ORIGINAL R ESEARCH AR TICLE

Aspirin Attenuates Angiotensin II-induced Cardiomyocyte Hypertrophy by Inhibiting the Ca2+/Calcineurin-NFAT Signaling Pathway Zheyu Yin,1 Xiaoyun Wang,2 Lan Zhang,2 Hongfeng Zhou,3 Li Wei2 & Xiaoqiu Dong1 1 Department of Ultrasonography, The Fourth Hospital of Harbin Medical University, Nangang District, Harbin, China 2 Department of Cardiology, The Fourth Hospital of Harbin Medical University, Nangang District, Harbin, China 3 The Third Affiliated Hospital of Harbin Medical University, Harbin, China

Keywords Angiotensin II; Aspirin; Calcineurin–NFAT; Cardiac hypertrophy. Correspondence X. Dong, Department of Ultrasonography, The Fourth Hospital of Harbin Medical University, 31 Yinhang Street, Nangang District, Harbin 150081 China. Tel.: +86-451-82576705; Fax: +86-451-82576705; E-mail: [email protected]

doi: 10.1111/1755-5922.12164

SUMMARY Introduction: In this study, we examined whether aspirin could inhibit cardiac hypertrophy. Methods: We utilized cultured neonatal mouse cardiomyocytes and mice for the study and subjected to cardiomyocyte immunochemistry, qRT-PCR, and immunoblotting analysis. The cardiac function was measured using M-mode echocardiography. Results: Ten lM aspirin significantly inhibited Ang II-induced increase in cardiomyocyte size, the mRNA, and protein levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and b-myosin heavy chain (b-MHC) (P < 0.05). Meantime, consistent with the result in vitro, the increase in HW/BW ratio, the mRNA, and protein levels of ANP, BNP, and b-MHC could be reduced by aspirin in vivo (P < 0.05). Analysis of cardiac function revealed that mouse hearts treated with Ang II displayed thickening of the ventricular walls, left ventricular end-diastolic dimensions, and left ventricular end-systolic dimensions were significantly decreased (P < 0.05), whereas interventricular septal thickness at end-diastole, interventricular septal thickness at end-systole, posterior wall thickness in diastole, and posterior wall thickness in systole were markedly increased (P < 0.05), which could be reversed by aspirin (P < 0.05). Moreover, aspirin blunted the increase inCa2+ and inhibited the calcineurin activity and NFAT dephosphorylation caused by Ang II (P < 0.05). Conclusions: Aspirin inhibited cardiac hypertrophy in vitro and in vivo through inhibition of the Ca2+/calcineurin–NFAT signaling pathway. Therefore, these findings suggested that aspirin might become a therapeutic option to reduce cardiac hypertrophy.

Introduction Cardiac hypertrophy is a common adaptive response of the heart to a variety of physiological as well as pathophysiological stimuli [1]. The reactivation of fetal cardiac genes and increased cell size are typical features for cardiac hypertrophy. Cardiac hypertrophy initially occurs as a compensatory response, and it can eventually lead to decompensation accompanied by heart failure, arrhythmias, and sudden death [2–4]. Because of the grave outcome of cardiac hypertrophy, many scientific studies attempt to find the underlying mechanisms of pathological hypertrophy and find method to reverse its deleterious aspect. Although the pathogenesis of cardiac hypertrophy is not completely understood, and is believed to be multifactorial, studies have demonstrated several medications, such as statins, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and spironolactone, have been shown to play a role in prevention of cardiac hypertrophy in certain subgroups of patients [5–8]. These medications have anti-inflammatory and antioxidant properties, which are thought to be responsible for their

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antihypertrophic potential. The cardioprotective properties of triflusal (2-acetoxy-4-trifluoromethylbenzoic acid) which is a nonsteroidal anti-inflammatory drug structurally related to the salicylate group of compound, has been established recently in cardiac hypertrophy [9]. Aspirin exhibits anti-inflammatory activity by its effects on cyclooxygenase (COX) activity, which is linked to inflammation as well as by inhibiting IL-4 and nuclear factor kappa B gene expression in non-COX-dependent pathways [10]. In recent study, low-dose aspirin therapy is associated with a significant reduction in mortality and morbidity risk of heart failure during long-term follow-up. These results suggest that low-dose aspirin may have a continuing role in prevention in heart failure [11]. There have been major advances in the identification of aspirin involved in cardioprotective process, but the overall complexity of hypertrophic remodeling suggests that additional regulatory mechanisms remain poorly understood. The aims of this study were to determine the role of aspirin in Ang II-induced cardiac hypertrophy. In this study, we evaluated the antihypertrophic effects of aspirin and disclosed the

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underlying mechanisms, which was involved in regulation of the calcineurin–NFAT signaling pathway in cardiomyocyte hypertrophy in vitro and in vivo.

Methods Animal Care All animal experimental procedures were approved by the ethical committee of Harbin Medical University, China and were in accordance with the National Institutes of Health Guidelines for the care and use of experimental animals (NIH Publication No. 85–23, revised 1996).

Cardiomyocyte Culture and Cell Models for Hypertrophy The procedure of myocyte dissociation was similar to that described previously [12]. In brief, neonatal mouse ventricular myocytes were isolated and cultured from the ventricles of 1-to-3-day-old kunming mice. The ventricles were excised, washed, and cut into small pieces in serum free Dulbecco’s modified Eagle’s medium/medium (DMEM) medium, then digested with a 0.25% solution of trypsin in a CO2 incubator at 37°C. To enrich cardiomyocytes and deplete nonmyocytes, the cells were centrifugated by differential preplating. After purified, cardiomyocytes were suspended in DMEM supplemented with 10% fetal bovine serum (FBS). Culture medium was renewed after 48 h and cardiomyocytes were further cultured for 24 h. Then the culture medium was changed to serum-free DMEM and cardiomyocytes were pretreated with 2.5–10 lM aspirin (Sigma, St. Louis, MO, USA) or phosphate buffer saline (PBS) for 1 h and subsequently stimulated with 1 lM Ang II (Sigma) for 48 h [13,14].

Animal Experiments Eight-week-old male kunming mice (23–27 g body weight) were supplied by the Medical Experimental Animal Center of Harbin Medical University, China. The mice were kept under standard animal room conditions (temperature 24  1°C; humidity 55–60%). Food and water were freely available throughout the experiments. The animals were randomly distributed into three groups as follows: control group (control, n = 6), Ang II-stimulation mice without aspirin pretreatment (Ang II, n = 6), and Ang II stimulation mice with aspirin pretreatment (Ang II + aspirin, n = 6). One week before the Ang II-stimulation, the mice received 7 days pretreatment with 0.9% salt solution (control group) or aspirin 5 mg/kg/day by oral gavage. After 7 days pretreatment with salt solution or aspirin, the model of cardiac hypertrophy was established by administration of Ang II at 1 mg/kg/day for 7 days [3] using osmotic mini-pumps (1007D; Alza Corp., Mountain View, CA USA) implanted subcutaneously in mice. After drugs administration, the mice received food and water ad libitum and hearts were collected after 2 weeks with anesthesia. The heart weight/body weight (HW/BW) ratio was calculated and the heart samples were frozen and stored in liquid nitrogen.

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Cardiomyocyte Immunochemistry and Cell Surface Area Analysis Cardiomyocytes were plated at a density of 1 9 105 cells/mL to obtain individual cells. After Ang II treatment for 48 h, the cardiomyocytes were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS, followed by blocking with 5% goat serum for 1 h at room temperature. The cardiomyocytes were incubated with monoclonal antibody against sarcomeric a-actinin (1:200 dilution; Sigma) at 4°C overnight. Nuclear staining was performed with 40 , 6-diamidino-2-phenylindole (DAPI; Sigma) at dilutions of 1:20. Using laser scanning confocal microscopy (FV300; Olympus, Tokyo, Japan) captured the cell picture. The cardiomyocyte surface was determined with ImagePro Plus software (Version 6.0; Media Cybernetics, Inc., Silver Spring, MD, USA.

Quantitative Reverse Transcription-PCR (qRT-PCR) Total RNA samples from cultured cardiomyocytes and cardiac tissues were isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocols. Total RNA (0.5 lg) was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to obtain cDNA. The SYBR Green PCR Master Mix Kit (Applied Biosystems) was used in qRT-PCR to quantify the RNA levels of the hypertrophic markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and b-myosin heavy chain (b-MHC) in cardiomyocytes, with GAPDH as an internal control. The qRT-PCR was performed on 7500 FAST Real-Time PCR System (Applied Biosystems) for 40 cycles. The sequences of primers used for amplification were as follows: ANP, 50 -CTCCGATAGA TCTGC CCTCTTGAA-30 and 50 -GGTACCGGAAGCTGTTGCAGCCTA-30 ; BNP, 50 -TGATTCTGCTCCTGCTTTTC-30 and 50 -GTGGATTGTTCTGGAGACTG-30 ; b-MHC, 50 -CAGCA GCCCAGTACCTCCGA-30 and 50 - TGTCATCAGGCACGAA GCAC-30 ; GAPDH, 50 -AAGAATGGTGAAGCAGGC-30 and 50 - TCCACCACCAG TTGCTGTA-30 .

Western Blot Analysis Total proteins were extracted from cultured cardiomyocytes from neonatal mouse and mouse hearts. The protein concentrations were determined with a BCA protein assay kit using bovine serum albumin as the standard. Protein samples (100 lg) were separated by SDS-PAGE and transferred onto PVDF membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then probed with the anticalcineurin, anti-NFAT and p-NFAT (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-ANP (1:200 dilution; Abcam, Cambridge, MA, USA), anti-BNP (1:200 dilution; Abcam), anti-b-MHC (1:1000 dilution; Cell Signaling Technology), and anti-GADPH (1:5000 dilution; Cell Signaling) antibodies, overnight at 4°C. Following incubation with the primary antibodies, membranes were incubated with secondary antibody (1:5000 dilution; Alexa Fluor 700 goat anti-mouse IgG (H+L) or Alexa Fluor 800 goat anti-rabbit IgG (H+L), Invitrogen) in PBS at room temperature for 2 h. Western blot bands were captured by using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lin-

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coln, NE, USA) and quantified with Odyssey v1.2 software (LICOR Biosciences) by measuring the band intensity (area 9 OD) in each group and normalizing to GAPDH as an internal control. Each western blot experiments were repeated six times.

Histology After being fixed, tissues specimens were embedded in paraffin. Cross-sectional slices along the minor axis were obtained with a microtome and then stained using Mayer’s Hematoxylin and Eosin. Cardiomyocyte hypertrophy was quantitated by measuring the diameter of 100 randomly sectioned (transverse) cardiomyocytes per microscopic field. Masson’s trichrome staining was used to evaluate collagen deposition. Sections were imaged at 2009 magnification by bright-field microscopy (IX71 Olympus). All quantitative evaluations were carried out by ImagePro Plus software (version 6.0; Media Cybernetics).

Echocardiography Fourteen days after drug treatment, mice were anesthetized with 2.5% (v/v) isoflurane and placed on the mouse board. Transthoracic echocardiography was performed on control, Ang II-stimulation, and Ang II stimulation mice with aspirin pretreatment, using a Vevo 770 ultrasound imaging system (Visualsonics Inc., Toronto, Canada) with a 30-MHz transducer. Two-dimensional guided Mmode tracings were recorded from the parasternal long-axis view at the mid papillary muscle level [15]. When the picture was stabilized, ventricular parameters including left ventricular end-diastolic dimensions (LVEDD), left ventricular end-systolic dimensions (LVESD), interventricular septal thickness at end-diastole (IVSTD), interventricular septal thickness at end-systole (IVSTS), posterior wall thickness in diastole (PWTD), and posterior wall thickness in systole (PWTS) were measured. All the measurements were made from more than three beats and averaged. After functional measurements, mice were killed and the hearts were collected in 4% paraformaldehyde or liquid nitrogen for use.

Flow Cytometry Analysis The intracellular calcium was measured by flow cytometry using the calcium-sensitive dyes, fluo-3, and Fura Red (Molecular Probes). Myocytes were resuspended in medium supplemented with 1% FBS, then stained with 4 lM Fluo-3 and 10 lM Fura Red for 30 min at 37°C. Fluo-3 fluorescence at 530 nm increases with increasing Ca2+ binding, whereas Fura-Red fluorescence at 670 nm decreases with increasing Ca2+ binding, allowing ratiometric measurement of Ca2+ [16]. The fluo-3/Fura Red fluorescence ratio was read on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Statistical Analysis All quantitative data were presented as the means  SEM and analyzed by SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Statistical evaluation of the data was performed by two-tailed unpaired Student’s t-tests and one-way ANOVA. Differences were considered as statistically significant when P < 0.05.

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Aspirin Inhibits Cardiomyocyte Hypertrophy

Results Aspirin Attenuates Ang II-induced Cardiac Hypertrophy in Neonatal Mouse Cardiomyocytes As previous research, cardiac hypertrophy could be induced by Ang II [17–19]. To investigate whether aspirin could attenuates Ang II-induced cardiac hypertrophy, mouse cardiomyocytes were challenged with aspirin (10 lmol/L) and Ang II (1 lmol/L) stated in methods section. Accordingly, we excitingly observed that Ang II markedly increased cell area of cardiomyocytes (2.38  0.19 vs. 1.00  0.07, P < 0.01) using cardiomyocyte immunochemistry and cell surface area analysis, whereas aspirin could reversed the effects of Ang II (1.35  0.12 vs. 2.38  0.19, P < 0.01) (Figure 1A, B). To further investigate the effect of aspirin on cardiac hypertrophy, we study the effect of aspirin on the markers of cardiac hypertrophy in different concentration. The feature of cardiac hypertrophy was reflected by increased protein contents such as ANP, BNP, and b-MHC. Therefore, we firstly investigated the effects of Ang II (1 lM) on these proteins in cardiomyocytes isolated from neonatal mice, which had been often used for the experiments. Ang II induced increases of ANP, BNP, and b-MHC expression in mRNA (P < 0.05 vs. control group) (Figure 2A–C). In contrast, aspirin (2.5–10 lM) significantly attenuated the Ang II-induced ANP, BNP, and b-MHC expression (P < 0.05 vs. Ang IIstimulated cardiomyocyte group). Moreover, Figure 2D–F show increase in ANP, BNP, and b-MHC protein expression induced by Ang II in neonatal mouse cardiomyocytes. These changes were inhibited by aspirin (2.5–10 lM) in the culture medium. These results indicated that the Ang II-Induced hypertrophy in cardiac myocytes could be attenuated by aspirin in vitro.

Aspirin Significantly Inhibits Mouse Cardiac Hypertrophy Induced by Ang II To determine the antihypertrophic role of aspirin in Ang IIinduced cardiac hypertrophy further, we then turned to investigate the beneficial effects of aspirin in vivo conditions. As shown in Figure 3A–C, Ang II induced cardiac hypertrophy revealed by HW and HW/BW ratio (P < 0.01), which could be successfully inhibited by aspirin pretreatment (P < 0.01). As demonstrated in Figure 3A, in the control groups, cardiomyocytes contained compactly arranged fibers with no intercellular space under the light microscopy, whereas in the Ang II group, cardiomyocytes were hypertrophic and cardiac muscle fibers severely ruptured. The situation was reversed after aspirin treatment. Ang II exacerbated any cardiac interstitial fibrosis in mice. CVF in the heart of normal mice was 4.24  0.20%, and this increased to 20.89  1.01% (P < 0.01, vs. control group) in the heart of Ang II-treated mice. Aspirin treatment reduced CVF to 6.99  1.87% (P < 0.01, vs. Ang II-treated group) (Figure 3D). Moreover, the protein expression level of ANP, BNP, and b-MHC in mouse heart were examined after treatment with drugs for 2 weeks. As shown in Figure 3E–G, Ang II induced increases of ANP, BNP, and b-MHC protein expression (P < 0.01). In contrast, aspirin significantly decreased the Ang II-induced ANP, BNP, and MHC expression (P < 0.01).

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Figure 1 Ang II-induced cardiomyocyte hypertrophy was reversed by aspirin. (A) Cardiomyocytes were stained for a-actinin proteins and DAPI (Original magnification, 9200). (B) Quantification of cell surface area from conditions in A using immunochemistry and cell surface area analysis, n = 100 independent experiments for each condition. Data are expressed as means  SEM. **P < 0.01 versus control; ##P < 0.01 versus Ang II group.

Figure 2 Ang II-induced cardiomyocyte hypertrophy was attenuated by aspirin in neonatal mouse cardiac myocytes. (A–C) The mRNA expression level of ANP, BNP, and b-MHC by qRT-PCR analysis in cardiac myocytes exposed to control (PBS), Ang II 1 lM, and Ang II + aspirin (2.5–10 lM). (D–F) Protein levels of ANP, BNP, and b-MHC. n = 6 independent experiments for each condition. Data are expressed as means  SEM. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus Ang II group.

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Figure 3 Aspirin significantly inhibits Mouse Cardiac Hypertrophy Induced by Ang II in vivo. (A) Gross hearts in the top row, heart sections stained with hematoxylin and eosin in the middle row and Masson’s trichrome staining in the bottom row. (B) Heart weight in different groups, n = 5 independent experiments for each condition. (C) Heart weight to body weight ratios (HW/BW) in different groups, n = 5 independent experiments for each condition. (D) Collagen deposition was quantified with an automated image analyzer and expressed as percentage of tissue area, n = 5 independent experiments for each condition. (E–G) Protein levels of ANP, BNP, and b-MHC by western blot experiments. n = 6 independent experiments for each condition. Data are expressed as means  SEM. **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus Ang II group.

Aspirin Significantly Reversed the Change of Mouse Cardiac Function Induced by Ang II In mouse heart, cardiac function was investigated using echocardiography after treatment with drugs. Analysis of cardiac function by M-mode echocardiography revealed that mouse hearts treated with Ang II displayed thickening of the ventricular walls, whereas aspirin could reverse the change (Figure 4A). Echocardiography examination showed that in Ang II-treated mouse heart LVEDD and LVESD were significantly decreased, whereas IVSTD, IVSTS, PWTD, and PWTS were markedly increased, indicating the cardiac hypertrophy (P < 0.05). Meantime, aspirin played antihypertrophic role in vivo and reversed Ang II-induced cardiac hypertrophy (P < 0.05; Figure 4B).

Aspirin Inhibits Ca2+/Calcineurin–NFAT Activation in Ang II-induced Cardiac Hypertrophy It has been confirmed that calcineurin–NFAT signaling channel plays an important role in cardiac hypertrophy [20,21]. Ca2+ is required for calcineurin–NFAT signaling channel [21]. Thus, we

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measured the intracellular Ca2+ by assessing Ang II-induced Ca2+ release in cultured cardiomyocytes at 48 h. The Fura-3/Fura Red ratio at 530/670 nm was not significantly changed in control group. Ca2+ increased 1.5-fold by Ang II treatment compared to the control group (P < 0.05). In contrast, the elevation of Ca2+ was significantly reduced by aspirin in comparison to Ang II-treated group (P < 0.05) (Figure 5A). Our result indicates that aspirin directly decreases the Ca2+ level elevated by Ang II in cardiomyocyte, which raised the possibility that aspirin might affect calcineurin–NFAT signaling. To test this hypothesis, we measured the protein expression of calcineurin in cardiomyocytes stimulated with Ang II. The protein expression of calcineurin significantly increased in cardiomyocytes in response to Ang II treatment. Moreover, aspirin blunted the increase in calcineurin expression induced by Ang II (Figure 5B). Recent studies found that activated calcineurin directly binds to NFAT transcription factors, resulting in NFAT dephosphorylation and nuclear translocation [22,23]. NFAT was significantly dephosphorylated by Ang II in cardiomyocytes. Meantime, NFAT dephosphorylation was altered by Ang II in cardiomyocytes that were co-treatment with aspirin (Figure 5C, D). These data indicated that aspirin inhibited

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Figure 4 Aspirin significantly reversed the change in mouse cardiac function induced by Ang II. (A) Representative M-mode echocardiographs from control, Ang II, Ang II + aspirin group. (B) LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; IVSTD, interventricular septal thickness in diastole; IVSTS, interventricular septal thickness in systole; PWTD, posterior wall thickness in diastole; PWTS, posterior wall thickness in systole. n = 6 independent experiments for each condition. Data are expressed as means  SEM. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus Ang II group.

Ang II-induced increases in Ca2+, which inhibited calcineurin and NFAT activation. To confirm the effect of aspirin on calcineurin– NFAT signaling channel, we then tested the change in calcineurin–NFAT signaling channel induced by Ang II or/and aspirin in vivo conditions. As shown in Figure 6A–C, aspirin also inhibited the activation of calcineurin and NFAT signaling channel.

Discussion In this study, we demonstrated that aspirin, which was currently used as an antiplatelet agent, inhibited cardiac hypertrophy induced by Ang II in vitro and vivo. Furthermore, our results indicated that the antihypertrophic effect of aspirin was mediated through the Ca2+/calcineurin-NFAT pathway. The previous study found that cardiac hypertrophy was both as an intermediate step in and a determinant of heart failure. This is a paramount significance to discovery of molecular, cellular mechanisms, and their signaling pathways underlying hypertrophic remodeling and the identification of potential therapeutic approaches for treating heart failure. Aspirin is an irreversible inhibitor for COX, and it can reduce the production of TXA2 in platelets. In addition, aspirin have antiinflammatory properties. Recent studies have indicated that aspirin is effective in the prevention and treatment of HF and

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acute pulmonary embolism (APE) [24]. Based on these studies, it was indicated that the application of aspirin could alleviate the inflammatory responses induced by HF and APE, and aspirin could be considered as an effective approach for the treatment of cardiac hypertrophy in clinical practice. Through inhibiting or blocking prohypertrophic pathways during cardiac hypertrophy could exert an antihypertrophic effect. It was reported that pretreatment of atorvastatin could prevent the increase in the phosphorylation of Akt and GSK-3b caused by cardiac hypertrophy, and this effect correlated with an increase in protein levels of PTEN, which negatively regulated the phosphoinositide-3 kinase/ Akt pathway [25]. Moreover, the study elucidated that atorvastatin inhibited cardiac hypertrophy and prevented the decrease in the protein levels of PPARa and PPARb/d and suppressed NF-jB activation during cardiac hypertrophy [26]. In this study, we excitingly observed that aspirin could reverse increased cell area of cardiomyocytes induced by Ang II in vitro. To further investigate whether aspirin have effect of anticardiac hypertrophy, we study the effect of aspirin on the markers of cardiac hypertrophy in different concentration (2.5-10 lM). We found that the mRNA and protein levels of ANP, BNP and b-MHC were obviously elevated with incubation of Ang II. Moreover, 2.5–10 lM aspirin significantly decreased the Ang II-induced ANP, BNP, and MHC expression in a dose-dependent manner,

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Figure 5 Ang II-induced Ca2+/calcineurin–NFAT activation was inhibited by aspirin in vitro. (A) Elevation of [Ca2+] in cardiomyocytes induced by Ang II was inhibited by aspirin 10 lM administered to cardiomyocytes 1 h before Ang II exposure. (B) Elevated calcineurin level in cardiac myocytes cultured in the presence of Ang II was inhibited by aspirin. (C, D) Aspirin blocked p-NFAT decrease induced by Ang II. Total NFAT was not changed under the indicated condition. n = 6 independent experiments for each condition. Data are expressed as means  SEM. **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus Ang II group.

Figure 6 Ang II-induced Ca2+/calcineurin–NFAT activation was inhibited by aspirin in vivo. (A–C) Aspirin blocked p-NFAT decrease induced by Ang II. Total NFAT was not changed under the indicated condition. n = 6 independent experiments for each condition. Data are expressed as means  SEM. **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus Ang II group.

which indicated the antihypertrophic effect of aspirin during hypertrophy. This antihypertrophic property of aspirin was further substantiated in vivo. The Ang II-treated mice showed cardiac hypertrophy that was greater than control group, and also showed a rapid progression to heart failure after 2 weeks. As shown in Figure 3A, aspirin inhibited the Ang II-induced hypertrophy revealed by HW/BW ratio (P < 0.01). We found in the Ang II group, cardiomyocytes were hypertrophic and cardiac muscle fibers severely ruptured. The situation was reversed after aspirin treatment. Ang

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II exacerbated any cardiac interstitial fibrosis in mice. Aspirin treatment reduced increased CVF induced by Ang II (P < 0.01, vs. Ang II-treated group; Figure 3C). Moreover, as shown in Figure 3D–F, Ang II induced increases of ANP, BNP, and b-MHC protein expression could be reversed by aspirin (P < 0.01).The results were consistent with the cellular experiment. Echocardiography examination showed that in Ang II treatment mouse hearts LVEDD and LVESD were significantly decreased, whereas IVSTD, IVSTS, PWTD, and PWTS were markedly increased, indicating the cardiac hypertrophy (P < 0.05) (Figure 4B). However, aspirin

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could reverse the Ang II-induced cardiac hypertrophy in vivo. The above results indicated that aspirin significantly reversed the change in mouse cardiac function induced by Ang II. How does aspirin lead to antihypertrophy? It is known that cardiac myocyte function is dependent on the Ca2+ in the cell. The inhibitory effect of aspirin on Ang II-induced hypertrophy is likely at least partially due to its effect on abnormal Ca2+-handling in hypertrophied myocytes. As shown in Figure 5 the level of Ca2+ measured in NRVMs of Ang II group was significantly increased compared with control group. In contrast, Ang II + aspirin (10 lM) treated myocytes displayed a significant lower Ca2+ concentration in myocytes compared with Ang II group, which indicates that aspirin blunted the effect of Ang II-induced increase in abnormal Ca2+-handling in hypertrophied myocytes. Recently, the calcineurin–NFAT signaling pathway was found to play an essential role in cardiac hypertrophy [27]. Cardiovascular diseases such as hypertension and myocardial infarction raised the contractile stress by producing increases in myocyte [Ca2+] on the heart [28,29], which activated calcineurin, a Ca2+/calmodulindependent phosphatase. Calcineurin plays an essential role in gene expression and cardiomyocyte growth by promoting dephosphorylation and nuclear translocation of NFAT [23], which is proved to induce the pathological cardiac hypertrophy. This was likely to be through Ca2+ action on calcineurin–NFAT signaling as we demonstrated above. As illustrated in Figures 5 and 6, Ang II activated calcineurin, which in turn dephosphorylated and inactivated a transcription factor called NFAT, which is essential for activation of the hypertrophic genes such as ANP,

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Limitations In this study, we only investigated the effects of aspirin on Ang IIinduced cardiomyocyte hypertrophy and observed a potential protection for aspirin against cardiomyocyte hypertrophy. Meantime, the study indicated that the antihypertrophic effect of aspirin was likely due to its effect on Ca2+/calcineurin-NFAT signaling. However, we did not detect the mechanisms involved in the effect of aspirin on Ca2+ in the cell. In addition, although aspirin has therapeutic potential, its mechanisms need to be further studied.

Conclusion We found that aspirin inhibited cardiac hypertrophy in vitro and in vivo through a mechanism that might involve inhibition of the Ca2+/calcineurin–NFAT signaling pathway, an important intracellular mediator of this process. Therefore, these findings suggested that aspirin might become a therapeutic agent to reduce cardiac hypertrophy.

Conflict of Interest The authors declare no conflict of interest.

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BNP, and b-MHC. Meantime, application of aspirin reduced Ca2+/ calcineurin–NFAT signaling activation, which indicated that the antihypertrophic effect of aspirin was likely due to its effect on Ca2+/calcineurin–NFAT signaling.

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Calcineurin-NFAT Signaling Pathway.

In this study, we examined whether aspirin could inhibit cardiac hypertrophy...
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