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Cardiovascular Research 2006 70(3):566-577; doi:10.1016/j.cardiores.2006.02.005
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Copyright © 2006, European Society of Cardiology

Combined therapy with PPAR{alpha} agonist and L-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency

Toru Asai, Kenji Okumura*, Ryotaro Takahashi, Hideo Matsui, Yasushi Numaguchi, Hisashi Murakami, Ryuichiro Murakami and Toyoaki Murohara

Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

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

Received 6 July 2005; revised 23 January 2006; accepted 3 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPAR{alpha} by fenofibrate, a PPAR{alpha} agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD.

Methods Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine+0.2% fenofibrate containing chow (CA+FE) from 4weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis.

Results At 8weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA+FE than by CA or FE alone. CA+FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n – 7,9) and 18:2(n – 6) fatty acids compared with that in WT mice. CA+FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C β2 in JVS mice. Furthermore, CA+FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400days after birth: 89 vs. 0%, P<0.0001).

Conclusions PPAR{alpha} activation, in addition to L-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.

KEYWORDS Cardiomyopathy; Hypertrophy; Lipid metabolism; Lipid signaling; Energy metabolism


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Fatty acids are the main source of energy production in the heart [1]. Disorders of cardiac energy metabolism, including defects in fatty acid oxidation (FAO), are an important cause of inherited cardiomyopathies [2,3]. Recent studies have also suggested that excess myocardial lipid accumulation due to an imbalance between fatty acid import and utilization, referred to as lipotoxicity, promotes inherited and acquired forms of cardiomyopathies [4].

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 lipid and energy metabolism [5]. Primary systemic carnitine deficiency (SCD) is a potentially lethal, inherited disorder characterized by progressive infantile-onset cardiomyopathy, weakness and recurrent hypoglycemic hypoketotic encephalopathy [6]. Impaired renal re-absorption of carnitine resulting from homogenous gene mutations in OCTN2, which is a sodium-dependent carnitine transporter, leads to a systemic carnitine defect and disorders of FAO in many tissues, including the heart [7]. SCD induces hypertrophic cardiomyopathy, which is characterized by cardiomyocyte hypertrophy associated with the deposition of intracellular lipid droplets, indicating lipotoxicity [8,9]. Although early diagnosis and treatment with high-dose L-carnitine supplementation is life-saving and reverses the pathology of SCD, not all cases are protected from cardiomyopathy [10,11].

The juvenile visceral steatosis (JVS) mouse was established as an excellent murine model of SCD [12]. Systemic carnitine deficiency in JVS mice is also caused by decreased renal re-absorption of carnitine due to mutations of the renal carnitine transporter gene, OCTN2, as has been suggested in human SCD as well [7]. JVS mice develop marked cardiac hypertrophy with lipid accumulation due to disorders of FAO [13,14]. A reduction in the capacity for myocyte FAO could lead to an increase in upstream lipid intermediates capable of activating cellular signaling pathways. 1,2-Diacylglycerol (DAG) is one such lipid second messenger that activates protein kinase C (PKC) [15]. It has been considered that the DAG-PKC pathway is one of the important pathways mediating cardiac hypertrophy [16–18]. We have reported that distinct species of DAG were increased in the hypertrophied heart of JVS mice [19,20].

Peroxisome proliferators-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and they have been shown to regulate the expression of a number of genes. PPAR{alpha}, one of the PPAR isoforms, is highly expressed in most tissues with an elevated capacity for FAO, including liver, heart, kidney and skeletal muscle. It is now known that PPAR{alpha} regulates fatty acid transport, esterification and oxidation via the transcriptional activation of genes encoding key enzymes at each step of cellular fatty acid utilization. Therefore, PPAR{alpha} is recognized as a key regulator of cardiac fatty acid metabolism [21,22]. The fibrate class of hypolipidemic drugs including fenofibrate is considered to act as specific activators of PPAR{alpha}[23]. Recently, Djouadi et al. reported that PPAR{alpha} activator corrects the impaired FAO capacity in cultured skin fibroblasts from patients with carnitine palmitoyl transferase 2 deficiency, one of the most common inborn errors of FAO [24].

In this study, we hypothesized that the activation of PPAR{alpha}, in addition to conventional L-carnitine supplementation, may exert beneficial effects on lipotoxic cardiomyopathy due to SCD. To test this hypothesis, we investigated the effects of combined therapy with fenofibrate, one of the synthetic PPAR{alpha} agonists and L-carnitine supplementation on JVS mice.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Animal preparation
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 Institute of Health. The JVS mice used were of the C3H strain [13,14] and were kindly donated by the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center, Kanazawa University Graduate 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 5days after birth. We treated the homozygous mutants with daily subcutaneous injections of L-carnitine (5µmol/mouse) from 11 to 21days after birth in order to prolong their lives according to a previously reported method [13]. Wild-type (WT) animals of the C3H strain were used as controls.

Both WT and JVS mice were divided into four groups according to diet: (1) a normal chow (control), (2) a 0.2% fenofibrate/kg containing chow (FE), (3) a 0.1% L-carnitine/kg containing chow (CA), which is identical to the dose of L-carnitine in clinical use in human, and (4) a 0.1% L-carnitine/kg+0.2% fenofibrate/kg containing chow (CA+FE). The estimated daily intake of L-carnitine and fenofibrate in both wild-type and JVS mice were approximately 2.6mg/day and 5.2mg/day, respectively. Each group was maintained on its respective diet from 4weeks of age, and was studied at 8weeks of age (hypertrophy stage) and 16weeks of age (dilated cardiomyopathy stage) [25]. Furthermore, long-term survival was assessed in each group. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis. The total number of animals used in this study was approximately 210.

2.2 Hemodynamic and echocardiographic measurement
On the day of sacrifice, the systolic blood pressure and heart rate were determined by a tail-cuff detection system, Softron BP-98A (Softron, Tokyo, Japan). Left ventricular (LV) function was evaluated by transthoracic echocardiography using the SONOS 7500 (Philips Medical Systems, Andover, MA, USA) with a 10-MHz imaging transducer [19].

2.3 Blood and tissue sampling
Each animal was anesthetized with diethyl ether. Blood samples were obtained from the heart 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.

2.4 Morphological study
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 staining with hematoxylin–eosin. The ventricular wall thickness and myocyte width were measured with NIH Image analysis software (National Institutes of Health, Bethesda, MD, USA). To detect neutral lipids, frozen sections were stained with oil red O.

2.5 Real-time RT-PCR analysis
Heart tissues were homogenized in liquid nitrogen and total RNA was extracted using the Qiagen RNeasy Mini kit according to the recommendation of the manufacturer protocol (Qiagen, Valencia, CA, USA). The first cDNA strand was synthesized using the SuperScriptTM First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The quantitative real-time PCR was performed using the LightCyclerTM System (Roche Diagnostics, Mannheim, Germany) and QuantiTectTM SYBR Green PCR kit (Qiagen) using 18S rRNA as an internal control. The sequences of the sense and antisense primers used for amplification were: r18S, 5'-GTAACCCGTTGAACCCCATT-3' and 5'-CCATCCAATCGGTAGTAGCG-3'; product size: 151bp, PPAR{alpha}, 5'-CAGCAACAACCCGCCTTTT-3' and 5'-CCTCTGCCTCTTTGTCTTCGA-3'; product size: 110bp, muscle carnitine palmitoly transferase 1 (mCPT-1), 5'-ATGGTCATCTTCTCCACCGGA-3' and 5'-ACGGACACAGATAGCCCAGATC-3'; product size: 126bp, medium chain acyl-CoA dehydrogenase (MCAD), 5'-GATGTGGCGGCCATTAAGA-3' and 5'-AGAACGCGCCAACAAGAAA-3'; product size: 120bp. PPAR{alpha}, mCPT-1 and MCAD mRNAs were normalized to the r18S and relatively quantified by standard curve analysis. The slope values of r18S, PPAR{alpha}, mCPT-1 and MCAD were – 3.619, – 3.764, – 3.836 and – 3.948, respectively.

2.6 Measurement of myocardial ATP content
Animals were anesthetized with diethyl ether and the ventricles were collected within 15s after incision and were snap-frozen with liquid nitrogen. The frozen tissue was homogenized in ice-cold 0.5mol/L perchloric acid for deproteinization, and then neutralized with 2mol/L K2CO3. The ATP levels in the neutralized extracts were determined spectrophotometrically by enzymatic assay as described previously in detail [14].

2.7 Myocardial lipid analysis
Simultaneous quantitation of triglycerides, DAG and ceramide in the myocardium was performed by the thin layer chromatography and flame ionization detection (TLC/FID) method [26], and the fatty acid composition of myocardial DAG was determined by gas chromatograph, as previously described [19,20].

2.8 Western blot analysis of PKC isoforms
Membranous and cytosolic fractions of protein extracts from the myocardium were prepared as described previously [27]. All samples (20µg) were subjected to immunoblot analysis with the use of the antibodies against PKCβ2 and PKC{varepsilon} (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as reported previously [27].

2.9 Statistics
All results are expressed as mean±S.E.M. Survival analysis was performed by the Kaplan–Meier method and between-group difference in survival was tested by the log-rank test. One-way ANOVA was used to test for mean differences in myocardial ATP content and Western blot analysis of PKC isoforms. Two-way ANOVA factoring by genotype and treatment was performed for all other data. When appropriate, Scheffé's post hoc test was followed. Because of skewed distributions, plasma and myocardial triglyceride levels were log transformed for analysis. Statistical significance was defined as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Survival
During a follow-up period of 400days, all WT mice survived. In contrast, JVS mice treated with a normal chow all died by 160days (mean survival period: 118±9 days) (Fig. 1). Whereas FE and CA treatment modestly but significantly prolonged the survival period (mean survival period: 166±15 and 211±10 days, respectively), eight of nine CA+FE-treated JVS mice survived during the follow-up period. Thus, CA+FE treatment dramatically reduced the high mortality of JVS mice.


Figure 1
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  		Fig. 1 Kaplan–Meier survival curves in JVS mice. JVS mice were randomized to normal chow (control), 0.2% fenofibrate containing chow (FE), 0.1% L-carnitine containing chow (CA) and 0.1% L-carnitine+0.2% fenofibrate containing chow (CA+FE). All WT mice survived during the follow-up period of 400days (data not shown). *P<0.05 vs. JVS-control, {dagger}P<0.0001 vs. all other groups.

 
3.2 Cardiac hypertrophy, hemodynamics and cardiac function
As shown in Fig. 2, at 8weeks of age, a significant reduction in body weight was evident only in FE-treated JVS mice (16.6±0.6g, P<0.05 vs. other groups). The ventricular weight in JVS mice was markedly greater than that in WT mice (133.7±2.3 vs. 76.9±0.5mg, P<0.05), was partially reduced by CA treatment (121.2±1.7mg, P<0.05 vs. JVS mice), and was even more reduced by CA+FE treatment (104.1±1.8mg, P<0.05 vs. CA-treated JVS mice). The ventricular weight-to-body weight ratio, an index of cardiac hypertrophy, was 1.9-fold greater in JVS mice than in WT mice (7.05±0.15 vs. 3.80±0.05mg/g, P<0.05), was partially reduced by FE (6.53±0.15mg/g, P<0.05 vs. JVS mice) and CA (6.01±0.08mg/g, P<0.05 vs. JVS mice), and was even more reduced by CA+FE treatment (5.16±0.08mg/g, P<0.05 vs. CA-treated JVS mice). Thus, CA+FE treatment markedly attenuated the cardiac hypertrophy in JVS mice.


Figure 2
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  		Fig. 2 Body weight and ventricular weight of WT and JVS mice at 8weeks of age. Values are mean±S.E.M. n=8 each group. Body weight (A), ventricular weight (B), ventricular weight-to-body weight ratio (C). *P<0.05 vs. all WT mice groups, {dagger}P<0.05 vs. JVS-control, {ddagger}P<0.05 vs. JVS-CA, §P<0.05 vs. all other groups.

 
Although there was no significant difference in heart rate among the eight groups, the systolic blood pressure in JVS mice, FE-treated JVS mice and CA-treated JVS mice was significantly lower than that in WT mice (P<0.01) (Table 1). Serial echocardiographic measurements were performed at 8 and 16 weeks of age in the eight groups. At 8weeks of age, echocardiography revealed that left ventricular fractional shortening (LVFS) was lower in JVS mice than in WT mice and was more improved by CA+FE than CA alone (Table 1). On the other hand, FE treatment had no beneficial effects on the LVFS in JVS mice at all. At 16weeks of age, JVS mice and FE-treated JVS mice exhibited marked LV dilatation and impaired LV contractility. The lowered LVFS in JVS mice was partially ameliorated by CA. CA+FE dramatically improved the deterioration of LVFS and LV dilatation in JVS mice to WT mice levels. Representative M-mode echocardiograms are displayed in Fig. 3A.


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  		Table 1 Hemodynamics and cardiac function in WT and JVS mice

 

Figure 3
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  		Fig. 3 (A) Representative M-mode echocardiograms obtained from WT and JVS mice. (B) Morphology of the hearts in WT and JVS mice. (a–e) Representative hematoxylin–eosin-stained myocardial sections (x 400) from WT and JVS mice. The bar indicates 10µm. (f–o) Representative hematoxylin–eosin-stained cross sections at the level of papillary muscles (x 10) from WT and JVS mice at 8 and 16weeks of age. The bar indicates 400µm.

 
As shown above, treatment in WT mice exerted no effects on physical parameters, survival period, and cardiac function. In JVS mice, although fenofibrate treatment without L-carnitine supplementation exhibited a modest inhibitory effect on cardiac hypertrophy and improvement in survival rate, it had no effects on cardiac function and was not so beneficial as L-carnitine supplementation alone. Therefore, we did not include all the treated-animal groups for further investigation.

3.3 Morphological analysis
As shown in Table 2 and Fig. 3B, at 8weeks of age, morphological analysis showed marked increases in ventricular wall thickness and myocyte width in JVS mice compared with those in WT mice, as reported previously [20]. These increases observed in JVS mice were significantly reduced by CA+FE treatment.


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  		Table 2 Morphological analysis of the hearts in WT and JVS mice at 8weeks of age

 
3.4 Biochemical characteristics of blood
As shown in Table 3, JVS mice revealed severe hypoglycemia and markedly increased levels of plasma TG and FFA compared to WT mice. Severe hypoglycemia in JVS mice was corrected by CA and CA+FE treatment. The increased plasma TG and FFA levels in JVS mice were partially reduced by CA treatment and were further reduced by FE and CA+FE treatment. The triglyceride levels were also reduced in WT mice by FE and CA+FE treatment.


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  		Table 3 Biochemical characteristics of blood in WT and JVS mice at 8weeks of age

 
3.5 Real-time RT-PCR
As shown in Fig. 4, the expression of PPAR{alpha}, mCPT-1 and MCAD mRNA in the ventricle of JVS mice was slightly increased without significance compared to WT mice. CA+FE treatment significantly increased the expression of PPAR{alpha} and MCAD mRNA in JVS mice, and the expression of mCPT-1 mRNA in JVS mice with CA+FE treatment was significantly higher than that in WT mice. In contrast, treatment did not exert any effects on the expression of these genes in WT mice.


Figure 4
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  		Fig. 4 Relative expression levels of PPAR{alpha} (A), mCPT-1 (B) and MCAD (C) mRNA in the ventricular tissue from WT and JVS mice at 8weeks of age. Relative mRNA abundance was normalized against r18S mRNA level in each sample. Results are presented as fold changes versus the values in WT-control mice. Values are mean±S.E.M. n=5 each group. *P<0.01, {dagger}P<0.05 vs. all WT mice groups, {ddagger}P<0.05 vs. JVS-control.

 
3.6 Myocardial ATP content
To assess the effects of treatment on energetic status, we determined the myocardial ATP content (Fig. 5). At 8weeks of age, the myocardial ATP content in JVS mice was markedly lower than that in WT mice (1.74±0.16 vs. 2.91±0.24mol/g, P<0.01). CA+FE treatment increased the myocardial ATP content to nearly WT levels in JVS mice (2.76±0.11mol/g in CA+FE-treated JVS mice, P<0.01 vs. JVS mice) [14].


Figure 5
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  		Fig. 5 Myocardial ATP levels in WT and JVS mice at 8 weeks of age. Values are mean±S.E.M. n=6 each group. *P<0.01 vs. WT mice, {dagger}P<0.01 vs. JVS-control, {ddagger}P<0.05 vs. JVS-CA.

 
3.7 Myocardial lipid contents
Coincident with cardiac dysfunction and hypertrophy, myocardial lipid analysis revealed striking myocardial accumulation of triglycerides in JVS mice compared with the levels observed in WT mice at 8weeks of age (Fig. 6). Myocardial lipid analysis showed that myocardial triglyceride levels in JVS mice were 15-fold higher than those of the WT mice (92.5±7.1 vs. 6.2±0.4µg/mg dry weight, P<0.01) (Fig. 6B). Thus, substantial cardiac steatosis was evident at 8weeks in JVS mice. The increased triglyceride levels in JVS mice were reduced in CA-treated JVS mice (48.3±2.6µg/mg dry weight, P<0.01 vs. JVS mice). CA+FE treatment dramatically reduced the accumulation of triglycerides in JVS mice (15.3±0.8µg/mg dry weight, P<0.01 vs. CA-treated JVS mice). In fact, neutral lipid accumulation within cardiomyocytes in JVS mice and CA-treated JVS mice, as determined by oil red O staining, was markedly greater than that in WT mice. This lipid accumulation was clearly reduced in CA+FE-treated JVS mice (Fig. 6A). On the other hand, the myocardial DAG levels in JVS mice was 2.7-fold higher than that in WT mice. However, the DAG levels were not significantly altered by CA or CA+FE treatment. In addition, there were no differences in the ceramide levels among the six groups.


Figure 6
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  		Fig. 6 (A) Representative oil red O-stained myocardial sections (x 400) from WT and JVS mice at 8 weeks of age. (B) Myocardial 1,2-diacylglycerol, triglyceride and ceramide levels in WT and JVS mice at 8 weeks of age. Values are mean±S.E.M. n=8 each group. *P<0.01 vs. WT-control, {dagger}P<0.01 vs. JVS-control, {ddagger}P<0.01 vs. JVS-CA.

 
3.8 Fatty acid composition of myocardial DAG
As shown in Fig. 7, the fatty acid composition of DAG in JVS mice was significantly different from that in WT mice at 8weeks of age. In JVS mice, the percentage of 16:0 fatty acid was lower, whereas percentages of 18:1(n – 7,9) and 18:2(n – 6) fatty acids were higher than in WT mice. Although CA treatment had no effect on these alterations in fatty acid species of DAG, CA+FE treatment significantly reduced the percentages of 18:1(n – 7,9) and 18:2(n – 6) fatty acids of DAG in JVS mice. As a result, CA+FE treatment significantly altered the fatty acid composition of myocardial DAG without changing the total levels.


Figure 7
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  		Fig. 7 Fatty acid composition of myocardial 1,2-diacylglycerol in WT and JVS mice at 8 weeks of age. Values are mean±S.E.M. n=8 each group. *P<0.01 vs. WT-control, {dagger}P<0.01, {ddagger}P<0.05 vs. JVS-control.

 
3.9 Western blot analysis of PKC isoforms
Fig. 8 shows representative immunoblots and calculated membrane-to-cytosol fraction ratios of cardiac PKCs β2 and {varepsilon} at 8weeks of age. There were no significant differences among groups in terms of the membrane-to-cytosol fraction ratio of PKC{varepsilon}. The membrane-to-cytosol ratios of PKCβ2 in both JVS and CA-treated JVS mice were higher than those in WT mice, and were significantly reduced by CA+FE treatment. Thus, the activation of PKCβ2 shown by membrane translocation in JVS mice was significantly inhibited by CA+FE treatment.


Figure 8
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  		Fig. 8 Representative immunoblots of cardiac PKCβ2 (A) and PKC{varepsilon} (B) in WT and JVS mice at 8 weeks of age. The calculated membrane-to-cytosol fraction ratio of each isoform is also shown. Values are mean±S.E.M. n=6 each group. *P<0.01 vs. WT mice, {dagger}P<0.01 vs. JVS-control, {ddagger}P<0.01 vs. JVS-CA.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
To our knowledge, this is the first study to demonstrate the efficacy of PPAR{alpha} agonist administration for cardiomyopathy due to a FAO disorder in vivo. The JVS mouse used in the present study serves as an excellent clinical, biochemical and molecular model for human SCD. The results of the present study suggest that fenofibrate administration, when only provided in addition to standard L-carnitine supplementation, may further attenuate cardiac hypertrophy, improve cardiac dysfunction and prolong survival in SCD. Furthermore, fenofibrate is one of the fibrate groups of hypolipidemic drugs that have been proven efficacy and long-term safety for use in the treatment of hypertriglyceridemia in humans. We propose here that combined therapy with a PPAR{alpha} agonist and L-carnitine supplementation could be a potent option in the treatment of human SCD.

Several lines of circumstantial evidence have suggested that SCD may be one of the causes of sudden infant death syndrome [28,29]. If diagnosed early and treated promptly with high-dose oral L-carnitine supplementation (100mg/kg/day divided into four daily doses), SCD is treatable with improvement of pathologic phenotype including cardiomyopathy [10,11,30]. Nevertheless, in the clinical setting, several problems remain to be resolved. Sufficient amelioration of the clinical manifestations requires the stable maintenance of serum carnitine concentrations, which depends on good compliance with the supplementation of oral L-carnitine. Since the gastrointestinal tolerance of high-dose L-carnitine is poor, the absolute bioavailability after an oral dose of 1–6 g L-carnitine is 5–18% [31]. Thus, patients must take a large daily dose of oral L-carnitine [32]. It has also been reported that the response to L-carnitine supplementation varies between individuals and not all cases are protected from cardiomyopathy [10,11]. The results of the present study indicate that combined therapy with a PPAR{alpha} agonist and L-carnitine supplementation may solve these problems.

As shown in Fig. 4, combined treatment with fenofibrate and L-carnitine significantly increased the myocardial expression of PPAR{alpha}, mCPT-1 and MCAD mRNA in JVS mice but did not exert any effects on that in WT mice. Therefore, the effects of combined therapy on gene expression of PPAR{alpha}, mCPT-1 and MCAD may be exerted only in energetically compromised hearts of JVS mice. The upregulation of PPAR{alpha} and its regulated genes by combined therapy may have resulted in an enhancement of the FAO capacity and subsequent improvement in the energetic status of the hearts in JVS mice, as evidenced by the increase in myocardial ATP content (Fig. 5). We and other groups have reported that the pharmacological inhibition of FAO leads to cardiac hypertrophy [19,33], suggesting that an imbalance in energy substrate utilization is associated with the mechanism of cardiomyopathy [34]. Due to a defect in FAO, cardiomyocytes of JVS mice strongly depend on glucose as an energy substrate. We suggest that the enhancement of FAO capacity and correction of imbalance in energy substrates by combined therapy contributed to the beneficial effects observed in the hearts of JVS mice. Accumulation of excess lipids in non-adipose tissues leads to cell dysfunction or cell death. This phenomenon, known as lipotoxicity, may play an important role in the pathogenesis of diabetes and heart failure in humans and animal models [4]. Several recent studies reported the detrimental effects of cardiac lipid accumulation in transgenic mice [35,36]. In animal models of obesity [37] and in diabetes [38], increased triglyceride accumulation in cardiomyocytes was observed and this accumulation has been proposed to contribute to heart failure. Thus, it has been suggested that a mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of lipid intermediates, which may exert cardiotoxic effects. In the present study, JVS mice exhibited marked myocardial triglyceride accumulation and LV dysfunction. Combined treatment with fenofibrate and L-carnitine may have not only accelerated the utilization of intracellular fatty acids by improving FAO capacity, but may also have inhibited the influx of long-chain fatty acids into cardiomyocytes by decreasing serum triglyceride and FFA levels. As a result, the accumulation of triglycerides in the myocardium was dramatically decreased in this model. Our findings therefore indicate that the reduction of lipid accumulation may lead to the dramatic improvement in LV function and to the attenuation of cardiac hypertrophy in the lipotoxic heart of JVS mice.

Intracellular lipid moieties are excellent candidates for mediating metabolic signals leading to myocyte growth. We and other groups have demonstrated that DAG, one of the lipid second messengers that activate PKC, was increased in the hypertrophied heart [19,20,39–41]. It has also been reported that the particular molecular species of DAG, rather than the total level, is related to the activation of PKC [42,43]. Recently, we have reported that dietary treatment with n – 3 polyunsaturated fatty acids attenuated cardiac hypertrophy, altered the distinct fatty acid composition of myocardial DAG and inhibited the membrane translocation of PKC isoenzymes in JVS mice [27]. These results suggest that the distinct molecular species composition of DAG contribute to the PKC activation in JVS mice. In the present study, combined treatment with L-carnitine and fenofibrate did not reduce the increased levels of myocardial DAG in JVS mice, but significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of PKCβ2. The alteration of the fatty acid composition of myocardial DAG in JVS mice treated with combined therapy may be due to the modification of plasma fatty acid composition. It has been reported that the expression of PKCβ was increased in failed human heart [44] and that cardiac-specific overexpression of the PKCβ2 isoform caused pathological cardiac hypertrophy [45,46]. We therefore suggest that the alteration of the molecular species composition of DAG and consequent inhibition of PKCβ2 redistribution, at least in part, lead to the attenuation of pathological cardiac hypertrophy in JVS mice.

The role of PPAR{alpha} in the pathogenesis of various heart diseases has not been well defined. In PPAR{alpha}-null mice, altered expression of PPAR{alpha}-regulated FAO enzymes led to age-dependent cardiac damage [47]. Moreover, metabolic stress caused by inhibition of cellular fatty acid flux resulted in massive cardiac and hepatic lipid accumulation and death [48]. On the other hand, mice with cardiac overexpression of PPAR{alpha} exhibited signs of diabetic cardiomyopathy including ventricular hypertrophy and dysfunction [36]. In a murine model of pressure overload-induced cardiac hypertrophy, reactivation of PPAR{alpha} with an agonist resulted in severe depression of cardiac power and efficiency [49]. It was also reported that fenofibrate worsened cardiac function in transgenic mice that overexpressed human lipoprotein lipase and developed lipotoxic cardiomyopathy [50]. However, fenofibrate inhibited myocardial inflammation and fibrosis in angiotensin II-infused rats [51] and inhibited left ventricular hypertrophy in aortic banded rats [52]. In mice, activation of PPAR{alpha} by an agonist exhibited protective effects on the heart from acute ischemia/reperfusion injury [53] but worsened cardiac contractile function and induced microinfarctions in a model of ischemic cardiomyopathy induced by repetitive ischemia/reperfusion [54]. The controversy surrounding the role of PPAR{alpha} in the heart suggests that the function of this transcription factor differs in various pathological states and that the activation of PPAR{alpha} is not always beneficial. In JVS mice, although fenofibrate treatment without L-carnitine supplementation exhibited a modest inhibitory effect on cardiac hypertrophy and improvement in survival rate, it had no effects on cardiac function and was not so beneficial as L-carnitine supplementation alone. Therefore, we did not include the experimental group of JVS mice treated with fenofibrate alone for further investigation. The present study suggests that the beneficial effects of PPAR{alpha} activation can be obtained only when combined with L-carnitine supplementation in lipotoxic cardiomyopathy due to SCD.

Previous studies have assessed the effects of PPAR{alpha} activation on the heart in normal animals. Although WY-14,643, a PPAR{alpha} agonist, increased cardiac mRNA levels of PPAR{alpha}-regulated enzymes such as MCAD and pyruvate dehydrogenase kinase 4 (PDK4) in normal rats, there were no changes in oxygen consumption, fatty acid oxidation, glucose oxidation, cardiac power and cardiac efficiency [49]. Fenofibrate exerted no effects on heart weight-to-body weight ratio or cardiac function assessed by echocardiography in normal rats [51]. In the present study, although fenofibrate treatment reduced the plasma triglyceride levels in WT mice, it had no effects on physical parameters, survival period and cardiac function. Therefore, we did not include the experimental group of WT mice treated with fenofibrate treatment alone for further investigation. Taken together, the net effects of PPAR{alpha} activation do not seem to affect the cardiac phenotype in normal animals.

In the present study, JVS mice developed marked cardiac hypertrophy accompanied with impaired contractility at 8 weeks of age. LV dilatation was not apparent in 8-week-old JVS mice, but progressive LV dilatation and dysfunction, so called LV remodeling, developed at 16 weeks of age. We consider the pathological cardiac hypertrophy as a maladaptive process leading to LV remodeling in JVS mice. Indeed, we confirmed that high-dose L-carnitine supplementation reduced the myocardial DAG with partial correction of its fatty acid composition and prevented cardiac hypertrophy at 8 weeks of age [19] and subsequent LV dilatation in JVS mice (unpublished data). We have also reported that modification of fatty acid composition of myocardial DAG by n – 3 polyunsaturated fatty acids attenuated cardiac hypertrophy at 8 weeks of age and subsequent cardiac dysfunction in JVS mice [27]. In the present study, combined therapy with fenofibrate and L-carnitine attenuated the pathological cardiac hypertrophy at 8weeks of age and completely prevented the LV remodeling at 16weeks of age. Therefore, we consider that analysis at 8weeks of age provides key findings for elucidating the pathophysiology of cardiomyopathy in JVS mice and that intervention to inhibit the pathological hypertrophy at 8weeks of age leads to the prevention of detrimental LV remodeling.

In conclusion, PPAR{alpha} activation, in addition to L-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism. Although cardiomyopathies due to FAO disorders such as SCD are minor contributors to the broad spectrum of heart diseases, it still remains a clinical issue requiring improved understanding and treatment. The present study not only provides powerful therapeutic options for the clinical setting, but also suggests a key to the elucidation of the mechanisms leading to the development of cardiomyopathies due to FAO disorders.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors would like to thank Dr. Masahiko Nishimura, 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 34 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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