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Am J Physiol Heart Circ Physiol 291: H835-H845, 2006. First published February 24, 2006; doi:10.1152/ajpheart.01365.2005
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Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta1 and phosphorylated JNK

Nobutake Shimojo,1 Subrina Jesmin,1 Sohel Zaedi,1 Seiji Maeda,1 Masaaki Soma,2 Kazutaka Aonuma,1 Iwao Yamaguchi,1 and Takashi Miyauchi1

1Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki; and 2Pharmacovigilance, Mochida Pharmaceutical, Tokyo, Japan

Submitted 23 December 2005 ; accepted in final form 20 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 µM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal ({alpha}-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of {alpha}-actinin and signaling molecule [transforming growth factor-beta1 (TGF-beta1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.

neonatal cardiomyocyte; transforming growth factor-beta1; c-Jun NH2-terminal kinase

CARDIAC HYPERTROPHY, which is defined by an increase in heart size and/or myofibrillar volume without a change in myocyte number, occurs in response to physiological and pathophysiological stimulation (15). Although cardiac hypertrophy is typically viewed as a compensatory response that normalizes ventricular wall stress, sustained hypertrophy is correlated with an increase in the incidence of and mortality from cardiovascular disease (26). Thus pathological cardiac hypertrophy is different from physiological cardiac hypertrophy. When the myocardial adaptations are unable to satisfy the increased demands or able to meet the increased demands only at the expense of normal function (35), the hypertrophy is considered to be pathological. On the other hand, when the adaptations are sufficient to fulfill the requirements while normal function is maintained, the hypertrophy is physiological (35). Primarily pathological and physiological states of left ventricular hypertrophy (LVH) have been considered on the basis of normal chamber performance and oxygen delivery as well as reversibility of the hypertrophy on removal of the overload (35). Moreover, both states are also characterized by the nature of the imposed load and the resulting myocardial adaptations (35). Only pathological LVH has been shown to be a risk factor for cardiovascular events (24) such as myocardial infarction, ischemia, arrhythmia, and sudden death (47).

Pathological cardiac hypertrophy is caused by a prolonged exposure of cardiomyocytes to external stimuli, such as hemodynamic overload and neurohumoral factors (47). One of the most important vascular hormones that contribute to the development of hypertrophy is endothelin (ET)-1. ET-1, which was initially identified and purified from porcine aortic endothelial cell cultures, is a vasoactive peptide that contains 21 amino acids and has 2 intramolecular disulfide bonds (48). We know that a variety of cells, including cardiomyocytes, produce this peptide (22) and that, in addition to its vasoconstrictive effects, it has potent mitogenic effects on a number of cells, including cardiomyocytes (42). In vitro studies have shown that ET-1-induced cardiac hypertrophy involves various hypertrophic signaling cascades mediated by ET type A (ETA) receptors, such as Raf-1 and MAP kinases, in the neonatal rat (46). Other signaling pathways mediating ET-1-induced cardiac hypertrophy include phospholipase C, PKC, ERK1 and ERK2, c-Fos and c-Jun, c-Jun NH2-terminal kinase (JNK), and p38 (22, 41). The role of ET-1 in cardiac hypertrophy has also been well documented in the recent past by in vivo studies (17, 23). ET-1 is markedly increased in the hypertrophied heart and the failing heart (37), conditions that are, interestingly, significantly inhibited by chronic treatment with ETA receptor antagonists (37). Taken together, these data strongly suggest a significant role for ET-1 in the development of cardiac hypertrophy in vitro and in vivo. Thus, whereas the effects of ET-1 on cardiac hypertrophy are well documented, little is known about the possible therapeutic interventions and their underlying signaling pathways.

The transforming growth factor (TGF)-beta superfamily comprises a set of regulatory peptides, such as TGF-beta1, that have multiple effects on cell growth and differentiation (5). Studies have shown altered expression and biological activity of TGF-beta1 in hypertrophic cardiomyocytes (5), an effect that may be mediated by ET-1 (47). However, our knowledge of the relation and interaction between TGF-beta1 and ET-1 in the pathophysiology of hypertrophic cardiomyocytes is incomplete. Specifically, the effect of ET-1 on TGF-beta1 expression in hypertrophied cardiomyocytes is not known.

Although there is no overwhelming consensus concerning the protective role of dietary fish oil in coronary artery disease (36), a significant number of experimental studies and clinical intervention trials have shown a cardioprotective effect of dietary fish and fish oil intake (19, 43). Among them, {omega}-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have recently been shown to have potential beneficial effects on cardiovascular diseases (8, 11). Studies demonstrate that dietary supplementation of fish oil and {omega}-3 long-chain polyunsaturated fatty acids (PUFAs) improves survival in patients with acute myocardial infarction (7, 40) and that use of fish oil reduces cardiac arrhythmias (18) and angina pectoris (40). The cardioprotective effects of fish consumption are thought to be beneficial through modulation of lipid and lipoprotein metabolism (45), regulation of blood pressure (4), improvement of vascular endothelial function (32), reduction of neutrophil and monocyte cytokine production (2), inhibition of thrombogenesis and the inflammatory response (9), and an antiarrhythmic effect (28). Little is known about the role of dietary fish oils in cardioprotection and the effects of fish oils on growth factors associated with cardiac hypertrophy. Siddiqui et al. (39) demonstrated the inhibitory effects of DHA on phenylephrine (PE)-induced cardiac hypertrophy and on Ras, Raf-1, ERK1/2, and p90rsk pathways. More recently, DHA has been shown to modulate activation of extracellular signaling pathways, such as PKC and MAPK (13). Thus it is reasonable to postulate that EPA, another major {omega}-3 PUFA in fish, may act collectively or singly in cardioprotection through alteration of hypertrophic responses in cardiomyocytes.

The present study was designed to examine whether pretreatment of cardiomyocytes with EPA could prevent ET-1-induced hypertrophy. To unravel potential mechanisms underlying this process, we investigated whether TGF-beta1 expression could be induced by ET-1 in neonatal cardiomyocytes and, if so, whether EPA can alter this effect. The effect of EPA on ET-1-induced JNK and c-Jun activation was studied in neonatal cardiomyocytes. Finally, expression levels of atrial and brain natriuretic peptides (ANP and BNP), classical molecular markers of cardiac hypertrophy, were examined in ET-1-treated cardiomyocytes with or without EPA pretreatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myocyte Isolation and Culture

Ventricular cardiomyocytes were isolated from 2- to 3-day-old Sprague-Dawley rats, as described previously (22), and incubated on fibronectin-coated dishes in DMEM/Ham's F-12 medium supplemented with 0.1% fatty acid-free BSA (Sigma, St. Louis, MO). The cells were cultured for 3 days after differential adhesion and then used for further experiments. Myocytes were exposed to vehicle (control, in 95% air-5% CO2), treated with 10 µM EPA, treated with 0.1 nM ET-1, or pretreated with 10 µM EPA and then treated with 0.1 nM ET-1 on day 5 of culture for 24 h and harvested for analysis. For determination of the optimal dose, 10–8, 10–9, 10–10, and 10–11 M ET-1 and 1, 3, 10, 30, and 100 µM EPA were used for dose-response experiments.

Cardiomyocyte Surface Area

To determine changes in cell size, the peripheries of cell images captured by a charge-coupled device camera (Olympus, Tokyo, Japan) were traced and analyzed using NIH Image software. These values were doubled to account for the portion of the cell surface in contact with the dish. All cells from randomly selected fields in two or three dishes were examined for each experimental group. A total of 60 cells were examined in each experimental group.

Protein Synthetic Rate

The effect of EPA on the rate of protein synthesis in ET-1-treated cardiomyocytes was investigated as described previously (22). Briefly, [14C]leucine incorporation into acid-insoluble cellular material was measured to assess protein synthesis in cultured neonatal rat ventricular myocytes. The cells were plated in 24-well dishes at a density of 105 cells/well. After treatment with ET-1 alone or EPA and ET-1 for 24 h, [14C]leucine (0.1 µCi/ml) was added, and the cells were incubated for 24 h. The cells were washed twice with cold PBS, and 5% trichloroacetic acid was added for 10 min. The cells were then incubated with 0.25% trypsin at 37°C for 30 min, and cell residues were solubilized in 0.5 N NaOH for 10 min. Aliquots were counted with a scintillation counter (model LS-6500, Beckman Coulter, Fullerton, CA).

Immunocytochemistry and Immunofluorescence

For immunocytochemistry and immunofluorescence staining, the cells were permeabilized with 0.2% Triton X-100 (Wako, Osaka, Japan), fixed with 3% formaldehyde or acetone for 10 min at room temperature in PBS or for 30 min at 40°C, reacted for 8 h with primary antibodies for {alpha}-actinin (Sigma), TGF-beta1, and phosphorylated JNK (R & D Systems, Minneapolis, MN), and then treated with peroxidase-conjugated secondary antibody (Amersham, Buckinghamshire, UK) and 3,3'-diaminobenzidine tetrahydrochloride for visualization. Hematoxylin was used for counterstaining. For immunofluorescence, after incubation with primary antibodies, the cells were rinsed in PBS and then exposed to the fluorescence secondary antibody Cy3-conjugated AffiniPure goat anti-rabbit IgG or fluorescein-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h according to the manufacturer's instructions. Sections processed without primary antibodies served as negative controls. For phosphorylated JNK, 4',6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Santa Cruz, CA) was used for counterstaining. The coverslips were mounted with Immunon (Thermo Shandon, Pittsburgh, PA). Immunofluorescent images were viewed using the laser scanning confocal imaging system (model MRC-1024, Bio-Rad Laboratories).

Enzyme-Linked Immunosorbent Assay

Levels of TGF-beta1 and phosphorylated JNK in cardiomyocytes were determined using ELISA kits [R & D Systems (TGF-beta1) and Sigma (phosphorylated JNK)] according to the manufacturers' instructions.

Western Blot Analysis

Cardiomyocytes were plated at a field density of 2 x 106 cells/cm2 on 60-mm culture dishes with 2 ml of culture medium. Cardiomyocytes of different groups were lysed on ice with buffer (10 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate, and 0.1% SDS). Protein concentration of the supernatant was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL). The samples were run on SDS-PAGE with 7.5–10% polyacrylamide gel and electrotransferred to a polyvinylidene difluoride (PVDF) filter membrane. To reduce nonspecific binding, the PVDF membrane was blocked for 2 h at room temperature with 5% nonfat milk in PBS containing 0.1% Tween 20 (TPBS). Thereafter, the PVDF membrane was incubated overnight at 4°C with specific antibodies for {alpha}-actinin (Sigma), TGF-beta1 (R & D Systems), JNK (Cell Signaling), phosphorylated JNK (Cell Signaling), c-Jun (Ser73; Cell Signaling), and phosphorylated c-Jun (Ser73; Cell Signaling) in TPBS; washed three times in TPBS; and then incubated with horseradish peroxidase-conjugated anti-rabbit (Amersham), anti-mouse (Amersham), or anti-goat (Santa Cruz Biotechnology) antibody diluted 1:2,000–10,000 in TPBS at room temperature for 60 min. The blots were visualized with the enhanced chemiluminescence detection system (Amersham, Pharmacia Biotech), exposed to X-ray film, and analyzed using NIH Image software. Antisera against TGF-beta1, {alpha}-actinin, phosphorylated c-Jun (Ser73), c-Jun, and JNK were purchased from Cell Signaling Technology (Beverly, MA).

RNA Preparation and Real-Time Quantitative PCR

Total RNA from cardiomyocytes was isolated using RNeasy (Qiagen, Tokyo, Japan). After isolation, DNase I treatment, and quantification, RNA was reverse transcribed to cDNA by Omniscript RT using a first-strand cDNA synthesis kit (Qiagen). The reaction was performed at 37°C for 60 min.

The mRNA expression levels of target genes were analyzed by real-time quantitative PCR with TaqMan probe using an ABI Prism 7700 sequence detector (Perkin-Elmer Applied Biosystems, Foster, CA). The gene-specific primers and TaqMan probes were synthesized from Primer Express version 1.5 software (Perkin-Elmer) according to the published cDNA sequences for each gene.

The expression of GAPDH mRNA was used as an internal control. The PCR mixture (25 µl total volume) consisted of forward and reverse primers for each gene (Perkin-Elmer) at 450 nM each, FAM-labeled primer probes (Perkin-Elmer) at 200 nM, and TaqMan Universal PCR Master Mix (Perkin-Elmer). Each PCR amplification was performed in triplicate as follows: 1 cycle at 95°C for 10 min and 40 cycles at 94°C for 15 s and 60°C for 1 min. The quantitative values of target mRNAs were normalized by GAPDH mRNA, because GAPDH mRNA expressions were more stable among all the samples than other internal controls such as beta-actin and 18S ribosomal RNA. Primers and probes are as follows: c-Jun [5'-GAGCCAAGAACTCGGACCTT-3' (forward) and 5'-CCATTGCTGGACTGGATGATCAG-3' (reverse)], c-Jun probe (5'-CTGCTCAAGCTGGCGTC-3'), TGF-beta1 [5'-CGGGAAGCAGTGCCAGAA-3' (forward) and 5'-TGCTCCACAGTTGACTTGAATCTC-3' (reverse)], TGF-beta1 probe (5'-CAGAGCTGCGCCTGCA-3'), ANP [5'-TGATGGATTTCAAGAACCTGCTAGAC-3' (forward) and 5'-GGAGGCATGACCTCATCTTCT-3' (reverse)], ANP probe (5'-CCGGCATCTTCTCCTCC-3'), BNP [5'-ACAATCCACGATGCAGAAGCT-3' (forward) and 5'-TCTCTGAGCCATTTCCTCTGACT-3' (reverse)], BNP probe (5'-CTGGAGCTGATAAGAGAAA-3'), GAPDH [5'-GTGCCAAAAGGGTCATCATCTC-3' (forward) and 5'-GGTTCACACCCATCACAAACATG-3' (reverse)], and GAPDH probe (5'-TTCCGCTGATGCCCC-3').

Statistical Analysis

Values are means ± SE. Data were compared using one-way ANOVA. Post hoc comparisons were made with Fisher's protected least significance t-test for multiple comparisons. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Preliminary Experimental Data

ET-1 dose-response study. Our previous data showed that 10–8 M ET-1 induced cardiomyocyte hypertrophy (22). Here, we determined the effects of various ET-1 doses on cardiomyocyte surface area (Fig. 1A). All the ET-1 doses used in the dose-response studies induced significant increases in cardiomyocyte surface area compared with the vehicle-treated (control) cells (Fig. 1A). However, there was no significant difference in cell surface area among the ET-1-treated groups. In addition, when we evaluated the effects of various ET-1 doses on cellular protein synthesis as determined by leucine uptake (Fig. 1B), 10–10 M ET-1 was the optimal dose (Fig. 1B). On the basis of these findings, 10–10 M ET-1 was used for subsequent experiments.


Figure 1
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  		Fig. 1. A: cell surface area of control (Con) and endothelin (ET)-1-treated cardiomyocytes. B: [14C]leucine incorporation in control and ET-1-treated cardiomyocytes. Values (means ± SE) are expressed relative to control. *P < 0.0001 vs. control.

 
Cardiomyocyte Response to Varying Doses of EPA After 10–10 M ET-1

Inasmuch as 3–300 µM EPA has been shown to upregulate nitric oxide production in endothelial cells (10, 34), we used 1–100 µM EPA to generate a response curve in the present study. In our preliminary experiments, we used 1–100 µM (specifically, 1, 3, 10, and 30 µM) EPA to conduct an EPA dose-response study. EPA at 1, 3, 10, and 30 µM significantly arrested increases in cell surface area induced by 10–10 M ET-1 (Fig. 2A). The cell surface area of groups treated with 30 and 100 µM EPA decreased compared with control (Fig. 2A). In addition, to determine the optimal dose of EPA for suppression of ET-1-induced cardiomyocyte hypertrophy, we performed a leucine uptake test (Fig. 2B). EPA at 1 and 3 µM could not exert significant inhibitory effects on the ET-1-induced increase in protein synthesis rate (Fig. 2B). Although 10 and 30 µM EPA prevented augmentation of protein synthesis, 30 µM EPA inhibited protein synthesis compared with control (Fig. 2B). On the basis of these findings, 10 µM EPA was used for subsequent experiments.


Figure 2
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  		Fig. 2. A: cell surface area of control, ET-1-treated, and eicosapentaenoic acid (EPA)-pretreated ET-1-treated cardiomyocytes. B: [14C]leucine incorporation in control, ET-1-treated, and EPA-pretreated ET-1-treated cardiomyocytes. Values (means ± SE) are expressed relative to control. *P < 0.05 vs. control. **P < 0.0001 vs. control. #P < 0.001 vs. ET-1 alone.

 
Effects of EPA on ET-1-Induced Cardiomyocyte Hypertrophy

Cell surface area. To examine whether EPA treatment could block ET-1-induced cardiac hypertrophy, cardiomyocytes were pretreated with EPA (Fig. 3A). After ET-1 administration, the cardiomyocytes clearly exhibited hypertrophy, which was prevented by pretreatment with EPA (Fig. 3A). Calculation of the cell surface area revealed that administration of ET-1 induced a 1.97-fold increase in cardiomyocyte surface area (P < 0.0001), an effect that was reduced to 1.41-fold in EPA-pretreated cells compared with control (P < 0.0001; Fig. 3B). When control cells were treated with EPA, there was no change in cell surface area (Fig. 3, A and B).


Figure 3
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  		Fig. 3. A: representative charge-coupled device camera images of control (Con), EPA-treated (Con + EPA), ET-1-treated (ET-1), and EPA-pretreated ET-1-treated (EPA + ET-1) cardiomyocytes. Magnification x400. B and C: surface area and leucine incorporation in control, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. Values are means ± SE. *P < 0.01 vs. Con. **P < 0.0001 vs. Con. #P < 0.0001 vs. ET-1.

 
[14C]leucine uptake. [14C]leucine uptake by cardiomyocytes was examined after ET-1 administration. ET-1 induced a 1.72-fold increase (P < 0.0001), which was remarkably reduced (1.28-fold) in EPA-pretreated cells compared with control (P < 0.0001; Fig. 3C). EPA treatment of control cells did not alter the rate of protein synthesis (Fig. 3C).

Expression of {alpha}-actinin protein. Immunofluorescence staining showed strong expression of {alpha}-actinin in ET-1-treated cardiomyocytes, an effect attenuated by EPA (Fig. 4A). Immunoblotting revealed a 72% increase (P < 0.001) in {alpha}-actinin expression after ET-1 administration, an effect that was also attenuated or normalized (P < 0.05) by EPA pretreatment (Fig. 4B). As predicted, EPA treatment of control cardiomyocytes did not cause any change in {alpha}-actinin expression (Fig. 4).


Figure 4
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  		Fig. 4. Protein expression levels of {alpha}-actinin in Con, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. A: immunofluorescence. Magnification x400. B: typical Western blot and intensity of the bands. In each experiment, Con value is normalized as 1.0. Values are means ± SE of 5–7 separate experiments. *P < 0.001 vs. Con. #P < 0.05 vs. ET-1.

 
mRNA expression of ANP and BNP. ET-1 treatment caused a 1.54-fold (P < 0.001) and a 1.65-fold (P < 0.001) increase in ANP and BNP mRNA expression in cardiomyocytes, respectively, as revealed by quantitative real-time PCR; these increases were remarkably suppressed by pretreatment with EPA (P < 0.05; Fig. 5). In control cardiomyocytes treated with EPA alone, expression of ANP and BNP was not altered (Fig. 5).


Figure 5
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  		Fig. 5. Gene expression levels of atrial and brain natriuretic peptides (ANP and BNP) by real-time PCR in Con, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. Values are means ± SE. *P < 0.001 vs. Con. #P < 0.05 vs. ET-1.

 
Effects of EPA on TGF-beta1 Protein and mRNA Expression in ET-1-Induced Cardiomyocyte Hypertrophy

Immunohistochemistry of TGF-beta1 showed a strong immunoreactivity of TGF-beta1 in ET-1-treated cardiomyocytes that largely was prevented by pretreatment with EPA (Fig. 6A). TGF-beta1 protein expression was highly upregulated in cardiomyocytes in which hypertrophy had been induced by ET-1, as demonstrated by ELISA (54% increase, P < 0.0001) and immunoblotting (2.5-fold increase, P < 0.0001), compared with the control group; this upregulation was greatly prevented by pretreatment with EPA (P < 0.0001 vs. ET-1; Fig. 6, B and C). The protein expression of TGF-beta1 corresponded to the gene expression level. The mRNA expression of TGF-beta1 by real-time PCR was significantly upregulated in neonatal cardiomyocytes after ET-1 administration (1.39-fold, P < 0.001 vs. control); this upregulation was arrested by pretreatment with EPA (P < 0.05 vs. ET-1; Fig. 6D). Interestingly, when control cardiomyocytes were treated with EPA only, there was no change in TGF-beta1 expression (Fig. 6).


Figure 6
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  		Fig. 6. Protein and gene expression levels of TGF-beta1 in Con, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. A: protein expression by immunofluorescence. Magnification x400. B: protein expression by ELISA. C: protein expression by immunoblot analysis (typical Western blot and intensity of the bands). In each experiment, the band obtained with control is normalized as 1.0. D: mRNA expression by real-time PCR. Values are means ± SE of 5–7 separate experiments. *P < 0.001 vs. Con; **P < 0.0001 vs. Con; #P < 0.0001 vs. ET-1 (in B and C). *P < 0.001 vs. Con; #P < 0.05 vs. ET-1 (in D).

 
Effects of EPA on ET-1-Induced Expression of Phosphorylated and Total JNK

The expression level of total JNK, a subfamily of MAPK, activated by ET-1 in cardiomyocytes (22) was significantly enhanced in ET-1-treated cardiomyocytes (59% increase, P < 0.0001 vs. control) compared with control (Fig. 7A). Results obtained by ELISA and the representative immunofluorescence photomicrographs also revealed an increase in levels of JNK that is dually phosphorylated at Thr183 and Tyr185 (Fig. 7, A and B). Nuclear localization of phosphorylated JNK in cardiomyocytes was confirmed by 4',6-diamidino-2-phenylindole counterstaining (Fig. 7C). Moreover, a slight immunoreactivity of phosphorylated JNK was also seen in cellular cytoplasm. The increases in expression of total JNK and phosphorylated JNK in ET-1-treated cardiomyocytes were greatly reversed by EPA pretreatment (Fig. 7). When control cardiomyocytes were treated with EPA only, there was no change in JNK expression or JNK activity (Fig. 7).


Figure 7
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  		Fig. 7. Protein expression and activity of JNK in Con, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. A: total JNK protein expression by immunoblot analysis (typical Western blot and intensity of the bands). In each experiment, the band obtained with control is normalized as 1.0. B: phosphorylated JNK quantitation by ELISA. Values are means ± SE of 5–7 separate experiments. C: phosphorylated JNK expression by immunofluorescence. Red, phosphorylated JNK (a); blue, 4',6-diamidino-2-phenylindole (b). Images are merged in c. Magnification x200. *P < 0.0001 vs. Con; #P < 0.0001 vs. ET-1 (in A). *P < 0.01 vs. Con; #P < 0.01 vs. ET-1 (in B).

 
Effects of EPA on ET-1-Induced Expression of Phosphorylated and Total c-Jun

Because ET-1 induced activation of JNK, as well as c-Jun, an important downstream molecule of JNK in cardiomyocytes, we determined expression patterns of c-Jun in the present experimental setting (Fig. 8). Immunoblot analyses demonstrated a significant increase in expression of c-Jun (phosphorylated and total), which is also involved in the signal transduction pathway associated with ET-1 stimulation in cardiomyocytes (22), in ET-1-treated cardiomyocytes (Fig. 8, A and B). The increase in c-Jun transcripts in ET-1-treated cardiomyocytes was quantitatively confirmed by real-time PCR (Fig. 8C). The increases in protein and mRNA expression levels of c-Jun were decreased by pretreatment with EPA in cardiomyocytes in which hypertrophy had been induced by ET-1. Treatment with EPA alone did not induce any alteration in c-Jun expression in control cardiomyocytes (Fig. 8).


Figure 8
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  		Fig. 8. Protein and gene expression levels of total c-Jun and activity of c-Jun in Con, Con + EPA, ET-1, and EPA + ET-1 cardiomyocytes. A and B: immunoblot analysis of phosphorylated and total c-Jun (typical Western blots and intensity of the bands). In each experiment, the band obtained with control is normalized as 1.0. C: real-time PCR analysis of c-jun mRNA expression. Values are means ± SE of 5–7 separate experiments. *P < 0.01 vs. Con; #P < 0.05 vs. ET-1 (in A and C). *P < 0.05 vs. Con; **P < 0.0001 vs. Con; #P < 0.001 vs. ET-1 (in B).

 
Effect of EPA on Prepro-ET-1 mRNA Expression Induced by ET-1

ET-1 (10–10 M) increased the ET-1 mRNA level in cardiomyocytes (153.0 ± 15.7% vs. control, P < 0.01); this increase was inhibited by pretreatment with 10 µM EPA (99.0 ± 9.3% vs. ET-1, P < 0.01), whereas treatment of cardiomyocyte with EPA alone did not affect ET-1 mRNA level (Table 1).


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  		Table 1. Effect of EPA on prepro-ET-1 mRNA expression in ET-1-induced hypertrophied cardiomyocytes

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The findings of the present study are important, in that they demonstrate for the first time that EPA pretreatment suppresses ET-1-induced neonatal ventricular cardiomyocyte hypertrophy in vitro. We show that pretreatment with EPA prevents ET-1-induced cardiac hypertrophy at the morphological and signal levels (ANP and BNP mRNA expression). Moreover, we also showed that ET-1 caused an upregulation of TGF-beta1 and JNK, one of the important components of the MAPK system, in neonatal ventricular cardiomyocytes. EPA pretreatment regressed TGF-beta1 and JNK upregulations and, importantly, also attenuated ET-1-induced hypertrophic changes in neonatal ventricular cardiomyocytes.

The cardiovascular system undergoes structural and functional adaptations, most notably cardiac hypertrophy, induced by various elements, such as mechanical factors and neurohormones (47). Initially, cardiac hypertrophy plays a beneficial physiological role that leads to improved cardiac contractile force (26, 47). However, if left unchecked, it can lead to contractile dysfunction and, subsequently, heart failure (26). ET-1 has been well documented in different studies in vivo and in vitro as one of the crucial factors for the development of cardiomyocyte hypertrophy. Consistent with other reports, in the present study, ET-1 also caused cardiomyocyte hypertrophy. In this study, we characterized hypertrophy of cardiomyocytes by monitoring cell size, protein synthesis, expression of ANP and BNP, and cytoskeletal organization of {alpha}-actinin. Our data indicate that cardiomyocyte surface area and protein synthesis were significantly increased 1.7- to 2.0-fold after stimulation with ET-1. In these cells, {alpha}-actinin organization was also increased. A growing body of evidence suggests ANP and BNP upregulation in cardiac hypertrophy, as well as in heart failure, as an adaptive mechanism. In the present investigation, we also found that ET-1 induced cardiomyocyte hypertrophy. Expression levels of ANP and BNP as revealed by real-time PCR were upregulated. Whereas the role of ET-1 in cardiomyocyte hypertrophy is well established and the present data, showing ET-induced cardiac hypertrophy with the upregulation of ANP, BNP, and {alpha}-actinin, are consistent with these earlier studies, less is known about the possible inhibitory action of the fish oil extract EPA on ET-1-induced cardiac hypertrophy. The present study is the first to provide such evidence. This finding is important, in that it may potentially provide insights that may lead to development of alternative approaches to heart failure therapies that are largely aimed at antagonizing factors that induce hypertrophic responses (i.e., angiotensin II and ET) (47). Indeed, recent prospective randomized studies indicate improved survival in patients treated with {omega}-3 PUFAs after myocardial infarction (40) and beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction (1, 12). The exact mechanisms underlying these effects are unknown. It is possible, on the basis of the present data, that EPA, among other factors, may be one of the active ingredients responsible for the improved prognosis. This speculation is supported by several human and animal model studies and by a recent study showing that fish oil rich in the {omega}-3 fatty acids EPA and DHA not only has an antihypertensive effect in spontaneously hypertensive rats, but it also decreases LVH (6, 40, 44).

The exact mechanism(s) underlying the prevention of cardiac hypertrophy by dietary substances has not been well clarified. It is possible that EPA and DHA, among other factors, may be involved, in that they regulate expression of some gene clusters likely to be implicated in the pathogenesis of cardiac hypertrophy, such as cytokines, signal transduction, transcription, cell cycle defense and repair, apoptosis, cell adhesion, the cytoskeleton, and hormones. Li et al. (27) reported an important modification of the lipid composition of membrane lipid rafts and T cell function by EPA supplementation, which suggests that EPA can enter directly into the cell membrane and directly affect the cell signal pathway and expression of various genes. Moreover, in human breast cancer cells, {omega}-3 PUFAs decrease cell proliferation, possibly by decreasing signal transduction through the Akt-nuclear factor-{kappa}B cell survival pathway (38). Recently, it was reported that DHA inhibited PE-induced cardiomyocyte hypertrophy via the ERK pathway (39). Collectively, these data suggest that EPA may diffuse through the cell membrane and affect the cell signal pathway and gene expression.

TGF-beta1, a locally generated cytokine, plays a pivotal role in the development of cardiac hypertrophy and heart failure, and its influence on ET-1 expression in nonmyocyte heart cells (fibroblasts) and rat liver stellate cells has been demonstrated (16, 20). However, the effect of ET-1 on TGF-beta1 expression and hypertrophy in cardiomyocytes is unknown. Because ET-1 is known to regulate TGF-beta1 in extracardiac cells, such as fetal skin-derived cultured mast cells (30), and ET-1 and TGF-beta1 are implicated in cardiac hypertrophy, it is likely that ET-1 regulates TGF-beta1 expression in cardiac cells. Consistent with our hypothesis, the present study found increased levels of TGF-beta1 mRNA and protein in ET-1-induced cardiomyocyte hypertrophy, suggesting a role for ET-1 in the regulation of TGF-beta1 expression. The effects of ET-1 on TGF-beta1 are likely to be regulated by the ETA receptor, the levels (mRNA and protein) of which are upregulated in cardiomyocytes (data not shown). This observation is consistent with studies showing that ETA receptor antagonism prevents elevation of cardiac TGF-beta1 mRNA and protein levels in deoxycorticosterone acetate-salt hypertensive rats (3). Importantly, the present study demonstrates that 10 µM EPA significantly suppresses the overexpressed ET-1-induced TGF-beta1 at protein and mRNA levels; these findings are consistent with earlier studies showing significant inhibition of TGF-beta1 expression (33, 49). In vascular smooth muscle cells from spontaneously hypertensive rats, 20 µM EPA significantly inhibited expression of TGF-beta1 mRNA (25). Moreover, EPA, which inhibits procarcinogenic PKC-betaII activity and colon carcinogenesis, inhibits the TGF-beta signaling axis and induces a hyperproliferative state within the colonic epithelium (49). Although the present study does not explain the mechanism by which EPA inhibited ET-1-induced TGF-beta1 upregulation in hypertrophied cardiomyocytes, EPA pretreatment of ET-1-treated cardiomyocytes could downregulate the increase in ETA receptor expression (unpublished observation). Thus, on the basis of the present findings, one might speculate that the inhibitory action of EPA on ET-1-induced upregulation of TGF-beta1 in cardiomyocytes may be partly mediated through the ETA receptor.

We previously demonstrated an upregulation of JNK in ET-1-induced hypertrophied cardiomyocytes and a regression of this upregulated JNK by peroxisome proliferator-activated receptor-{alpha} agonist administration (22). Here, we further show the upregulation of JNK and c-Jun in ET-1-induced hypertrophied cardiomyocytes, in agreement with earlier studies that showed ET-1-induced activation of the JNK pathway in smooth muscle cells and cardiomyocytes (22) and others that showed a signaling pathway linking the ETA receptor to JNK (14, 22). JNK, which is thought to be the principal factor involved in c-Jun upregulation (22), is activated by its dual phosphorylation motif, which results in phosphorylation of serine residues in the NH2-terminal region of c-Jun and, thereby, an increase in the transcriptional-activating activity of c-Jun (22). Moreover, it has been shown that activation of the JNK pathway contributes to transcriptional and morphological responses to Gq receptor-coupled hypertrophic agonists, such as ET-1 and PE (14). Collectively, these data, along with the present findings, suggest that upregulation of the JNK signaling pathway could further amplify the ET-1-induced cellular response. EPA may arrest the ET-1-induced cardiomyocyte hypertrophic progression through the inhibition of hypertrophic factors other than TGF-beta1, and MAPK may be one of the factors involved. Accordingly, in this study, we demonstrated that EPA interrupted the earliest ET-1-induced events, i.e., JNK activation, c-Jun phosphorylation, and c-Jun induction in cardiomyocytes. A growing body of evidence has focused on the effect of EPA on different MAPK signalings. EPA can inhibit ultraviolet-induced matrix metalloproteinase-1 expression by inhibiting the MEK1-ERK-c-Fos and SEK1-JNK-c-Jun pathways in human dermal fibroblasts (25) and by decreasing LPS-induced c-Jun phosphorylation, protein levels, and JNK activation in human monocytic THP-1 cells (50). Treatment of hippocampal neurons with EPA abrogated the LPS-induced increases in phosphorylation of JNK and c-Jun (29). EPA and DHA, other PUFAs, significantly suppressed IL-6 superinduction by deoxynivalenol and impaired deoxynivalenol-induced ERK1/2 and JNK1/2 phosphorylation in spleens of mice (31). In our preliminary experiment, EPA could not suppress the ET-1-induced upregulation of ERK in cardiomyocytes. Therefore, the inhibitory effect of EPA on ET-1-related hypertrophic responses might be through interference in the JNK, rather than the ERK, pathway. Moreover, as shown in Table 1, ET-1-induced ET-1 mRNA (prepro-ET-1) expression in cardiomyocytes was suppressed by pretreatment with EPA, which further suggests that EPA may suppress ET-1-induced cardiomyocyte hypertrophy through the inhibition of JNK signaling.

To gain more insights into the antihypertrophic action of EPA on cardiomyocytes, one should look at the effects of EPA on angiotensin II-induced cardiomyocyte hypertrophy. In addition, the effects of another important fish oil, DHA, on the ET-1-induced cardiomyocyte hypertrophy should be studied. Although DHA was able to inhibit PE-induced cardiomyocyte hypertrophy, EPA could not suppress the increased protein synthesis in PE-induced cardiomyocyte hypertrophy (39). The differential protective role of different fish oils, such as EPA and DHA, should be investigated at a more molecular level to clarify the mechanism of antihypertrophic action of EPA and DHA on cardiomyocyte hypertrophy caused by various hypertrophy-inducing factors.

In the present study, we have investigated the effects of EPA pretreatment on ET-1-induced hypertrophy of neonatal ventricular cardiomyocytes in vitro and deciphered their possible signaling pathways. Here, we report that EPA limits ET-1-induced cardiomyocyte hypertrophy. In isolated cardiomyocytes, EPA inhibits the important hypertrophic pathway involving TGF-beta1 as induced by the vascular peptide ET-1. We also found that ET-1 stimulates additional hypertrophic signaling involving JNK and c-Jun and that EPA inhibits this signaling. Finally, EPA has the ability to downregulate the enhanced gene expression of ANP and BNP induced by ET-1. Thus EPA modulates multiple signal inputs to prevent cardiomyocyte hypertrophy in vitro.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ministry of Education, Science, Sports, and Culture of Japan Grants-in-Aid for Scientific Research 15390077 and 15650130 and a grant from the Miyauchi Project of Tsukuba Advanced Research Alliance at the University of Tsukuba.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Motoyuki Iemitsu, Naoto Yamaguchi, and Masahiro Hagiwara for constructive suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Div., Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan (e-mail: t-miyauc{at}md.tsukuba.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Albert CM, Campos H, Stampfer MJ, Ridker PM, Manson JE, Willett WC, and Ma J. Blood levels of long-chain n-3 fatty acids and the risk of sudden death. N Engl J Med 346: 1113–1118, 2002.[Abstract/Free Full Text]
  2. Alexander JW. Immunonutrition: the role of {omega}-3 fatty acids. Nutrition 14: 627–633, 1998.[CrossRef][ISI][Medline]
  3. Ammarguellat F, Larouche I, and Schiffrin EL. Myocardial fibrosis in DOCA-salt hypertensive rats: effect of endothelin ETA receptor antagonism. Circulation 103: 319–324, 2001.[ISI][Medline]
  4. Bao DQ, Mori TA, Burke V, Puddey IB, and Beilin LJ. Effects of dietary fish and weight reduction on ambulatory blood pressure in overweight hypertensives. Hypertension 32: 710–717, 1998.[Abstract/Free Full Text]
  5. Brand T and Schneider MD. The TGF-beta superfamily in myocardium: ligands, receptors, transduction, and function. J Mol Cell Cardiol 27: 5–18, 1995.[ISI][Medline]
  6. Brandle M and Jacob R. Effects of a diet rich in {omega}-3 fatty acids on left ventricular geometry and dynamics in spontaneously hypertensive rats. Eur J Appl Physiol 61: 177–181, 1990.[CrossRef]
  7. Burr ML, Fehily AM, Gilbert JF, Rogers S, Holliday RM, Sweetnam PM, Elwood PC, and Deadman NM. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 2: 757–761, 1989.[ISI][Medline]
  8. Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 36: 1007–1024, 2001.[ISI][Medline]
  9. Chen LY, Jokela R, Li DY, Bavry AA, Sandler H, Sjoquist M, Saldeen T, and Mehta JL. Effect of stable fish oil on arterial thrombogenesis, platelet aggregation, and superoxide dismutase activity. J Cardiovasc Pharmacol 35: 502–505, 2000.[CrossRef][ISI][Medline]
  10. Chisaki K, Okuda Y, Suzuki S, Miyauchi T, Soma M, Ohkoshi N, Sone H, Yamada N, and Nakajima T. Eicosapentaenoic acid suppresses basal and insulin-stimulated endothelin-1 production in human endothelial cells. Hypertens Res 8: 655–661, 2003.[CrossRef]
  11. Das UN. Beneficial effects of n-3 fatty acids in cardiovascular diseases: But, why and how? Prostaglandins Leukot Essent Fatty Acids 63: 351–362, 2000.[CrossRef][ISI][Medline]
  12. Daviglus ML, Stamler J, Orencia AJ, Dyer AR, Liu K, Greenland P, Walsh MK, Morris D, and Shekelle RB. Fish consumption and the 30-year risk of fatal myocardial infarction. N Engl J Med 336: 1046–1053, 1997.[Abstract/Free Full Text]
  13. Denys A, Aires V, Hichami A, and Khan NA. Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid. FEBS Lett 564: 177–182, 2004.[CrossRef][ISI][Medline]
  14. Finn SG, Dickens M, and Fuller SJ. c-Jun N-terminal kinase-interacting protein 1 inhibits gene expression in response to hypertrophic agonists in neonatal rat ventricular myocytes. Biochem J 358: 489–495, 2001.[CrossRef][ISI][Medline]
  15. Frey N and Olson EN. Cardiac hypertrophy: the good, the bad and the ugly. Annu Rev Physiol 65: 45–79, 2003.[CrossRef][ISI][Medline]
  16. Gandhi CR, Kuddus RH, Uemura T, and Rao AS. Endothelin stimulates transforming growth factor-beta1 and collagen synthesis in stellate cells from control but not cirrhotic rat liver. Eur J Pharmacol 406: 311–318, 2000.[CrossRef][ISI][Medline]
  17. Giannessi D, Del Ry S, and Vitale RL. The role of endothelins and their receptors in heart failure. Pharmacol Res 43: 111–126, 2001.[CrossRef][ISI][Medline]
  18. Grimsgaard S, Bonaa KH, Hansen JB, and Myhre ES. Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on hemodynamics in humans. Am J Clin Nutr 68: 52–59, 1998.[Abstract]
  19. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 354: 447–455, 2001.
  20. Harada M, Itoh H, Nakagawa O, Ogawa Y, Miyamoto Y, Kuwahara K, Ogawa E, Igaki T, Yamashita J, Masuda I, Yoshimasa T, Tanaka I, Saito Y, and Nakao K. Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes. Circulation 96: 3737–3744, 1997.[ISI][Medline]
  21. Hu FB, Bronner L, Willett WC, Stampfer MJ, Rexrode KM, Albert CM, Hunter D, and Manson JE. Fish and {omega}-3 fatty acid intake and risk of coronary heart disease in women. JAMA 287: 1815–1821, 2002.[Abstract/Free Full Text]
  22. Irukayama-Tomobe Y, Miyauchi T, Sakai S, Kasuya Y, Ogata T, Takanashi M, Iemitsu M, Sudo T, Goto K, and Yamaguchi I. Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-{alpha} partly via blockade of c-Jun NH2-terminal kinase pathway. Circulation 109: 904–910, 2004.[CrossRef][ISI][Medline]
  23. Ito H. Endothelins and cardiac hypertrophy. Life Sci 61: 585–593, 1997.[CrossRef][ISI][Medline]
  24. Jones JV, James MA, and MacConnell TJ. Left ventricular hypertrophy. J Hum Hypertens 5 Suppl 2: 35–40, 1991.
  25. Kim HH, Shin CM, Park CH, Kim KH, Cho KH, Eun HC, and Chung JH. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts. J Lipid Res 46: 1712–1720, 2005.[Abstract/Free Full Text]
  26. Levy D, Garrison RJ, Savage DD, Kannel WB, and Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med 322: 1561–1566, 1990.[Abstract]
  27. Li Q, Tan L, Wang C, Li N, Li Y, Xu G, and Li J. Polyunsaturated eicosapentaenoic acid changes lipid composition in lipid rafts. Eur J Nutr 45: 144–151, 2006.[CrossRef][ISI][Medline]
  28. Li Y, Kang JX, and Leaf A. Differential effects of various eicosanoids on the production or prevention of arrhythmias in cultured neonatal rat cardiac myocytes. Prostaglandins 54: 511–530, 1997.[CrossRef][ISI][Medline]
  29. Lonergan PE, Martin DS, Horrobin DF, and Lynch MA. Neuroprotective actions of eicosapentaenoic acid on lipopolysaccharide-induced dysfunction in rat hippocampus. J Neurochem 91: 20–29, 2004.[CrossRef][ISI][Medline]
  30. Matsushima H, Yamada N, Matsue H, and Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells. Eur J Immunol 34: 1910–1919, 2004.[CrossRef][ISI][Medline]
  31. Moon Y and Pestka JJ. Deoxynivalenol-induced mitogen-activated protein kinase phosphorylation and IL-6 expression in mice suppressed by fish oil. J Nutr Biochem 14: 717–726, 2003.[CrossRef][ISI][Medline]
  32. Mori TA, Watts GF, Burke V, Hilme E, Puddey IB, and Beilin LJ. Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men. Circulation 102: 1264–1269, 2000.[Abstract/Free Full Text]
  33. Nakayama M, Fukuda N, Watanabe Y, Soma M, Hu WY, Kishioka H, Satoh C, Kubo A, and Kanmatsuse K. Low dose of eicosapentaenoic acid inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats through suppression of transforming growth factor-beta. J Hypertens 17: 1421–1430, 1999.[CrossRef][ISI][Medline]
  34. Okuda Y, Kawashima K, Sawada T, Tsurumaru K, Asano M, Suzuki S, Soma M, Nakajima T, and Yamashita K. Eicosapentaenoic acid enhances nitric oxide production by cultured human endothelial cells. Biochem Biophys Res Commun 232: 487–491, 1997.[CrossRef][ISI][Medline]
  35. Richey PA and Brown SP. Pathological vs. physiological left ventricular hypertrophy: a review. J Sports Sci 16: 129–141, 1998.[CrossRef][ISI][Medline]
  36. Sacks FM, Stone PH, Gibson CM, Silverman DI, Rosner B, and Pasternak RC. Controlled trial of fish oil for regression of human coronary atherosclerosis. HARP Research Group. J Am Coll Cardiol 25: 1492–1498, 1995.[Abstract]
  37. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, and Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature 384: 353–355, 1996.[CrossRef][Medline]
  38. Schley PD, Jijon HB, Robinson LE, and Field CJ. Mechanisms of {omega}-3 fatty acid-induced growth inhibition in MDA-MB-231 human breast cancer cells. Breast Cancer Res Treat 92: 187–195, 2005.[CrossRef][ISI][Medline]
  39. Siddiqui RA, Shaikh SR, Kovacs R, Stillwell W, and Zaloga G. Inhibition of phenylephrine-induced cardiac hypertrophy by docosahexaenoic acid. J Cell Biochem 92: 1141–1159, 2004.[CrossRef][ISI][Medline]
  40. Singh RB, Niaz MA, Sharma JP, Kumar R, Rastogi V, and Moshiri M. Randomized, double-blind, placebo-controlled trial of fish oil and mustard oil in patients with suspected acute myocardial infarction: the Indian experiment of infarct survival. 4. Cardiovasc Drugs Ther 11: 485–491, 1997.[CrossRef][ISI][Medline]
  41. Sugden PH. Signalling pathways in cardiac myocyte hypertrophy. Ann Med 33: 611–622, 2001.[ISI][Medline]
  42. Suzuki T, Hoshi H, and Mitsui Y. Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium. FEBS Lett 268: 149–151, 1990.[CrossRef][ISI][Medline]
  43. Takeo S, Nasa Y, Tanonaka K, Yabe K, Nojiri M, Hayashi M, Sasaki H, Ida K, and Yanai K. Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats. Mol Cell Biochem 188: 199–208, 1998.[CrossRef][ISI][Medline]
  44. Vaskonen T, Laakso J, Mervaala E, Sievi E, and Karppanen H. Interrelationships between salt and fish oil in stroke-prone spontaneously hypertensive rat. Blood Press 5: 178–189, 1996.[Medline]
  45. Weber P and Raederstorff D. Triglyceride-lowering effect of {omega}-3 LC-polyunsaturated fatty acids. Nutr Metab Cardiovasc Dis 10: 28–37, 2000.[ISI][Medline]
  46. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, and Yazaki Y. Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem 271: 3221–3228, 1996.[Abstract/Free Full Text]
  47. Yamazaki T, Komuro I, Shiojima I, and Yazaki Y. The molecular mechanism of cardiac hypertrophy and failure. Ann NY Acad Sci 30: 38–48, 1999.
  48. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415, 1988.[CrossRef][Medline]
  49. Yu W, Murray NR, Weems C, Chen L, Guo H, Ethridge R, Ceci JD, Evers BM, Thompson EA, and Fields AP. Role of cyclooxygenase 2 in protein kinase C betaII-mediated colon carcinogenesis. J Biol Chem 278: 11167–11174, 2003.[Abstract/Free Full Text]
  50. Zhao Y and Chen LH. Eicosapentaenoic acid prevents lipopolysaccharide-stimulated DNA binding of activator protein-1 and c-Jun N-terminal kinase activity. J Nutr Biochem 16: 78–84, 2005.[CrossRef][ISI][Medline]



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