nardosinone protects h9c2 cardiac cells from angiotensin ii-induced hypertrophy

J Huazhong Univ Sci Technol [Med Sci] 33(6):2013

822

Nardosinone Protects H9c2 Cardiac Cells from Angiotensin Ⅱ-induced Hypertrophy*

Meng DU (杜萌)1, 2†, Kun HUANG (黄坤)1, 2†, Lu GAO (高路)2, Liu YANG (杨柳)2, Wen-shuo WANG (王文硕)3, Bo WANG (王博)3, Kai HUANG (黄恺)1, 2#, Dan HUANG (黄丹)1, 2# 1Clinical Center for Human Genomic Research, 2Department of Cardiovascular Diseases, 3Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2013

Summary: Pathological cardiac hypertrophy induced by angiotensin Ⅱ (AngⅡ) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngⅡ-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosi-none (25, 50, 100, and 200 μmol/L) or AngⅡ (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngⅡ. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngⅡ-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang Ⅱ-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways. Key words: nardosinone; cardiac hypertrophy; H9c2 cells; PI3K/Akt; MEK/ERK

Cardiac hypertrophy is an adaptive response of the heart to various stimuli, and can predispose individuals to heart failure, arrhythmia or even sudden death[1]. It is characterized by several molecular and phenotypic changes including the reactivation of fetal genes, such as atrial natriuretic peptide (ANP), B-type natriuretic pep-tide (BNP) and β-myosin heavy chain (β-MHC)[2]. Al-though signal transduction pathways are inherently com-plex and abundant, studies in animal models have re-vealed several important mediators of cardiac hypertro-phy, such as phosphatidylinositol 3-kinase (PI3K)/Ser-ine-threonine kinase Akt pathway[3]. It has been proved that Ang played a critical role in the dⅡ evelopment of cardiac hypertrophy[4], as it not only causes cardiac hy-pertrophy through activation of PI3K/Akt pathway and the downstream target proteins, such as the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K), but also leads to the mitogenesis of cardiac cells via the mitogen-activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) pathway[5].

Nardosinone is isolated from the roots of Nar- Meng DU, E-mail: [email protected]; Kun HUANG, E-mail: [email protected] †The authors contributed equally to this work. #Corresponding authors, Kai HUANG, E-mail: [email protected]; Dan HUANG, E-mail: joy_huangdan@ yahoo.com.cn *This project was supported by the grants from the National Natural Science Foundation of China (No. 30971245 and No. 81000112).

dostachys chinensis[6]. Previous studies have shown that nardosinone can potently inhibit NO production in LPS-stimulated RAW 264.7 cells[7], suggesting that nar-dosinone has a protective effect on cells. However, the effect of nardosinone on cardiac hypertrophy remains largely unknown. In this study, we investigated the pro-tective effect of nardosinone on H9c2 cells exposed to Ang Ⅱ. We showed that nardosinone could inhibit Ang Ⅱ-induced hypertrophy by repressing PI3K/Akt and MEK/ERK signaling pathways.

1 MATERIALS AND METHODS 1.1 Cell Culture

The H9c2 cell line was ordered from Cell Bank of the Chinese Academy of Sciences, Shanghai, China. The structure of nardosinone (C15H22O3, 23720-80-1, Shaanxi Sciphar Hi-Tech Industry Co., China) was previously reported by Schulte et al[8], and it was dissolved in ddH2O and stored at a concentration of 200 μmol/L. The cells were cultured in Dulbecco’s modified Eagle me-dium (DMEM, GIBCO, USA) supplemented with 10% fetal bovine serum (FBS, Shanghai ExCell Biology, China), penicillin (100 U/mL) and streptomycin (100 mg/mL) (GIBCO, USA), and grown in an atmosphere of 5% CO2 in a humidified incubator. The cells were seeded at a density of 1×106 per well into six-well culture plates and at a density of 5.0×103 per well in 96-well plates, then cultured for 24 h. After the cells were starved for 12 h, different concentrations of nardosinone (25, 50, 100, and 200 μmol/L) followed by Ang Ⅱ (1 μmol/L) (A9525, Sigma, USA) and Ang Ⅱ alone were added to the me-

33(6):822-826,2013J Huazhong Univ Sci Technol [Med Sci]

DOI 10.1007/s11596-013-1205-9


823

dium, respectively. The cells in six-well plates were for protein and mRNA extraction and those in 96-well plates for measurement of cell surface area. 1.2 Measurement of Cell Surface Area

The H9c2 cells were seeded on sterile glass cover-slips in a 96-well plate. The cells were fixed and perme-ablized with 0.1% Triton X-100 in phosphate buffered saline (PBS). The coverslips were washed 3 times with PBS and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Triton X-100 for 1 h at room temperature. Cells were stained with anti-sarcomeric actin antibody (ab11008, Abcam, USA) at a dilution of 1:100 in 1% goat serum (ab7481, Abcam, USA). After rinsing with TBS, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen, USA). Finally, SlowFade Gold antifade reagent with DAPI (S36939, Invitrogen, USA) was used for counterstaining. A single cell was measured with a quantitative digital image analysis system (Image Pro-Plus, version 6.0). At least 50 cells per well were measured in 3 independent experiments respectively. 1.3 Quantitative Real-time PCR

Total RNA was extracted from the H9c2 cells with TRIzol reagent (D9108A, Takara Biotechnology, Japan), according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed using RNA PCR Kit (RR036A, Takara Biotechnology, Japan) and the result-ing cDNA was used as a PCR template. The mRNA lev-els were determined by real-time PCR with ABI PRISM 7900 Sequence Detector system (Applied Biosystem, USA) according to the manufacturer’s instructions. The real-time PCR primer sequences were as follows: for ANP, forward: 5'-ATACAGTGCGGTGTCCAACA-3', reverse: 5'-AGCCCTCAGTTTGCTTTTCA-3'; for BNP, forward: 5'-TTGGGCAGAAGATAGACCGGAT-3', reverse: 5'-GGTCTTCCTAAAACAACCTCA-3'; for β-MHC, forward: 5'-AACCTGTCCAAGTTCCGCAA- GGTG-3', reverse: 5'-GAGCTGGGTAGCACAAGAG- CTACT-3'. GAPDH was used as endogenous control and the primer sequences were forward: 5'-TTGCCATCAA- CGACCCCTTC-3' and reverse: 5'-TTGTCATGGATGA- CCTTGGC-3'. PCR reaction mixture contained the

SYBR GreenⅠ(RR420A, 4Takara Biotechnology, Ja-pan), cDNA and the primers. Relative gene expression level (the amount of target, normalized to endogenous control gene) was calculated using the comparative Ct method formula 2-ΔΔCt. 1.4 Western Blot Analysis

Protein was extracted from the H9c2 cells in different groups, and the protein concentration was measured with the BCA protein assay kit (23227, Thermo, USA). After denaturation and SDS-PAGE electrophoresis, separated proteins were transferred to nitrocellulose membranes. The membranes were then blocked with 5% nonfat milk in Tris-buffered saline (TBS) (10 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.6) for 3 h. After incubation with dif-ferent primary antibodies in TBS at 4°C overnight, the membranes were incubated with peroxidase-conjugated secondary antibody in TBS at room temperature for 2 h. Specific band was detected with Bio-rad imaging system. The expression levels of the proteins were normalized to GAPDH protein for total cell lysate. 1.5 Statistical Analysis

Values are shown as ±s of at least 3 independent experiments. The significance of differences was esti-mated by one-way ANOVA or Independent Samples T Test followed by Student-Newmann-Keuls multiple comparison tests. P<0.05 was considered statistically significant. All statistical analyses were performed with SPSS software (version 11.0, SPSS Inc).

2 RESULTS 2.1 Inhibitory Effect of Nardosinone on Cardiac Hy-pertrophy In Vitro

The H9c2 cells exposed to Ang (1 μmol/L) were Ⅱassayed by staining sarcomeric actin protein for assess-ing hypertrophic growth. As shown in fig. 1A, Ang Ⅱsignificantly increased cell surface area (P<0.05), which could be inhibited by pretreatment with nardosinone (25, 50, 100, and 200 μmol/L), especially at the concentration of 100 μmol/L (P<0.01, fig. 1B). This phenomenon sug-gested that nardosinone played a protective role in H9c2 cell hypertrophy induced by Ang .Ⅱ

Fig. 1 Effect of nardosinone on Ang Ⅱ-induced hypertrophy of H9c2 cells

A: nardosinone (25, 50, 100 and 200 μmol/L) inhibited Ang Ⅱ-induced hypertrophy, which was evaluated by the visualization of sarcomere organization; B: nardosinone (25, 50, 100 and 200 μmol/L) inhibited Ang Ⅱ-induced enlargement of cell surface area. Data are expressed as ±s for examination of 60 randomly selected cells from 3 individual experiments. *P<0.05 vs. control group; #P<0.05, ##P<0.01 vs. Ang Ⅱ-treated group. 1: AngⅡ-treated group; 2-5: nardosinone-treated groups (25, 50, 100 and 200 μmol/L, respectively)

2.2 Reversal of Ang Ⅱ-induced Upregulation of ANP, BNP and β-MHC mRNA Expression by Nardosinone

The upregulated ANP, BNP and β-MHC are often

used as molecular markers of cardiac hypertrophy[2]. The H9c2 cells were coincubated with four different concen-trations of nardosinone (25, 50, 100, and 200 μmol/L)


824

and AngⅡ(1 μmol/L) to investigate the effect of nar-dasinone on hypertrophy. Nardosinone in all groups could significantly reverse the Ang Ⅱ-induced reactiva-tion of ANP, BNP and β-MHC expression (P<0.05). And nardosinone used at a concentration of 100 μmol/L was more effective than that at other concentrations (P<0.01), as shown in fig. 2A. Then the H9c2 cells were coincu-

bated with nardosinone (100 μmol/L) and AngⅡ (1 μmol/L) for different time duration (6, 12 and 24 h). The result demonstrated that nardosinone was a considerable time-dependent inhibitor of AngⅡ-induced reactivation of ANP, BNP and β-MHC expression (P<0.05), as shown in fig. 2B–2D. These findings suggested that nardosinone could inhibit cardiac hypertrophy in vitro.

Fig. 2 Effect of nardosinone on Ang Ⅱ-induced up-regulation of ANP, BNP and β-MHC mRNA expression

A: H9c2 cells pretreated with nardosinone (25, 50, 100 and 200 μmol/L) exhibited a reduction of ANP, BNP and β-MHC mRNA expression compared with Ang Ⅱ-treated group. B–D: The up-regulation of ANP, BNP and β-MHC mRNA expression induced by Ang Ⅱ could be blocked by nardosinone (100 μmol/L) at different time duration. Data are expressed as ±s from 3 individual experiments. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. Ang Ⅱ-treated group.

2.3 Inhibitory Effects of Nardosinone on Phosphory-lation of PI3K/Akt and MEK/ERK Signaling Path-way Induced by Ang Ⅱ

Then the H9c2 cells were treated with Ang Ⅱ and nardosinone to explore the underlying mechanism. The result showed that the phosphorylation levels of PI3K, Akt, mTOR, p70S6K, MEK and ERK increased in re-

sponse to Ang Ⅱ in a time-dependent manner as shown in fig. 3A and 4A. nardosinone blocked Ang Ⅱ-induced phosphorylation of PI3K/Akt and MEK/ERK pathway in a time-dependent manner too. These results further dem-onstrated that nardosinone inhibited AngⅡ-induced hy-pertrophy mediated by PI3K/Akt/mTOR and MEK/ERK pathway.

Fig. 3 Effects of nardosinone on the phosphorylation of PI3K/Akt pathway activated by Ang Ⅱ

A: The phosphorylation level of PI3K/Akt/mTOR/p70S6K in AngⅡ-treated H9c2 cells in the absence or presence of nardosi-none (100 µmol/L) was evaluated by Western blotting. B: Ang Ⅱ-induced increase of phosphorylation level of PI3K/Akt/mTOR/p70S6K in H9c2 cells was reversed by nardosinone. Data are expressed as ±s from 3 individual experi-ments. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. Ang Ⅱ-treated group


825

Fig. 4 Effects of nardosinone on the phosphorylation of MEK/ERK pathway activated by Ang Ⅱ

A: The phosphorylation level of MEK/ERK in AngⅡ-treated H9c2 cells in the absence or presence of nardosinone (100 µmol/L) was evaluated by Western blotting. B: Ang Ⅱ-induced increase of phosphorylation level of MEK/ERK in H9c2 cells was reversed by nardosinone. Data are expressed as ±s from 3 individual experiments. *P<0.05, **P<0.01 vs. control group;

#P<0.05, ##P<0.01 vs. Ang Ⅱ-treated group

3 DISCUSSION

Pathological cardiac hypertrophy as a consequence

of the maladaptive alterations in many kinds of heart diseases is an important risk factor for subsequent car-diac morbidity and mortality[9]. It has been firmly con-firmed that sustained Ang Ⅱ infusion increased blood pressure and induced cardiac hypertrophy[10]. Ang Ⅱ can directly modulate myocardial contractility, metabolism and hypertrophic growth. Many studies have shown that activation of the PI3K/Akt and MEK/ERK pathways was involved in the progression of hypertrophy induced by Ang Ⅱ[11, 12]. Activated PI3K regulates multiple down-stream targets involved in a wide range of cellular proc-esses including growth, survival, metabolism and motil-ity. Akt is an immediate downstream target of PI3K and plays a critical role in the PI3K signaling pathway in cardiac hypertrophy. The transgenic mice with constitu-tively active PI3K in myocardiocytes possess increased protein synthesis and heart size[13–15]. The hypertrophic response following Akt activation is apparently associ-ated with its downstream targets mTOR and p70S6K, and accumulated evidence suggested that activation of mTOR and p70S6K mediated cardiac angiogenesis and fibrosis[16, 17]. The MEK/ERK signaling pathway is also involved in the cardiac hypertrophy and the importance of this pathway was further proved through the analysis of transgenic mice that expressed activated MEK. These mice developed stable concentric hypertrophy[18]. Mechanistically, activation of the MEK/ERK pathway induces cardiac hypertrophy by enhancing the transcrip-tional activity of nuclear factor of activated T cells (NFAT), which induces crosstalk with the cal-cineurin/NFAT circuit[19].

The roots and rhizomes of Nardostachys chinensis is a kind of traditional Chinese medicine because of its antimalarial and antinociceptive effect. Recently, the Nardostachys jatamansi extract showed an inhibitory effect on the cytokine-induced NF-κB activation[20]. nar-dosinone, one of the principal extracts from Nar-dostachys chinensis[8, 21, 22], has been proved to have a beneficial effect on the neurite outgrowth induced by nerve growth factor in PC12D cells via activating the signaling pathway of mitogen-activated protein kinase

and protein kinase C[6, 23, 24]. Thus we hypothesize that nardosinone might play a protective role in other patho-physiological processes. In the present study, H9c2 cells induced by Ang Ⅱ as a cellular model of cardiac hyper-trophy were applied to analyze the effect of nardosione. Considering the protective role of nardosinone, it was not surprising to find that pretreatment with H9c2 cells with nardosinone could protect H9c2 cells from Ang Ⅱ-induced hypertrophy, which was confirmed by the decrease of cell surface area and mRNA expression of ANP, BNP and β-MHC. Therefore, nardosinone may be clinically beneficial for patients with cardiac hypertrophy induced by Ang Ⅱ, which yields new understanding of the therapeutic effects of nardosinone on pathological cardiac hypertrophy.

The present study first demonstrated that nardosi-none could be a potential new therapeutic agent to pre-vent cardiac hypertrophy induced by Ang Ⅱ. And we further found that the protective effect of nardosinone was mediated by inhibition of PI3K/Akt and MEK/ERK pathways. More importantly, our results provided ex-perimental evidence for the application of nardosinone on the treatment of cardiac hypertrophy. While, future clinical trials are required to prove the new potential clinical therapeutic use of nardosinone.

Conflict of Interest Statement

The authors declare that there is no conflict of interest with any financial organization or corporation or individual that can inappropriately influence this work. REFERENCES 1 Heineke J, Molkentin Jd. Regulation of cardiac hyper-

trophy by intracellular signalling pathways. Nat Rev Mol Cell Biol, 2006,7(8):589-600

2 Finckenberg P, Mervaala E. Novel regulators and drug targets of cardiac hypertrophy. J Hypertens, 2010,28 (Suppl 1):S33-S38

3 Aoyagi T, Matsui T. Phosphoinositide-3 kinase signaling in cardiac hypertrophy and heart failure. Curr Pharm Des, 2011,17(18):1818-1824

4 Hunyady L, Turu G. The role of the AT1 angiotensin re-ceptor in cardiac hypertrophy: angiotensin Ⅱ receptor or stretch sensor? Trends Endocrinol Metab, 2004,15(9): 405-408


826

5 Liu X, Xie R, Liu S, et al. Rat parathyroid hormone 1-34 signals through the MEK/ERK pathway to induce cardiac hypertrophy. J Int Med Res, 2008,36(5):942-950

6 Li P, Matsunaga K, Yamamoto K, et al. Nardosinone, a novel enhancer of nerve growth factor in neurite out-growth from PC12D cells. Neurosci Lett, 1999,273(1): 53-56

7 Hwang JS, Lee SA, Hong SS, et al. Inhibitory constitu-ents of nardostachys chinensis on nitric oxide production in raw 264.7 macrophages. Bioorg Med Chem Lett, 2012,22(1):706-708

8 Schulte, KE, Glauch G, Rucker G. Nardosinone, a new constituent of Nardostachys chinensis Batalin. Tetrahe-dron Lett (German), 1965(35):3083-3084

9 Maulik SK, Kumar S. Oxidative stress and cardiac hyper-trophy: a review. Toxicol Mech Methods, 2012,22(5): 359-366

10 Obayashi M, Yano M, Kohno M, et al. Dose-dependent effect of ANG II-receptor antagonist on myocyte remod-eling in rat cardiac hypertrophy. Am J Physiol, 1997,273(4 Pt 2):H1824-H1831

11 Carneiro-Ramos MS, Diniz GP, Nadu AP, et al. Blockage of angiotensin II type 2 receptor prevents thyrox-ine-mediated cardiac hypertrophy by blocking Akt activa-tion. Basic Res Cardiol, 2010,105(3):325-335

12 He KL, Zheng QF, Mu SC, et al. Changes of mito-gen-activated protein kinase activity in cardiac tissues, Ang II and cardiac hypertrophy in spontaneously hyper-tensive rats. Sheng Li Xue Bao (Chinese), 1998,50(5): 539-542

13 Liu L, Zhao X, Pierre SV, et al. Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes. Am J Physiol Cell Physiol, 2007, 293(5):C1489- C1497

14 Braz JC, Gill RM, Corbly AK, et al. Selective activation of PI3Kalpha/Akt/GSK-3beta signalling and cardiac compensatory hypertrophy during recovery from heart failure. Eur J Heart Fail, 2009,11(8):739-748

15 Kim YK, Kim SJ, Yatani A, et al. Mechanism of en-hanced cardiac function in mice with hypertrophy in-

duced by overexpressed Akt. J Biol Chem, 2003,278(48): 47622-47628

16 Kemi OJ, Ceci M, Wisloff U, et al. Activation or inacti-vation of cardiac AKT/MTOR signaling diverges physio-logical from pathological hypertrophy. J Cell Physiol, 2008,214(2):316-21.

17 Medeiros C, Frederico MJ, da Luz G, et al. Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet-induced obesity rats. J Cell Physiol, 2011,226(3): 666-674

18 Bueno OF, De Windt LJ, Tymitz KM, et al. The MEK1-ERK1/2 signaling pathway promotes compen-sated cardiac hypertrophy in transgenic mice. Embo J, 2000,19(23):6341-6350

19 Sanna B, Bueno OF, Dai YS, et al. Direct and indirect interactions between calcineurin-NFAT and MEK1-extracellular signal-regulated kinase 1/2 signaling pathways regulate cardiac gene expression and cellular growth. Mol Cell Biol, 2005,25(3):865-878

20 Song MY, Bae UJ, Lee BH, et al. Nardostachys jatamansi extract protects against cytokine-induced beta-cell dam-age and streptozotocin-induced diabetes. World J Gastro-enterol, 2010,16(26):3249-3257

21 Rucker G, Kahrs KH, Hembeck HW, et al. The confor-mation of nardosinone. Arch Pharm (Weinheim) (Ger-man), 1975,308(11):858-862

22 Rucker G, Dyck E. Photochemical reactions of nardosi-none (author’s transl). Arch Pharm (Weinheim) (German), 1977,310(10):835-840

23 Li P, Matsunaga K, Yamakuni T, Ohizumi Y. Nardosinone, the first enhancer of neurite outgrowth-promoting activity of staurosporine and dibutyryl cyclic AMP in PC12D cells. Brain Res Dev Brain Res, 2003,145(2):177-183

24 Li P, Yamakuni T, Matsunaga K, et al. Nardosinone en-hances nerve growth factor-induced neurite outgrowth in a mitogen-activated protein kinase- and protein kinase C-dependent manner in PC12D cells. J Pharmacol Sci, 2003,93(1):122-125

(Received Feb. 26, 2013; revised Sep. 12, 2013)

nardosinone protects h9c2 cardiac cells from angiotensin ii-induced hypertrophy

Documents