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Cardiovascular Research 2005 68(2):213-223; doi:10.1016/j.cardiores.2005.05.018
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Copyright © 2005, European Society of Cardiology

Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency

Ryotaro Takahashia, Kenji Okumuraa,*, Toru Asaia, Toshihisa Hiraia, Hisashi Murakamia, Ryuichiro Murakamia, Yasushi Numaguchia, Hideo Matsuia, Masafumi Itob and Toyoaki Muroharaa

aDepartment of Cardiology, Nagoya University Graduate School of Medicine,65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
bDepartment of Pathology, Nagoya University Hospital, Nagoya, Japan

* Corresponding author. Tel.: +81 52 744 2168; fax: +81 52 744 2177. Email address: kenji{at}med.nagoya-u.ac.jp

Received 8 February 2005; revised 15 May 2005; accepted 18 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: 1,2-Diacylglycerol (DAG), a lipid second messenger that activates protein kinase C (PKC), is increased with a distinct fatty acid composition in the heart of the juvenile visceral steatosis (JVS) mouse, which develops pathological cardiac hypertrophy with lipid accumulation induced by the perturbation of fatty acid β-oxidation due to systemic carnitine deficiency. Fish oil (FO) may exert its beneficial effects on the cardiomyopathy in JVS mice by modifying the molecular species composition of myocardial DAG. To test this hypothesis, we investigated the effects of dietary FO on the hypertrophied hearts in JVS mice.

Methods: Both control and JVS mice were fed a FO diet (containing 10% FO) or a standard diet from 4 weeks of age.

Results: At 8 weeks of age, the ventricular-to-body weight ratio in JVS mice was 2.7-fold higher than that in controls (9.9 ± 0.1 vs. 3.7 ± 0.1 mg/g, P<0.01) and was reduced by dietary FO (7.7 ± 0.1 mg/g, P<0.01 vs. JVS mice). In JVS mice, myocardial DAG levels were elevated by 2.3-fold with a distinct fatty acid composition with increases in C18:1n-7,9 and C18:2n-6 fatty acids compared with controls; dietary FO had no effects on the total DAG levels but significantly altered the fatty acid composition of DAG with reduction of both fatty acid species. Immunoblot analysis showed that dietary FO prevented the membrane translocation of cardiac PKCs {alpha}, β2, and {varepsilon} in JVS mice. Dietary FO did not affect the plasma and myocardial total carnitine levels in JVS mice. Furthermore, dietary FO significantly improved the progressive left ventricular dysfunction and survival rate in JVS mice.

Conclusions: Dietary FO may attenuate cardiac hypertrophy with improvements in cardiac function and survival in JVS mice via modification of the molecular species composition of myocardial DAG and the consequent inhibition of PKC redistribution. These results suggest the implication of the molecular species composition of DAG in the pathogenesis of lipotoxic cardiomyopathy due to perturbations of fatty acid β-oxidation.

KEYWORDS Cardiomyopathy; Hypertrophy; Lipid signaling; Second messengers; Protein kinase C

This article is referred to in the Editorial by Stanley et al. (pages 175–177) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Carnitine is essential for the transport of long-chain fatty acids into the mitochondrial matrix for β-oxidation, and is known to play an important role in cellular lipid and energy metabolism [1]. Primary systemic carnitine deficiency (SCD) is a rare hereditary disease caused by decreased renal re-absorption of carnitine due to mutations of the carnitine transporter OCTN2 gene [2], and is associated with hypertrophic cardiomyopathy, which is treatable by L-carnitine supplementation [3,4]. It has been reported that autopsy from the hearts of SCD patients showed cardiomyocyte hypertrophy associated with the deposition of intracellular lipid droplets, which are indicative of lipotoxicity [5,6].

The juvenile visceral steatosis (JVS) mouse was established as an excellent murine model of SCD [7]. SCD in JVS mice is caused by decreased renal re-absorption of carnitine due to a spontaneous mutation in the OCTN2 gene, as has been reported in human SCD as well [2]. In JVS mice, carnitine levels in tissues including the myocardium are decreased and fatty acid β-oxidation is depressed [8]. As a result, JVS mice develop marked cardiac hypertrophy with lipid accumulation [8,9] and progressive cardiac dysfunction. Thus, the JVS mouse may serve as a murine model of lipotoxic cardiomyopathy induced by the perturbation of fatty acid β-oxidation. We have previously reported that 1,2-diacylglycerol (DAG), a lipid second messenger that activates protein kinase C (PKC) [10], was increased with a distinct fatty acid composition in the hypertrophied hearts of JVS mice [11,12]. The DAG–PKC pathway is known to be one of the important intracellular pathways mediating the hypertrophic response [13–15]. Several studies have demonstrated the acyl chain dependence of DAG in the activation of PKC in vitro [16,17]. Particular molecular species of DAG, more than its overall concentration, determine the activation of PKC isozymes in agonist-stimulated rat cardiomyocytes [18]. Therefore, the distinct molecular species composition of myocardial DAG may play a crucial role in the signaling pathway leading to pathological cardiac hypertrophy in JVS mice.

Fish oil (FO) or n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) have been shown in epidemiological and clinical trials to reduce the incidence of cardiovascular disease and sudden death [19]. n-3 PUFA and FO were shown to alter the fatty acid composition of DAG or phospholipids in cultured rat cardiomyocytes [20,21] and in the rat myocardium [22,23], respectively. n-3 PUFA and FO were also shown to inhibit the PKC activity in vitro [24–27] and in vivo [28,29], respectively. Moreover, it was recently reported that DHA prevented phenylephrine-induced hypertrophy in neonatal cardiomyocytes [30].

Since the distinct molecular species composition of myocardial DAG has been thought to play a crucial role in the development of lipotoxic cardiomyopathy in JVS mice, FO may exert its beneficial effects on pathological cardiac hypertrophy in JVS mice by modifying the fatty acid composition of DAG. To test this hypothesis, we investigated the effects of dietary FO on the fatty acid composition of myocardial DAG and pathological cardiac hypertrophy in JVS mice.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Animal model and treatment
All protocols described were approved by the Animal Ethics Committee of Nagoya University, Nagoya, Japan. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). The JVS mice used were of the C3H strain [7]. All animals were maintained under specific pathogen-free conditions. Homozygous mutants (jvs/jvs) had swollen fatty livers that were recognizable through the abdominal wall at 5 days after birth. We treated the homozygous mutants with daily subcutaneous injections of L-carnitine 5 µmol/mouse from 11 to 21 days after birth in order to prolong their lives according to a previously reported method [9]. Wild-type mice of the C3H strain were used as controls. Both control and JVS mice were divided into two dietary groups, those fed on a standard diet or those fed on a FO diet, and the animals were maintained on the respective diet starting at 4 weeks of age. The composition of the standard diet was based on an AIN-93G purified diet [31] containing 10 g of corn oil per 100 g of chow. The FO diet contained 10 g of fish oil (from menhaden, Sigma-Aldrich, St. Louis, MO, USA) per 100 g of chow in place of corn oil, and was otherwise identical to the standard diet. Carnitine was not included in the ingredients of either diet. The fatty acid composition of each diet, measured by gas chromatography, is shown in Table 1. The animals were allowed free access to water and to the respective diet throughout the study period. Both control and JVS mice were studied at 6 weeks of age (stage of developing hypertrophy), 8 weeks of age (stage of established hypertrophy), and 10 weeks of age (stage of dilated cardiomyopathy).


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  		Table 1 Fatty acid composition of diets

 
2.2 Morphological analysis
Cardiac tissue was examined by means of light microscopy. The tissue was fixed in 10% formaldehyde in phosphate buffer, and paraffin sections at a thickness of 4 µm were examined after they were stained with hematoxylin–eosin. The ventricular wall thickness and myocyte width of each sample were measured with NIH Image analysis software (National Institutes of Health, Bethesda, MD, USA) by an operator who was blinded to the study groups. Three fields were randomly chosen from the septal, posterior, and right ventricular wall at macro view and five largest cells from each field were chosen for the measurement of myocyte width.

2.3 Echocardiographic and hemodynamic measurements
On the day of sacrifice, the systolic blood pressure and heart rate were determined in each animal. The tail cuff method was used, employing a photoelectric tail cuff detection system, Softron BP-98A (Softron, Tokyo, Japan). Left ventricular function was evaluated by transthoracic echocardiography using SONOS 7500 (Philips Medical Systems, Andover, MA, USA) with a 10-MHz imaging transducer. Briefly, each animal was slightly sedated with 20 mg kg–1 i.p. sodium pentobarbital. M-mode images of the left ventricle at the midchordal levels were recorded, and left ventricular dimensions at end-diastole (EDD) and end-systole (ESD) were measured by the leading edge method. For each measurement, data from three cardiac cycles were averaged, and fractional shortening (FS) was calculated as FS (%)=(EDD–ESD)/EDD x 100.

2.4 Survival study
The survival analysis was performed in controls treated with the standard diet, controls treated with the FO diet, JVS mice treated with the standard diet, and JVS mice treated with the FO diet. During the study period, cages were inspected daily for animals that had died.

2.5 Blood and tissue sampling
Each animal was anesthetized with diethyl ether. Blood samples were obtained directly from the hearts. The hearts were rapidly excised and washed thoroughly with cold saline. After the atria were removed, the ventricles were snap-frozen in liquid nitrogen. Samples used for lipid analysis were lyophilized before measurement.

2.6 Myocardial total carnitine levels
The total carnitine levels in the myocardium was determined by an enzymatic method described elsewhere [32].

2.7 Myocardial lipid analysis
The lyophilized tissue samples were homogenized in 5 ml of a chilled chloroform–methanol mixture (2:1, v/v) containing 0.01% butylated hydroxytoluene as an antioxidant and cholesteryl acetate as an internal standard. Simultaneous quantitation of DAG, triglycerides, and ceramides was performed by the TLC–FID method, as previously described [33]. In brief, 1 µl of a lipid extract solution containing ceramides, neutral lipids, and free fatty acids was dissolved in chloroform and applied carefully to silica gel using 75-µm precoated thin-layer rods (Chromarod-SIII, Iatron Lab., Inc., Tokyo, Japan). The first development was carried out in a solvent system of chloroform–methanol–H2O (57:12:0.6, v/v). The second and the third developments were carried out in 1,2-dichloroethane–chloroform–acetic acid (46:6:0.05, v/v). The fourth development was carried out in n-hexane–diethyl ether–acetic acid (98:1:1, v/v). The Chromarods were then scanned in an Iatroscan MK-5 analyzer (Iatron Lab., Inc., Tokyo, Japan). The fatty acid composition of DAG obtained by TLC was determined by gas chromatography (model GC 14-A, Shimadzu, Kyoto, Japan) using an HR-SS-10 fused silica capillary column (30 m x 0.25 mm internal diameter, Shinwakakoh, Kyoto, Japan), as previously described [11,12].

2.8 PKC isozyme expression and translocation
PKC isozyme expression and translocation were measured by immunoblot analysis. The cytosolic fractions of the total cellular proteins were separated by 1-h centrifugation at 100,000 x g after homogenization of the snap-frozen tissue of the ventricle in sample buffer containing 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 10 mM EGTA, 10 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM dithiothreitol. The membrane fractions were homogenized from precipitation in sample buffer with 1.0% Triton X-100 and were centrifuged at 100,000 x g for 1 h. The protein concentration was determined by the method of Bradford. All samples (20 µg) were subjected to immunoblot analysis with the use of the antibodies against PKCs {alpha}, β2, {delta}, and {varepsilon} (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the enhanced chemiluminescence method (Amersham Biosciences, Buckinghamshire, UK).

2.9 Statistics
All results are expressed as mean ± S.E.M. The survival analysis was performed by the Kaplan–Meier method, and between-group differences in survival were tested by the log-rank test. Between-group comparisons were assessed by one-way ANOVA with Scheffé's post hoc test. A value of P<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Effects of dietary FO on cardiac hypertrophy in JVS mice
At 8 weeks of age, the body weight of JVS mice was significantly lower than that in controls (16.8 ± 0.2 vs. 22.8 ± 0.3 g, P<0.01) (Fig. 1A). However, dietary FO did not affect body weight in either group. The ventricular weight in JVS mice was significantly higher than that in controls (166.9 ± 1.3 vs. 85.4 ± 1.7 mg, P<0.01), and was reduced by dietary FO (134.7 ± 3.2 mg in JVS mice treated with the FO diet, P<0.01 vs. JVS mice treated with the standard diet) (Fig. 1B). The ventricular weight-to-body weight ratio, an index of cardiac hypertrophy, in JVS mice was 2.7-fold higher than that in controls (9.9 ± 0.1 vs. 3.7 ± 0.1 mg/g, P<0.01), and was significantly reduced by dietary FO (7.7 ± 0.1 mg/g in JVS mice treated with the FO diet, P<0.01 vs. JVS mice treated with the standard diet) (Fig. 1C). Therefore, JVS mice at 8 weeks of age exhibited marked cardiac hypertrophy, whereas treatment with dietary FO attenuated the cardiac hypertrophy.


Figure 1
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  		Fig. 1 Physical characteristics at 8 weeks of age. Ventricular hypertrophy in JVS mice was attenuated by dietary FO. Body weight (A), ventricular weight (B), and ventricular weight-to-body weight ratio (C). Values are mean ± S.E.M. n = 8 each group. *P<0.01 vs. controls treated with a standard diet; {dagger}P<0.01 vs. JVS mice treated with a standard diet.

 
3.2 Effects of dietary FO on morphology of the heart
Gross photographs of hematoxylin–eosin staining of the heart showed that JVS mice developed marked cardiac hypertrophy at 8 weeks of age, leading to ventricular dilatation at 10 weeks of age (Fig. 2). Dietary FO partially prevented cardiac hypertrophy and subsequent ventricular dilatation in JVS mice. Morphological analysis of the heart at 8 weeks of age exhibited significantly increased ventricular wall thickness and myocyte width in JVS mice, as compared with those of controls (Table 2). These increases were significantly reduced by dietary FO.


Figure 2
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  		Fig. 2 Morphology of the hearts. Dietary FO partially prevented cardiac hypertrophy and subsequent ventricular dilatation in JVS mice. Representative serial transverse sections at the level of papillary muscles (x 10). Hematoxylin–eosin stain. The bar indicates 400 µm.

 

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  		Table 2 Morphological analysis of the hearts

 
3.3 Effects of dietary FO on physiological parameters and survival rate
Serial echocardiography was performed at 6, 8, and 10 weeks of age (Fig. 3, A and B). JVS mice exhibited progressive dilatation of the left ventricle and a deterioration of fractional shortening throughout the study period. Dietary FO significantly reduced the dilatation of left ventricle and ameliorated the deterioration of fractional shortening in JVS mice. Dietary FO also improved the deterioration of systolic blood pressure in JVS mice (Fig. 3C). JVS mice showed lower heart rates than those of controls; however, dietary FO did not affect the heart rate in either group (Fig. 3D).


Figure 3
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  		Fig. 3 Serial echocardiography and physiological parameters. Dietary FO reduced the dilatation of left ventricle and ameliorated the deterioration of fractional shortening and systolic blood pressure in JVS mice. Left ventricular (LV) end diastolic dimension (A), fractional shortening (B), systolic blood pressure (C), and heart rate (D). Values are mean ± S.E.M. n = 6 each group. *P<0.01, {dagger}P<0.05 vs. controls treated with a standard diet at the same week of age; {ddagger}P<0.01, §P<0.05 vs. JVS mice treated with a standard diet at the same week of age.

 
Kaplan–Meier survival analysis revealed that dietary FO significantly improved the survival of JVS mice (mean survival period: 117 ± 3 days in JVS mice treated with the FO diet vs. 80 ± 3 days in JVS mice treated with the standard diet, P<0.0001) (Fig. 4).


Figure 4
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  		Fig. 4 Kaplan–Meier survival analysis of control and JVS mice treated with a fish oil diet. n = 20 each group. *P<0.0001 vs. controls treated with a standard diet; {dagger}P<0.0001 vs. JVS mice treated with a standard diet.

 
3.4 Plasma glucose and lipid levels
In JVS mice, plasma glucose levels were lower, whereas triglyceride, total cholesterol, and free fatty acid levels were higher than those of controls at 8 weeks of age (Table 3). Although dietary FO reduced the total cholesterol and the free fatty acid levels in controls, it had no effects on the increased lipid levels in JVS mice.


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  		Table 3 Plasma glucose and lipid levels and total carnitine levels in plasma and myocardium

 
3.5 Total carnitine levels in plasma and myocardium
The plasma and myocardial total carnitine levels in JVS mice were significantly lower than those of controls at 8 weeks of age (Table 3). Dietary FO reduced the plasma total carnitine levels in controls. However, dietary FO exerted no effects on the total carnitine levels in either the plasma or the myocardium in JVS mice. Therefore, the possibility of an FO-induced modulation of the total carnitine levels in JVS mice was excluded.

3.6 Myocardial lipid levels
In JVS mice, myocardial DAG and triglyceride levels were significantly higher than those of controls at 8 weeks of age (1.10 ± 0.02 vs. 0.48 ± 0.02 µg/mg dry weight, P<0.01; 33.2 ± 4.4 vs. 9.5 ± 0.8 µg/mg dry weight, P<0.01; respectively) (Fig. 5, A and B). Dietary FO markedly increased the triglyceride levels (69.0 ± 5.6 µg/mg dry weight, P<0.01 vs. JVS mice treated with the standard diet), but did not alter the levels of DAG (1.05 ± 0.09 µg/mg dry weight, P = NS) in JVS mice. No significant difference was found between controls and JVS mice as regards myocardial levels of ceramide, another lipid second messenger that, in contrast to DAG, exerts an inhibitory effect on PKC and is also a mediator of apoptosis [34] (Fig. 5C). Dietary FO did not affect the ceramide levels of either controls or JVS mice.


Figure 5
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  		Fig. 5 Myocardial lipid levels at 8 weeks of age. Dietary FO markedly increased the triglyceride levels but did not alter the levels of DAG in the hearts of JVS mice. 1,2-Diacylglycerol (A), triglycerides (B), and ceramides (C). Values are mean ± S.E.M. n = 7 each group. *P<0.01 vs. controls treated with a standard diet; {dagger}P<0.01 vs. JVS mice treated with a standard diet.

 
3.7 Fatty acid composition of myocardial DAG
The fatty acid composition of myocardial DAG differed significantly between controls and JVS mice at 8 weeks of age (Table 4). In JVS mice, the percentages of C16:1n-7, C18:1n-7,9, and C18:2n-6 fatty acids were significantly higher, whereas those of C14:0, C14:1n-5, C16:0, C18:0, C20:1n-9, and C22:0 fatty acids were significantly lower than those of controls. Dietary FO significantly altered the fatty acid composition of DAG in JVS mice: the percentages of C18:1n-7,9, C18:2n-6, and C20:4n-6 fatty acids were reduced, whereas those of C16:0, C16:1n-7, C20:5n-3 (EPA), C22:0, C24:0, C24:1n-9, and C22:6n-3 (DHA) fatty acids were increased.


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  		Table 4 Fatty acid composition of myocardial 1,2-diacylglycerol

 
3.8 Membrane translocation of cardiac PKC isozymes
Membrane translocation of PKC, defined as a redistribution of PKC from the cytosolic fraction to the membrane fraction, is known to be concomitant with increased PKC activity and is generally used as an index of PKC activation. Immunoblot analysis demonstrated that membrane-to-cytosol ratios of PKCs {alpha}, β2, {delta}, and {varepsilon} in JVS mice were significantly higher than those of controls at 8 weeks of age (Fig. 6, A through D). Dietary FO significantly inhibited the membrane translocation of PKCs {alpha}, β2, and {varepsilon}, but not {delta}, to control levels in JVS mice.


Figure 6
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  		Fig. 6 Representative immunoblots and membrane-to-cytosol ratios of cardiac protein kinase C (PKC) isozymes at 8 weeks of age. Dietary FO prevented membrane translocation of cardiac PKCs {alpha}, β2, and {varepsilon}, but not {delta}, in JVS mice. PKC{alpha} (A), PKCβ2 (B), PKC{delta} (C), and PKC{varepsilon} (D). Values are mean ± S.E.M. n = 5 each group. *P<0.01, {dagger}P<0.05 vs. controls treated with a standard diet; {ddagger}P<0.01, §P<0.05 vs. JVS mice treated with a standard diet.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
n-3 PUFA have been shown to alter the fatty acid composition of DAG and phospholipids in cardiomyocytes. Cultured neonatal rat cardiomyocytes in a DHA-supplemented medium exhibited fatty acid patterns of DAG and phospholipid different from controls [20]. It was also reported that cultured neonatal rat cardiomyocytes incubated in culture medium with EPA exhibited an altered fatty acid composition of phospholipids and a reduced rate of inositolphosphate production [21]. Furthermore, dietary FO significantly altered the fatty acid composition of phospholipids in the rat myocardium in vivo [22,23]. In the current study, we demonstrated for the first time that dietary FO modifies the fatty acid composition of myocardial DAG in vivo.

FO and n-3 PUFA have been shown to inhibit the activity of PKC in vitro [24–27]. Murray et al. reported that dietary FO inhibits colonic PKCβ2 activity in vivo and blocks PKCβ2-mediated hyperproliferation and carcinogenesis in the colonic epithelium of transgenic PKCβ2 mice [28]. It was also reported that dietary FO alters the T cell membrane microdomain lipid composition and suppresses the PKC{theta} signaling axis in mice [29]. To the best of our knowledge, the present study is the first to demonstrate the inhibition of PKC redistribution in the heart in vivo by dietary intervention with FO.

Previous studies have demonstrated the implication of the acyl chain of DAG in the activation of protein kinase C in vitro [16,17]. Eskildsen-Helmond et al. reported that particular molecular species of DAG, more than its total concentration, determine the activation of PKC isoenzymes in agonist-stimulated rat cardiomyocytes [18]. In the present study, dietary FO significantly altered the fatty acid composition of myocardial DAG in JVS mice. Although the myocardial triglyceride levels were markedly elevated by dietary FO, possibly due to the excess load of fatty acids in cardiomyocytes, the DAG levels remained unchanged. Therefore, the inhibitory effects of dietary FO on the membrane translocation of cardiac PKCs {alpha}, β, and {varepsilon} in JVS mice may be related to the alteration of the distinct fatty acid composition of DAG. In JVS mice, myocardial DAG with a distinct fatty acid composition may activate PKCs {alpha}, β, and {varepsilon}, and the modification of the fatty acid composition of DAG by FO may disrupt hypertrophic signaling via the inhibition of these PKC isoenzymes. In contrast to PKCs {alpha}, β, and {varepsilon}, dietary FO did not inhibit the membrane translocation of PKC{delta} in JVS mice. PKC{delta} may be activated by a mechanism independent of the molecular composition of DAG in JVS mice. The results of the present study also suggest that lipid-mediated signaling, rather than the intracellular accumulation of triglycerides, is implicated in the development of pathological cardiac hypertrophy in JVS mice. Indeed, Listenberger et al. proposed that cellular triglyceride accumulation itself is not initially toxic, and that the accumulation of excess fatty acids in triglyceride pools may instead serve as a buffer to protect against lipotoxicity [35]. We have previously confirmed that quinapril, an angiotensin converting enzyme inhibitor, had no effects on cardiac hypertrophy as well as cardiac function in JVS mice (unpublished data). Thus, the alteration of molecular species composition of DAG may be more essential than the activation of G protein-coupled receptors by endogenous regulators for the hypertrophic signaling in the lipotoxic hearts of JVS mice.

PKC is known to be one of the key molecules in the signaling pathway leading to cardiac hypertrophy [13–15]. However, large controversy exists in the literature with respect to isozyme-specific roles of PKC in the development of cardiac hypertrophy. A comparative analysis of PKC isozymes using adenovirus-mediated transfection of wild-type or dominant inhibitory forms of PKCs {alpha}, β2, {delta}, and {varepsilon} in neonatal rat cardiomyocytes suggested that only PKC{alpha} was sufficient to stimulate hypertrophy and only inhibition of PKC{alpha} inhibited agonist-mediated hypertrophy [36]. Similarly, adenovirus-mediated transfection of a dominant negative form of PKC{alpha} attenuated phorbol myristate actetate-induced hypertrophy in neonatal rat cardiomyocytes [37]. However, the results of in vivo studies showing gain-of-function by overexpression or translocation facilitation and loss-of-function by gene ablation or translocation inhibition agree that PKC{alpha} has minimal effects on cardiac hypertrophy but is a critical determinant of myocardial systolic function [38,39]. Cardiac-specific overexpression of PKCβ2 in mice led to pathological hypertrophy [40,41]. However, PKCβ-knockout mouse hearts were found to hypertrophy normally to phenylephrine or aortic banding, suggesting that PKCβ is not necessary for the hypertrophic response [42]. Both transgenic overexpression and translocation activation of PKC{varepsilon} in the hearts of mice resulted in mild concentric hypertrophy with normal cardiac performance, whereas the overexpression of a PKC{varepsilon} translocation inhibitor induced a dilated cardiomyopathy [43,44]. However, a recent study demonstrated that PKC{varepsilon}-knockout mice subjected to transverse aortic constriction showed preserved cardiac hypertrophy, suggesting that PKC{varepsilon} is not required for the development of pressure overload-induced hypertrophy [45]. Overexpression of neither constitutively active mutants of PKC{varepsilon} nor {delta} was sufficient to induce hypertrophy in neonatal rat cardiomyocytes, and only constitutively active mutants of PKC{delta} were sufficient to induce apoptosis [46]. However, cardiac-specific overexpression of a PKC{delta} translocation activator in mice induced non-pathological cardiac hypertrophy identical to the PKC{varepsilon} translocation activator [47]. Transgenic expression of a PKC{delta} translocation inhibitor in mice at high levels caused a myofibrillar cardiomyopathy characterized by the disruption of the cardiomyocyte cytoskeleton, whereas overexpression at low levels caused protection from postischemic myocardial damage [48]. In the present study, JVS mice showed increased membrane translocation of cardiac PKCs {alpha}, β2, {delta}, and {varepsilon}, and dietary FO inhibited the translocation of PKCs {alpha}, β2, and {varepsilon}, but not {delta}. Therefore, the attenuation of cardiac hypertrophy with an improvement of cardiac function in FO-treated JVS mice may have been due to the overall inhibitory effects of FO on the redistribution of PKCs {alpha}, β2, and {varepsilon}. However, direct modulation of PKC isozymes by pharmacological or transgenic approach is necessary to determine the specific roles of PKC isozymes in the development of cardiomyopathy in JVS mice.

To the best of our knowledge, this is the first report to demonstrate the inhibitory effects of FO on cardiac hypertrophy in vivo. The present study also demonstrated that modification of molecular species of DAG by FO modulates PKC redistribution in the heart in vivo. However, other mechanisms may also be involved in the attenuation of cardiac hypertrophy achieved with dietary FO in JVS mice. EPA and DHA have been shown to inhibit the activities of mitogen-activated protein kinase, cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II, and the transactivation of transcription factor activator protein 1 in vitro [24,25,49]. It was also reported that EPA and DHA inhibit cardiac sarcolemmal Na+/H+exchange [50]. Recently, Siddiqui et al. reported that DHA prevented phenylephrine-induced hypertrophy in neonatal cardiomyocytes; this outcome was possibly the result of an inhibition of the Ras–Raf-1–extracellularly regulated kinase 1 and 2–p90 ribosomal S6 kinase pathway [30]. Although we cannot exclude these effects of n-3 PUFA, our findings suggest that the disruption of lipid second messenger-mediated signaling by FO is the predominant mechanism in the attenuation of cardiac hypertrophy due to the perturbation of fatty acid β-oxidation in JVS mice. However, we consider that the effects of FO obtained in this study may only apply to cardiac hypertrophy due to SCD and not to other models of cardiac hypertrophy.

PUFAs including DHA and EPA have been shown as ligands to activate peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), a nuclear hormone receptor that regulates genes involved in fatty acid β-oxidation [51]. Although we have not examined the effects of FO on the PPAR{alpha} pathway and energy metabolism in the hearts in this study, FO may have exerted beneficial effects on cardiomyopathy in JVS mice by facilitating fatty acid β-oxidation and thereby improving energy metabolism through the activation of PPAR{alpha}. Indeed, we have confirmed that fenofibrate, a PPAR{alpha} agonist, in addition to conventional L-carnitine supplementation, ameliorated the cardiomyopathy with improvement in cardiac energy metabolism in JVS mice (unpublished data).

In conclusion, dietary FO attenuates cardiac hypertrophy with improvements in cardiac function and survival in JVS mice; these effects are at least in part the results of a modification of the molecular species composition of myocardial DAG and the consequent inhibition of PKC redistribution. FO consumption, in addition to conventional L-carnitine supplementation, may be recommended in treating SCD. The present study provides evidence that the molecular species composition of DAG plays a crucial role in the pathogenesis of lipotoxic cardiomyopathy due to perturbations of fatty acid β-oxidation.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors would like to thank Dr. Masahiko Nishimura of the Division for Research of Laboratory Animals, Center for Research of Laboratory Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine for caring for the animals.


   Notes
 
Time for primary review 15 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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