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Cardiovascular Research 2003 58(3):565-574; doi:10.1016/S0008-6363(03)00291-8
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Copyright © 2003, European Society of Cardiology

Impact of {alpha}-tocopherol on cardiac hypertrophy due to energy metabolism disorder: the involvement of 1,2-diacylglycerol

Ryotaro Takahashia, Kenji Okumuraa,*, Hideo Matsuia, Yoshihiro Saburia, Hiroki Kamiyaa, Kenichiro Matsubaraa, Toru Asaia, 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

kenji{at}med.nagoya-u.ac.jp

* Corresponding author. Tel.: +81-52-744-2168; fax: +81-52-744-2177.

Received 4 November 2002; accepted 26 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency, shows a disorder of fatty acid oxidation and develops cardiac hypertrophy associated with lipid accumulation. Recently, {alpha}-tocopherol was shown to decrease 1,2-diacylglycerol (DAG) levels. We investigated the involvement of DAG in cardiac hypertrophy due to energy metabolism disorder by evaluating the effects of {alpha}-tocopherol administration on the hearts of JVS mice. Methods: Both JVS and control mice were fed a high {alpha}-tocopherol diet or a standard diet from 4 to 8 weeks of age. Myocardial DAG levels and fatty acid composition were assessed at 8 weeks of age. Results: The ventricular to body weight ratio in the JVS mice was significantly higher than that in the control mice [11.2±0.1 (mean±S.E.M.) versus 3.8±0.1 mg/g, P<0.01], and was reduced by {alpha}-tocopherol treatment (9.7±0.2 mg/g, P<0.01 versus JVS mice). However, echocardiographic analysis showed the exaggeration of left ventricular dilatation in the {alpha}-tocopherol treated JVS mice (P<0.01 versus JVS mice). The myocardial thiobarbituric-acid-reactive substance level was not affected by {alpha}-tocopherol treatment. The myocardial DAG level was 2.5-fold higher in the JVS mice compared with that in the control mice (2004±136 versus 806±36 ng/mg dry weight, P<0.01) with a significant increase in 18:1 and 18:2 fatty acids. {alpha}-Tocopherol treatment reduced myocardial DAG levels in the JVS mice (1443±49 ng/mg dry weight, P<0.01 versus JVS mice) without any alteration of the fatty acid composition. Conclusions: {alpha}-Tocopherol treatment may partially reduce cardiac hypertrophy but it may also depress cardiac function in the JVS mice by decreasing the myocardial DAG level. An increase in DAG might be involved in the development of cardiac hypertrophy and in the maintenance of cardiac function in energy metabolism disorder of the heart.

KEYWORDS Cardiomyopathy; Hypertrophy; Lipid metabolism; Second messengers; Signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Fatty acids are the main source of energy production in the heart [1], and disorders of cardiac energy metabolism, which include defects in fatty acid β-oxidation, are the major causes of inherited cardiomyopathy [2]. Carnitine is essential for the transport of long-chain fatty acids into the mitochondrial matrix for β-oxidation, and plays an important role in cellular energy metabolism [3]. Primary systemic carnitine deficiency is a rare hereditary disease due to a renal carnitine transport defect, and is associated with hypertrophic cardiomyopathy [4]. However, the precise mechanism of the development of cardiac hypertrophy in primary systemic carnitine deficiency is poorly understood. Recently, the juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency, was established [5]. Systemic carnitine deficiency in the JVS mouse is caused by decreased renal reabsorption of carnitine due to mutations of the renal carnitine transporter gene [6]. As a result, carnitine levels in tissues including the myocardium are decreased and fatty acid β-oxidation is depressed [7]. The JVS mouse develops marked cardiac hypertrophy [8], characterized by enlarged myocytes and nuclei, ragged red fibers, an increased number of mitochondria and lipid droplets, and an absence of fibrosis [9].

Cardiac hypertrophy in the JVS mouse is associated with marked intracellular lipid accumulation in the myocardium [7]. Therefore, intracellular lipid moieties may be good candidates for mediating metabolic signals leading to cardiac hypertrophy in this model. A reduction in myocyte fatty acid catabolism within the mitochondrion could lead to an increase in upstream lipid intermediates capable of activating intracellular signaling pathways. 1,2-Diacylglycerol (DAG) is one such lipid second messenger and is an endogenous activator of protein kinase C (PKC) [10]. The DAG–PKC pathway has been implicated as one of the important intracellular signaling pathways mediating the hypertrophic response [11–13]. Thus, the assessment of DAG may be important to elucidate the pathogenesis of cardiac hypertrophy in the JVS mouse. We have reported that distinct species of myocardial DAG were increased in the JVS mice [14]. In the JVS mice, carnitine treatment prevented cardiac hypertrophy and reduced the increased level of myocardial DAG.

Recently, molecular functions of {alpha}-tocopherol, which are independent of its antioxidant or pro-oxidant properties, have been reported [15,16]. {alpha}-Tocopherol was shown to decrease the DAG level by the activation of DAG kinase both in vitro [17] and in vivo [18].

In the present study, we investigated the involvement of DAG in cardiac hypertrophy due to energy metabolism disorder by evaluating the effect of {alpha}-tocopherol administration on the hearts of JVS mice.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animal preparation
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 [5], and were kindly donated by the Institute for Experimental Animals, Kanazawa University School of Medicine, Kanazawa. 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 [8]. Wild-type animals of the C3H strain were used as controls. Both control and JVS mice were divided into three groups according to diet: (1) a standard diet containing 75 I.U. vitamin E/kg chow; (2) a high {alpha}-tocopherol diet containing 500 I.U. vitamin E/kg chow; (3) a high {alpha}-tocopherol diet containing 1000 I.U. vitamin E/kg chow. Each group was maintained on its respective diet for 4 weeks, from 4 to 8 weeks of age. The composition of the standard diet was based on an AIN-93G purified diet [19]. The ingredients of the high {alpha}-tocopherol diets, besides the vitamin E, were identical to the standard diet. Carnitine was not included in the ingredients of either diet. Both control and JVS mice were studied at 8 weeks of age. We have demonstrated that cardiac hypertrophy in the JVS mouse was established by 8 weeks of age and that the hypertrophy led to marked left ventricular dilatation with reduced wall thickness at 16 weeks of age [20]. At 8 weeks of age, left ventricular fractional shortening was already significantly decreased in the JVS mice compared with that of control mice. Therefore, the JVS mice at 8 weeks of age had established hypertrophy at the initial transitional stage from hypertrophy to heart failure.

2.2 Hemodynamic and echocardiographic measurement
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 the EUB 8000 CV (Hitachi, Tokyo, Japan) 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 level of the chordae tendineae were recorded, and left ventricular dimensions at end-diastole (EDD) and end-systole (ESD) were measured by means of the leading edge method. For each measurement, data from three cardiac cycles were averaged, and fractional shortening (FS) was calculated as FS (%)=(EDD–ESD)/EDDx100.

2.3 Blood and tissue sampling
Each animal was anesthetized with diethyl ether. Blood samples were obtained from the hearts, directly. The hearts were rapidly excised and washed thoroughly with cold saline. After the atria were removed, the ventricles were immediately frozen in liquid nitrogen and lyophilized.

2.4 Morphological study
Cardiac tissue was examined by means of light microscopy. Tissue was fixed in 10% formaldehyde in phosphate buffer, and paraffin sections at a thickness of 4 µm were examined after staining with hematoxylin–eosin methods. Ventricular wall thickness and myocyte width were measured with NIH IMAGE analysis software (US National Institutes of Health, USA). Myocyte width was measured from five randomly chosen cells in each of three randomly chosen fields of septal, posterior, and right ventricular wall from sections from four animals in each group.

2.5 Status of {alpha}-tocopherol in the heart
The {alpha}-tocopherol level in the heart was measured by means of high-performance liquid chromatography (HPLC) according to a previously described method [21], using a Jasco HPLC system (Jasco, Tokyo, Japan). Briefly, samples extracted in n-hexane were evaporated under N2 gas at 37°C. The residue was resuspended in 50 ml of n-hexane and then injected into the HPLC column (Unisil Q NH2, GL Science, Tokyo, Japan). The {alpha}-tocopherol peak was determined using an 821-FP fluorometer (excitation 298 nm, emission 325 nm, Jasco).

2.6 Analysis of thiobarbituric-acid-reactive substances
The amount of thiobarbituric-acid-reactive substances (TBARS) in the heart was determined with the use of a modified fluorescence method [22,23]. The sample was homogenized in 1.15% KCl containing 10 µM deferoxamine, 0.04% butylated hydroxytoluene, and 2% ethanol. The homogenates were incubated with TCA–TBA·HCl reagent [15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, 0.25 M hydrochloric acid], and 2% butylated hydroxytoluene at 95°C for 60 min in an oil bath. The sample was then centrifuged at 1000 g for 10 min and the absorbance of the supernatant was measured at 535 nm at 25°C. Commercially available malondialdehyde was used as a standard.

2.7 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, ceramide, and other lipid levels was performed by the TLC–FID method as previously described [24]. 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., Tokyo, Japan). The first development was carried out in a solvent system of chloroform–methanol–H2O (57:12:0.6, v/v) until the solvent front had migrated approximately 2.5 cm. The second development was carried out in 1,2-dichloroethane–chloroform–acetic acid (46:6:0.05, v/v) until the solvent had migrated approximately 9 cm. The third development consisted of a repetition of the second development. The fourth development was carried out in n-hexane–diethyl ether–acetic acid (98:1:1, v/v) until the solvent front had migrated approximately 11.5 cm. The Chromarods were then scanned in an Iatroscan MK-5 analyzer (Iatron Lab.). Each sample was analyzed with three Chromarods, and the results were averaged.

2.8 Measurement of the fatty acid composition of DAG
Following the quantitation of DAG and ceramides, the remaining lipids were separated using silica-gel plates (20x20 cm, Kieselgel 60 F254, Merck, Darmstadt, Germany). The area corresponding to DAG was identified by exposure of 1,2-diolein alone to iodine vapor, and the lipid spot was scraped into 2 ml chloroform–methanol (9:1, v/v). The extract was evaporated to dryness with a stream of N2 gas, and the fatty acyl moieties in this fraction were transmethylated with boron fluoride–methanol [25]. Methyl fatty acids were analyzed on a gas chromatograph (model GC 14-A, Shimadzu, Kyoto, Japan) equipped with a flame ionization detection system and an HR-SS-10 fused-silica capillary column (30 mx0.25 mm I.D., Shinwakakoh, Kyoto, Japan). Peaks were identified by comparison to standards (Nu-Chek-Prep, Elysian, MN, USA), and the peak areas were calculated.

2.9 Statistics
All results are expressed as mean±S.E.M. Between-group comparisons were assessed by one-way ANOVA with Scheffé's posthoc test. Statistical significance was defined as P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
The JVS mice at 8 weeks of age exhibited marked cardiac hypertrophy, whereas treatment with {alpha}-tocopherol for 4 weeks partially reduced this development of cardiac hypertrophy. The body weight of the JVS mice was significantly lower than that of the control mice. (16.3±0.3 g in the JVS mice versus 22.1±0.4 g in the control mice, P<0.01). However, {alpha}-tocopherol treatment did not affect body weight in either group. The ventricular weight of the JVS mice was significantly higher than that of the control mice (182.4±4.6 mg in the JVS mice versus 83.2±1.3 mg in the control mice, P<0.01), and was partially reduced by {alpha}-tocopherol treatment (157.8±2.7 mg in the JVS mice treated with a high {alpha}-tocopherol diet containing 1000 I.U. vitamin E/kg chow, P<0.01 versus JVS mice treated with a standard diet). Similarly, the ventricular weight to body weight ratio, an index of cardiac hypertrophy, of the JVS mice was significantly higher than that of the control mice (11.2±0.1 mg/g in the JVS mice versus 3.8±0.1 mg/g in the control mice, P<0.01), and was partially but significantly reduced by {alpha}-tocopherol treatment (9.7±0.2 mg/g in the JVS mice treated with a high {alpha}-tocopherol diet containing 1000 I.U. vitamin E/kg chow, P<0.01 versus JVS mice treated with a standard diet) (Fig. 1).


Figure 1
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  		Fig. 1 Characteristics of the juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol. Values are mean±S.E.M. of eight animals. *, P<0.01 versus control mice treated with a standard diet; {dagger}, P<0.01; {ddagger}, P<0.05 versus JVS mice treated with a standard diet.

 
3.1 Morphological analysis
Morphological analysis showed significantly increased ventricular wall thickness and myocyte width in the JVS mice compared with those in the control mice. These increases were also significantly reduced by {alpha}-tocopherol treatment (Table 1 and Fig. 2). Assessment of perivascular and interstitial fibrosis by Masson's trichrome stain showed that fibrosis was scarcely detectable in the JVS mice, regardless of {alpha}-tocopherol treatment (data not shown).


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  		Table 1 Morphological analysis of the hearts in control and juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol

 

Figure 2
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  		Fig. 2 Representative transverse sections through the hearts in control and juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol. Hematoxylin–eosin stain; original magnification x10. The bar indicates 400 µm.

 
3.2 Physical characteristics
In the JVS mice, systolic blood pressure, fractional shortening, and heart rate were significantly decreased, whereas left ventricular end-diastolic dimension was significantly increased, compared with those in the control mice. Treatment with {alpha}-tocopherol for 4 weeks reduced systolic blood pressure and fractional shortening, and exaggerated left ventricular dilatation significantly in the JVS mice (Table 2 and Fig. 3).


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  		Table 2 Physical characteristics in control and juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol

 

Figure 3
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  		Fig. 3 Representative M-mode echocardiographic tracings of the hearts of control and juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol.

 
3.3 Biochemical characteristics of blood
Plasma {alpha}-tocopherol levels in the JVS mice were significantly higher than those in the control mice. {alpha}-Tocopherol treatment significantly elevated plasma {alpha}-tocopherol levels in both control and JVS mice. Plasma total carnitine levels and glucose concentrations were significantly lower in the JVS mice than in the control mice. However, they were not affected by {alpha}-tocopherol treatment in either the control or the JVS mice. Plasma triglyceride, total cholesterol, and free fatty acid (FFA) levels were significantly higher in the JVS mice than in the control mice. After {alpha}-tocopherol treatment, plasma total cholesterol levels showed a modest but a significant decrease in both control and JVS mice. Plasma FFA levels also showed a modest but a significant decrease in the {alpha}-tocopherol treated JVS mice. In contrast, plasma triglyceride levels were not affected by {alpha}-tocopherol treatment in either the control or the JVS mice (Table 3).


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  		Table 3 Biochemical characteristics of blood in control and juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol

 
3.4 Myocardial {alpha}-tocopherol and TBARS levels
Myocardial {alpha}-tocopherol levels in the JVS mice were significantly higher than those in the control mice. After treatment with {alpha}-tocopherol for 4 weeks, myocardial {alpha}-tocopherol levels were significantly increased in both control and JVS mice.

The myocardial TBARS level, an index of oxidative stress, was significantly lower in the JVS mice than that in the control mice. However, the production of TBARS was not affected by {alpha}-tocopherol treatment in either the control or the JVS mice (Fig. 4).


Figure 4
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  		Fig. 4 Myocardial {alpha}-tocopherol and thiobarbituric acid reactive substance (TBARS) levels in the control and the juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol. Values are mean±S.E.M. of seven animals. *, P<0.01 versus control mice treated with a standard diet; {dagger}, P<0.01 versus JVS mice treated with a standard diet; #, P<0.01 versus JVS mice treated with a high {alpha}-tocopherol diet containing 500 I.U. vitamin E/kg chow.

 
3.5 Myocardial lipid contents
Fig. 5 shows the data regarding myocardial lipid contents. The myocardial DAG level was 2.5-fold higher in the JVS mice compared with that in the control mice (2004±136 ng/mg dry weight in the JVS mice versus 806±36 ng/mg dry weight in the control mice, P<0.01). {alpha}-Tocopherol treatment significantly reduced the myocardial DAG level in the JVS mice (1443±49 ng/mg dry weight in the JVS mice treated with a high {alpha}-tocopherol diet containing 1000 I.U. vitamin E/kg chow, P<0.01 versus JVS mice treated with a standard diet).


Figure 5
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  		Fig. 5 Myocardial 1,2-diacylglycerol, triglyceride, total cholesterol, and ceramide levels in the control and the juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol. Values are mean±S.E.M. of eight animals. *, P<0.01 versus control mice treated with a standard diet. {dagger}, P<0.01 versus JVS mice treated with a standard diet.

 
Myocardial triglyceride levels were significantly higher in the JVS mice than in the control mice. There was no difference in myocardial total cholesterol levels of the control and the JVS mice. No significant difference was found between the control and the JVS mice as regards myocardial levels of ceramide, another lipid second messenger which, in contrast to DAG, has an inhibitory effect on PKC [26]. {alpha}-Tocopherol treatment did not affect the myocardial triglyceride, total cholesterol, or ceramide levels in either the control or the JVS mice.

3.6 Fatty acid composition of myocardial DAG
The fatty acid composition of DAG differed significantly between the control mice and the JVS mice. The percentages of 18:1(n-9), 18:2(n-6) fatty acids were significantly higher, whereas those of 14:0, 16:0, 18:0, 20:0, 20:1(n-9), 20:3(n-6), 24:1(n-9) fatty acids were significantly lower in the JVS mice than those in the control mice. However, {alpha}-tocopherol treatment did not alter the fatty acid composition of DAG in either the control or the JVS mice (Fig. 6).


Figure 6
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  		Fig. 6 Fatty acid composition of myocardial DAG in the control and the juvenile visceral steatosis (JVS) mice treated with {alpha}-tocopherol. Values are mean±S.E.M. *, P<0.01; {dagger}, P<0.05 versus control mice treated with a standard diet.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we demonstrated the following findings in the JVS mice: (1) marked cardiac hypertrophy developed with an increase in the myocardial DAG level and elevation of 18:1 and 18:2 fatty acids, as reported previously [14]; (2) the myocardial {alpha}-tocopherol level was elevated, whereas the TBARS level was depressed, indicating increased antioxidant capacity; (3) {alpha}-tocopherol treatment partially reduced cardiac hypertrophy but exaggerated left ventricular dilatation and depressed cardiac function; (4) {alpha}-tocopherol treatment increased the myocardial {alpha}-tocopherol level without affecting the myocardial TBARS level; (5) {alpha}-tocopherol treatment reduced the myocardial DAG level without alteration of fatty acid composition. These results suggest that the effect of {alpha}-tocopherol treatment on cardiac hypertrophy in the JVS mice was related to the ability of {alpha}-tocopherol to reduce DAG levels, an effect which is independent of the antioxidant or pro-oxidant ability of {alpha}-tocopherol.

Recently, emerging evidence concerning the molecular function of {alpha}-tocopherol, independent of its antioxidant or pro-oxidant properties, has been reported [15,16]. {alpha}-Tocopherol was shown to decrease the DAG level by the activation of DAG kinase both in vitro and in vivo. DAG is rapidly converted to phosphatidic acid (PA) by the action of DAG kinase. Tran et al. reported that {alpha}-tocopherol suppressed the DAG level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity [17]. Koya et al. reported that prevention of glomerular dysfunction in diabetic rats could be achieved by {alpha}-tocopherol treatment through activation of DAG kinase and consequent inhibition of the DAG–PKC pathway [18]. Therefore, we considered that {alpha}-tocopherol might be a suitable candidate for the manipulation of myocardial DAG levels in the JVS mice.

Previously, we reported that cardiac hypertrophy in the JVS mice was accompanied with increased antioxidant capacity [27], as indicated in other animal models of cardiac hypertrophy due to pressure overload [28,29]. In the hearts of JVS mice at 4 weeks of age, superoxide dismutase activity was significantly higher and the TBARS level was significantly lower than those in the control mice. In the present study, in the JVS mice at 8 weeks of age, the myocardial {alpha}-tocopherol level was significantly higher and the myocardial TBARS level was significantly lower than that in the control mice. Since antioxidant activity was enhanced in the hypertrophied hearts of JVS mice, lipid peroxidation appeared to be fully reduced. Therefore, treatment with {alpha}-tocopherol in the JVS mice might not have affected the myocardial TBARS level.

It is well known that the DAG–PKC pathway is implicated as an important intracellular signaling pathway mediating the hypertrophic response [11–13]. In vitro, many experiments have demonstrated that the DAG level was raised by the stimulation of cultured cells. However, relatively few studies have reported the involvement of increased DAG levels in the hypertrophied hearts in vivo. We previously reported that the DAG level was increased in the hypertrophic hearts of Bio 14.6 hamsters [30]. We have also reported increased DAG production in the hypertrophied hearts of spontaneously hypertensive rats; it was found that enalapril treatment reduced the hypertrophy together with a decrease in the myocardial DAG level [31]. Milano et al. demonstrated that myocardial DAG in the hypertrophied hearts of transgenic mice with cardiac-specific expression of a constitutively active mutant {alpha}1B-adrenergic receptor was increased [32]. Moreover, Akhter et al. reported that myocardial DAG was increased in surgically induced pressure overload hypertrophied hearts of nontransgenic control mice, but not in those of transgenic mice with cardiac-specific inhibition of G{alpha}q-mediated signaling showing reduced left ventricular hypertrophy [33]. In the present study, we demonstrated that the JVS mice developed marked cardiac hypertrophy with an increase in the myocardial DAG level, and that {alpha}-tocopherol treatment reduced the hypertrophy in association with a reduction in the myocardial DAG level. Although physiological effects of {alpha}-tocopherol on the heart besides decreasing the DAG level cannot be denied, to our knowledge, this is the first report to demonstrate the involvement of myocardial DAG in cardiac hypertrophy by directly manipulating the myocardial DAG level in vivo. Dhalla et al. reported that although vitamin E therapy improved myocardial redox state and delayed the development of heart failure, it had no effect on cardiac hypertrophy in animal models of surgically induced pressure overload [34]. However, their report did not mention the effects of {alpha}-tocopherol on the DAG–PKC pathway. Taken together, we suggest that an increase in myocardial DAG may be involved in the pathology of cardiac hypertrophy due to energy metabolism disorder in the absence of pressure overload.

Treatment with {alpha}-tocopherol reduced, but did not completely prevent, the cardiac hypertrophy in the JVS mice. Several reasons for this can be considered. The dose of {alpha}-tocopherol administered in the present study did not completely reduce the increase in myocardial DAG in the JVS mice, indicating that the dose might not have been sufficient to prevent the development of cardiac hypertrophy. DAG is generated through several pathways, namely, de novo synthesis, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylcholine (PC), and other lipids such as phosphatidylethanolamine. Hydrolysis of PIP2 produces DAG by the action of phospholipase C (PLC) [12,35], whereas hydrolysis of PC by phospholipase D (PLD) is known to produce PA, which in turn is converted to DAG by the action of phosphatidate phosphohydrolase (PAP) [36–38]. Previous studies have demonstrated that in agonist-stimulated rat cardiomyocytes, DAG production derived from the PC–PLD pathway is slow in onset and sustained, in contrast to the rapid and more transient production of DAG from the PIP2–PLC pathway [39,40]. In a recent study, we suggested that DAG in the hypertrophied hearts of JVS mice may be derived from both the PC–PLD pathway followed by PAP activation and the PIP2–PLC pathway, judging from the fatty acid composition of DAG [14]. DAG generated from PIP2 hydrolysis is rapidly converted to PA by the action of DAG kinase, whereas DAG generated from PC hydrolysis is reported to be a poor substrate for DAG kinase [41]. Tran et al. reported that {alpha}-tocopherol caused an increased conversion of DAG to PA by activating DAG kinase activity without causing any change in the activities of PLD, PAP or PLC [17]. Thus, {alpha}-tocopherol treatment might have partially affected DAG accumulation in the hypertrophied hearts of JVS mice through the activation of DAG kinase. Moreover, other signaling pathways mediating cardiac hypertrophy may also have been involved in the JVS mice. In fact, we have recently reported that in the JVS mice, calcineurin activity was increased and treatment with calcineurin inhibitor FK506 partially attenuated the development of cardiac hypertrophy [42].

In the present study, although {alpha}-tocopherol treatment partially reduced cardiac hypertrophy, it exaggerated left ventricular dilatation and depressed cardiac function in the JVS mice. To our knowledge, no previous studies have reported adverse effects of {alpha}-tocopherol on cardiac function. Moreover, {alpha}-tocopherol is reported as not inductive of apoptosis [43]. Pi et al. demonstrated that DAG dose-dependently induced a strong positive inotropic effect in isolated adult rat ventricular myocytes using a light-sensitive caged DAG compound [44]. Several studies have shown that phorbor esters, which mimic the effects of DAG on PKC activation, induced a positive inotropic response in isolated hearts and myocytes [45–47]. Many neurohormones associated with DAG signaling initiate large positive inotropic responses. We have previously demonstrated that norepinephrine [48] and insulin [49] increased both the amount of DAG and cardiac contractility in isolated perfused rat hearts. Furthermore, we have reported that etomoxir, a carnitine palmitoyl transferase I inhibitor, ameliorated the myocardial dysfunction of diabetic rats and elevated the myocardial DAG level with a significant increase in 18:1 and 18:2 fatty acids [50]. Taken together, the deterioration of cardiac function by {alpha}-tocopherol treatment in the JVS mice could be attributed to the reduction in the myocardial DAG level. In the JVS mice, cardiac hypertrophy induced secondarily due to carnitine deficiency may be a compensatory response to maintain cardiac function. When cardiac hypertrophy is prevented by interventions such as a decrease in the DAG level, it is reasonable to expect that cardiac function might deteriorate. Although the precise mechanism of the deteriorated cardiac function induced by {alpha}-tocopherol in the JVS mice remains to be explored, an increase in DAG levels may be implicated in the maintenance of cardiac function in cases of cardiac hypertrophy due to energy metabolism disorder.

In conclusion, our results suggest that although {alpha}-tocopherol treatment partially reduces cardiac hypertrophy, it also depresses the cardiac function of JVS mice by decreasing the myocardial DAG level. An increase in DAG might be involved in the development of cardiac hypertrophy and in the maintenance of cardiac function in energy metabolism disorder of the heart.

Time for primary review 23 days.


   Acknowledgements
 
The authors would like to thank Dr. Masahiko Nishimura, Institute for Laboratory Animal Research, Nagoya University Graduate School of Medicine, for caring for the animals, and Mr. Toshiaki Suzuki for technical advice regarding the histological analysis. The JVS mice were a gift from the Institute for Experimental Animals, Kanazawa University School of Medicine, Kanazawa.


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

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