induced ROCK stimulation. Furthermore, we have provided evidence that the oxidative agent DTDP activated ROCK, which consequently induced hypertrophy. All the data implicate the activation of reactive oxygen species–RhoA/ ROCK signaling axis in the pathogenesis of hypertrophy and reactive oxygen species production might be the upstream of RhoA/ROCK activation It is well documented that PD has a strong anti-hypertrophic effect. The anti-oxidative mechanisms of PD are demonstrated to be related to improving mitochondrial function, increasing NO production and

rebalancing the intracellular nitro-oxidative system, and directly scavenging reactive oxygen species. These have been reported to be related to the therapeutic effects of PD on the treatment of hemorrhagic shock, ischemia–reperfusion injury and regulation of cardiac function. Consistent with previous studies, we found that polydatin decreased reactive oxygen species production, similar to the effect of reactive oxygen species scavenger MnTBAP. This appears to be the major mechanism underlying phenylephrine-induced activation of ROCK, for reactive oxygen species scavenger largely abolished the action of reactive oxygen species–RhoA/ROCK signaling pathway upon phenylephrine stimulation and PD reducing oxidative agent DTDP induced activation of reactive oxygen species–RhoA/ROCK. In this study, we found that PD significantly increased NO production in control and phenylephrine-treated cells. Meanwhile, the data show that NO had a remarkable effect on inhibition of phenylephrine-induced RhoA/ROCK activation. It appears that NO produced by PD inhibited RhoA/ROCK activation through two mechanisms. First, the increased NO reduced intracellular reactive oxygen species level, and subsequently inhibited RhoA/ROCK activation. Supporting evidence in that L-NAME which largely inhibited PD-induced NO production significantly suppressed the effect of PD inhibition of MDA production. Of note, a L-NAME's effect on PD inhibition of MDA production was modest. The increased NO had a much larger effect on suppression of the activated RhoA/ROCK signal than on MDA level. The data indicates the second possible mechanism: PD-induced increase of NO directly inhibits RhoA/ROCK signal. This is consistent with previous report that the anti-hypertrophic effects of NO are due, in part, to cGMP-dependent inhibition of the RhoA–ROCK–cofilin signaling pathway (Hunter et al., 2009). We have a diagram depicting the mechanisms of PD inhibition of RhoA/ROCK pathway through targeting on reactive oxygen species and NO and the interwoven of the two signals (Fig. 8). Previously, Gao et al. (2010) have proved the beneficial effects of polydatin on ventricular remodeling in vivo. But the duration of that study was only one month. In our current research, polydatin was

M. Dong et al. / European Journal of Pharmacology 746 (2015) 186–197

195

Sham

Sham

PD

TAC

TAC+PD

Fig. 7. Effect of polydatin on TAC-induced hypertrophy in left ventricular function. Animals were subjected to either TAC or sham operation, with or without daily polydatin treatment (100 mg/kg) for 13 weeks. (A) Short-axis biventricular M-mode images. (B) Quantification of end-diastolic and end-systolic LVID and LVPW, LVEF and FS. Data are shown as mean 7S.E.M. nPo 0.05 vs sham or sham plus PD; #Po 0.05 vs TAC, n ¼10. TAC indicates transverse aortic constriction; LVID, left ventricular internal diameter; PD, polydatin; LVIDD, end-diastolic LVID; LIVDS, end-systolic LVID; LVPWD, Left ventricular posterior wall, diastolic; LVPWS, Left ventricular posterior wall, systolic; LVEF, left ventricular ejection function; FS, fractional shortening.

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TAC

PD

ˉ

ˇ ˉ

Reactive Oxygen Species

↑

NO ˉ RhoA/ROCK↑ Sham

PD

TAC

TAC+PD

AMPK

pMBS tMBS ROCK1 ROCK2 ANP actin

Fig. 8. RhoA activity and protein expressions of ROCK activity, ROCK1, ROCK2 and ANP in animals of TAC or sham operation, with or without daily polydatin treatment (50 mg/kg) for 13 weeks. (A) RhoA activity, western blotting of pBMS, tMBS and ANP (B), ROCK activity was represented as the ratio of pMBS to tMBS; Statistics analysis of ROCK activity (C) and ANP (D). Data are shown as data are expressed as the mean 7S.E.M. nP o0.05 vs sham and PD. PD; #Po 0.05 vs phenylephrine. PD, polydatin; TAC, transverse aortic constriction.

treated in C57 mice for a longer period, around three months. TAC causes a decrease of blood flow which leads the hypoxia in cardiac myocytes. The lasting hypoxic exposure can cause a considerable induction of reactive oxygen species leading to adaptive cardiac hypertrophy. Using the TAC mice model, we proved that oral administration of polydatin reduced hypertrophy. The oral dose of polydatin was 50 mg/kg/day which had the optimal beneficial effect on this

Hypertrophy Fig. 9. Protective regulation mechanism of polydatin (PD) in myocardium hypertrophy. The RhoA/ROCK cascade inhibition by PD directly or indirectly via reactive oxygen species pathways. PD, polydatin; TAC, transverse aortic constriction; reactive oxygen species, reactive oxygen species; NO, nitric oxide; ROCK, Rho Kinase; AMPK, AMP-activated protein kinase.

model. Polydatin medication is a new therapy, and permission has been obtained for phase II clinical trials from the Chinese Food and Drug Administration. The dose used in our study is the middle of the dosing spectrum in vivo, which can range between 10 and 100 g/day. Moreover, the improved left ventricular function was associated with diminished hypertrophy determined by relative molecular markers. Regarding the development of heart failure, polydatin could prevent the hypertrophy from pressure overload in our study. Transition to heart failure developed 13 weeks after onset of pressure overload in mice, associated with a decrease in FS and LVEF. Polydatin clearly prevented this disease progression. Although mechanistic insights are more difficult to establish using in vivo approaches, the beneficial effect of polydatin seen in this model demonstrated substantial concordance with that in cultured myocytes. A previous study showed that the administration of polydatin can restore the pulse pressure and increase survival rate in irreversible shock and it can promote capillary reflow (Zhao, 2005). Recently, Zhao et al. demonstrated that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect on severe shock, which was better than resveratrol or cyclosporin A (Wang et al., 2012). Interestingly, the potential benefit derived from polydatin's anti-oxidative and anti-inflammation actions was supported by Zhang et al.'s (2012) study. Thus we believed that there should be other potential systemic effect of PD that may help alleviate the cardiac hypertrophy in vivo (Fig. 9). Finally, in our study we have demonstrated that polydatin has a potent antihypertrophic effect in vitro, as well as a highly effective ability to reduce hypertrophy and heart failure in vivo. Inhibition of reactive oxygen species production by polydatin may also reduce adverse remodeling and improve LV contractile function, and may therefore hold therapeutic potential for the treatment of chronic heart failure. However, the possibility that polydatin is acting through other or additional nonspecific mechanisms cannot be ruled out and this requires further studies. 4.1. Limitation Multiple signaling pathways such as calcineurin, MAPK or AKT participate in the pathogenesis of hypertrophy. In our recent study,

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polydatin (PD) could modulate Ca2 þ handling and excitation– contraction coupling in rat ventricular myocytes (Deng et al., 2012) which suggests PD might affect Calcineurin/NFAT signaling pathway in hypertrophy. RhoA/ROCK pathway should have crosstalk with calcineurin and MAPK. This is a possible limitation of this current report and it will be addressed in future studies. Because the current genome annotation revealed that the gene in the mouse genome is much closer to human genome than rat, mouse was used as animal model in this study. However, the heart of suckling mouse is too small to isolate the ventricular myocytes; neonatal rat myocytes were used for the cell culture experiment. It will be perfect if using the same animal species both in vitro and in vivo, despite rat cardiomyocytes and mouse hypertrophic model have been widely used in previous studies. Although rodents share much similarity in genes with human, it would be better to use cardiomyocytes generated from human induced pluripotent stem cell (Burridge et al., 2014) to study the polydatin hypertrophic protection mechanism in the future work. It is also interesting to verify polydatin in clinic in the future.

Funding This work was supported by Grant from National Natural Science Foundation of China (81202529 and 31171096) and Basic Research Foundation of SZ (Nos. JC201005250059A and JCYJ20120613115535998).

Acknowledgments We appreciated Prof. John Elsby Sanderson for kindly editing this manuscript. References Aikawa, R., Komuro, I., Nagai, R., Yazaki, Y., 2000. Rho plays an important role in angiotensin II-induced hypertrophic responses in cardiac myocytes. Mol. Cell. Biochem. 212, 177–182. Brown, J.H., Del Re, D.P., Sussman, M.A., 2006. The Rac and Rho hall of fame: a decade of hypertrophic signaling hits. Circ. Res. 98, 730–742. Burridge, P.W., Matsa, E., Shukla, P., Lin, Z.C., Churko, J.M., Ebert, A.D., Lan, F., Diecke, S., Huber, B., Mordwinkin, N.M., Plews, J.R., Abilez, O.J., Cui, B., Gold, J.D., Wu, J.C., 2014. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860. Cheng, Y., Zhang, H.T., Sun, L., Guo, S., Ouyang, S., Zhang, Y., Xu, J., 2006. Involvement of cell adhesion molecules in polydatin protection of brain tissues from ischemia–reperfusion injury. Brain Res. 1110, 193–200. Chi, A.Y., Waypa, G.B., Mungai, P.T., Schumacker, P.T., 2010. Prolonged hypoxia increases ROS signaling and RhoA activation in pulmonary artery smooth muscle and endothelial cells. Antioxid. Redox Signal. 12, 603–610. Deng, J., Liu, W., Wang, Y., Dong, M., Zheng, M., Liu, J., 2012. Polydatin modulates Ca2 þ handling, excitation–contraction coupling and beta-adrenergic signaling in rat ventricular myocytes. J. Mol. Cell. Cardiol. 53, 646–656. Dong, M., Liao, J.K., Fang, F., Lee, A.P., Yan, B.P., Liu, M., Yu, C.M., 2012. Increased Rho kinase activity in congestive heart failure. Eur. J. Heart Fail. 14, 965–973. Gao, J.P., Chen, C.X., Gu, W.L., Wu, Q., Wang, Y., Lu, J., 2010. Effects of polydatin on attenuating ventricular remodeling in isoproterenol-induced mouse and pressure-overload rat models. Fitoterapia 81, 953–960. Hamid, S.A., Bower, H.S., Baxter, G.F., 2007. Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am. J. Physiol. Heart Circ. Physiol. 292, H2598–H2606. Hunter, J.C., Zeidan, A., Javadov, S., Kilic, A., Rajapurohitam, V., Karmazyn, M., 2009. Nitric oxide inhibits endothelin-1-induced neonatal cardiomyocyte hypertrophy via a RhoA–ROCK-dependent pathway. J. Mol. Cell. Cardiol. 47, 810–818. Jernigan, N.L., Walker, B.R., Resta, T.C., 2008. Reactive oxygen species mediate RhoA/ Rho kinase-induced Ca2 þ sensitization in pulmonary vascular smooth muscle

197

following chronic hypoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L515–L529. Jiang, X., Liu, W., Deng, J., Lan, L., Xue, X., Zhang, C., Cai, G., Luo, X., Liu, J., 2013. Polydatin protects cardiac function against burn injury by inhibiting sarcoplasmic reticulum Ca2 þ leak by reducing oxidative modification of ryanodine receptors. Free Radic. Biol. Med. 60, 292–299. Knock, G.A., Snetkov, V.A., Shaifta, Y., Connolly, M., Drndarski, S., Noah, A., Pourmahram, G.E., Becker, S., Aaronson, P.I., Ward, J.P., 2009. Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2 þ sensitization. Free Radic. Biol. Med. 46, 633–642. Kuwahara, K., Teg Pipes, G.C., McAnally, J., Richardson, J.A., Hill, J.A., Bassel-Duby, R., Olson, E.N., 2007. Modulation of adverse cardiac remodeling by STARS, a mediator of MEF2 signaling and SRF activity. J. Clin. Investig. 117, 1324–1334. Lang, R.M., Bierig, M., Devereux, R.B., Flachskampf, F.A., Foster, E., Pellikka, P.A., Picard, M.H., Roman, M.J., Seward, J., Shanewise, J.S., Solomon, S.D., Spencer, K.T., Sutton, M.S., Stewart, W.J., Chamber Quantification Writing GroupAmerican Society of Echocardiography's GuidelinesStandards CommitteeEuropean Association of, Echocardiography, 2005. Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J. Am. Soc. Echocardiogr. 18, 1440–1463. Levy, D., Garrison, R.J., Savage, D.D., Kannel, W.B., Castelli, W.P., 1990. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N. Engl. J. Med. 322, 1561–1566. Liao, Y.H., Xia, N., Zhou, S.F., Tang, T.T., Yan, X.X., Lv, B.J., Nie, S.F., Wang, J., Iwakura, Y., Xiao, H., Yuan, J., Jevallee, H., Wei, F., Shi, G.P., Cheng, X., 2012. Interleukin-17A contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and neutrophil infiltration. J. Am. Coll. Cardiol. 59, 420–429. Liu, P.Y., Chen, J.H., Lin, L.J., Liao, J.K., 2007. Increased Rho kinase activity in a Taiwanese population with metabolic syndrome. J. Am. Coll. Cardiol. 49, 1619–1624. Pan, J., Singh, U.S., Takahashi, T., Oka, Y., Palm-Leis, A., Herbelin, B.S., Baker, K.M., 2005. PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes. J. Cell. Physiol. 202, 536–553. Phrommintikul, A., Tran, L., Kompa, A., Wang, B., Adrahtas, A., Cantwell, D., Kelly, D.J., Krum, H., 2008. Effects of a Rho kinase inhibitor on pressure overload induced cardiac hypertrophy and associated diastolic dysfunction. Am. J. Physiol. Heart Circ. Physiol. 294, H1804–H1814. Sah, V.P., Hoshijima, M., Chien, K.R., Brown, J.H., 1996. Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes. Dissociation of Ras and Rho pathways. J. Biol. Chem. 271, 31185–31190. Sano, M., Minamino, T., Toko, H., Miyauchi, H., Orimo, M., Qin, Y., Akazawa, H., Tateno, K., Kayama, Y., Harada, M., Shimizu, I., Asahara, T., Hamada, H., Tomita, S., Molkentin, J.D., Zou, Y., Komuro, I., 2007. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 446, 444–448. Sawyer, D.B., Siwik, D.A., Xiao, L., Pimentel, D.R., Singh, K., Colucci, W.S., 2002. Role of oxidative stress in myocardial hypertrophy and failure. J. Mol. Cell. Cardiol. 34, 379–388. Su, S.Y., Hsieh, C.L., 2011. Anti-inflammatory effects of Chinese medicinal herbs on cerebral ischemia. Chin. Med. 6, 26. Wang, X., Song, R., Bian, H.N., Brunk, U.T., Zhao, M., Zhao, K.S., 2012. Polydatin, a natural polyphenol, protects arterial smooth muscle cells against mitochondrial dysfunction and lysosomal destabilization following hemorrhagic shock. Am. J. Physiol. Regul. Integr. Comp. Physiol. 302, R805–R814. Wang, X., Song, R., Chen, Y., Zhao, M., Zhao, K.S., 2013. Polydatin—a new mitochondria protector for acute severe hemorrhagic shock treatment. Expert Opin. Investig. Drugs 22, 169–179. Zeidan, A., Javadov, S., Chakrabarti, S., Karmazyn, M., 2008. Leptin-induced cardiomyocyte hypertrophy involves selective caveolae and RhoA/ROCK-dependent p38 MAPK translocation to nuclei. Cardiovasc. Res. 77, 64–72. Zeidan, A., Javadov, S., Karmazyn, M., 2006. Essential role of Rho/ROCK-dependent processes and actin dynamics in mediating leptin-induced hypertrophy in rat neonatal ventricular myocytes. Cardiovasc. Res. 72, 101–111. Zhang, H., Yu, C.H., Jiang, Y.P., Peng, C., He, K., Tang, J.Y., Xin, H.L., 2012. Protective effects of polydatin from Polygonum cuspidatum against carbon tetrachlorideinduced liver injury in mice. PLoS One 7, e46574. Zhang, L.P., Ma, H.J., Bu, H.M., Wang, M.L., Li, Q., Qi, Z., Zhang, Y., 2009. Polydatin attenuates ischemia/reperfusion-induced apoptosis in myocardium of the rat. Sheng Li Xue Bao 61, 367–372. Zhang, L.P., Yang, C.Y., Wang, Y.P., Cui, F., Zhang, Y., 2008. Protective effect of polydatin against ischemia/reperfusion injury in rat heart. Sheng Li Xue Bao 60, 161–168. Zhao, K.S., 2005. Hemorheologic events in severe shock. Biorheology 42, 463–477. Zhao, K.S., Jin, C., Huang, X., Liu, J., Yan, W.S., Huang, Q., Kan, W., 2003. The mechanism of polydatin in shock treatment. Clin. Hemorheol. Microcirc. 29, 211–217.