Download Combined therapy with PPARα agonist and l

Cardiovascular Research 70 (2006) 566 – 577
www.elsevier.com/locate/cardiores
Combined therapy with PPARa 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, Toyoaki Murohara
Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Received 6 July 2005; received in revised form 23 January 2006; accepted 3 February 2006
Available online 28 February 2006
Time for primary review 34 days
Abstract
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 PPARa by fenofibrate, a
PPARa 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 4 weeks 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 8 weeks 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 h2 in JVS mice. Furthermore, CA + FE prevented the
progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%,
P < 0.0001).
Conclusions: PPARa 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.
D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Cardiomyopathy; Hypertrophy; Lipid metabolism; Lipid signaling; Energy metabolism
1. Introduction
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
* Corresponding author. Tel.: +81 52 744 2168; fax: +81 52 744 2177.
E-mail address: [email protected] (K. Okumura).
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 h-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 reabsorption of carnitine resulting from homogenous gene
mutations in OCTN2, which is a sodium-dependent carnitine
transporter, leads to a systemic carnitine defect and disorders
0008-6363/$ - see front matter D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2006.02.005
T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
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 lcarnitine 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. 1,2Diacylglycerol (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.
PPARa, 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
PPARa 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, PPARa 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 PPARa [23]. Recently, Djouadi et al.
reported that PPARa 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 PPARa,
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 PPARa
agonists and l-carnitine supplementation on JVS mice.
567
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 5 days after birth. We
treated the homozygous mutants with daily subcutaneous
injections of l-carnitine (5 Amol/mouse) from 11 to 21 days
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 lcarnitine 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 2.6 mg/day and
5.2 mg/day, respectively. Each group was maintained on its
respective diet from 4 weeks of age, and was studied at
8 weeks of age (hypertrophy stage) and 16 weeks 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
2. Materials and methods
2.1. Animal preparation
All protocols described were approved by the Animal
Ethics Committee of Nagoya University, Nagoya, Japan. The
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 Am were examined after staining with hematoxylin –
eosin. The ventricular wall thickness and myocyte width
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T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
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.
[27]. All samples (20 Ag) were subjected to immunoblot
analysis with the use of the antibodies against PKCh2 and
PKC( (Santa Cruz Biotechnology, Santa Cruz, CA, USA),
as reported previously [27].
2.5. Real-time RT-PCR analysis
2.9. Statistics
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 SuperScripti First-Strand
Synthesis System (Invitrogen, Carlsbad, CA, USA). The
quantitative real-time PCR was performed using the LightCycleri System (Roche Diagnostics, Mannheim, Germany)
and QuantiTecti 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, 5VGTAACCCGTTGAACCCCATT-3V and 5V-CCATCCAATCGGTAGTAGCG-3V; product size: 151 bp, PPARa,
5V-CAGCAACAACCCGCCTTTT-3V and 5V-CCTCTGCCTCTTTGTCTTCGA-3V; product size: 110 bp, muscle
carnitine palmitoly transferase 1 (mCPT-1), 5V-ATGGTCATCTTCTCCACCGGA-3V and 5V-ACGGACACAGATAGCCCAGATC-3V; product size: 126 bp, medium chain acyl-CoA
dehydrogenase (MCAD), 5V-GATGTGGCGGCCATTAAGA-3V and 5V-AGAACGCGCCAACAAGAAA-3V; product
size: 120 bp. PPARa, mCPT-1 and MCAD mRNAs were
normalized to the r18S and relatively quantified by standard
curve analysis. The slope values of r18S, PPARa, 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 15 s after incision and were
snap-frozen with liquid nitrogen. The frozen tissue was
homogenized in ice-cold 0.5 mol/L perchloric acid for
deproteinization, and then neutralized with 2 mol/L K2CO3.
The ATP levels in the neutralized extracts were determined
spectrophotometrically by enzymatic assay as described
previously in detail [14].
All results are expressed as mean T S.E.M. Survival
analysis was performed by the Kaplan– Meier method and
between-group difference in survival was tested by the logrank 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
3.1. Survival
During a follow-up period of 400 days, all WT mice
survived. In contrast, JVS mice treated with a normal chow
all died by 160 days (mean survival period: 118 T 9 days)
(Fig. 1). Whereas FE and CA treatment modestly but
significantly prolonged the survival period (mean survival
period: 166 T 15 and 211 T10 days, respectively), eight of
nine CA + FE-treated JVS mice survived during the followup period. Thus, CA + FE treatment dramatically reduced
the high mortality of JVS mice.
3.2. Cardiac hypertrophy, hemodynamics and cardiac function
As shown in Fig. 2, at 8 weeks of age, a significant
reduction in body weight was evident only in FE-treated
JVS mice (16.6 T 0.6 g, P < 0.05 vs. other groups). The
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
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,
.
P < 0.0001 vs. all other groups.
T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
569
Fig. 2. Body weight and ventricular weight of WT and JVS mice at 8weeks of age. Values are mean T 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, .P < 0.05 vs. JVS-control, -P < 0.05 vs. JVS-CA, ‘P < 0.05 vs. all
other groups.
ventricular weight in JVS mice was markedly greater than
that in WT mice (133.7 T 2.3 vs. 76.9 T 0.5 mg, P < 0.05),
was partially reduced by CA treatment (121.2 T 1.7 mg,
P < 0.05 vs. JVS mice), and was even more reduced by
CA + FE treatment (104.1 T1.8 mg, 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 T 0.15 vs. 3.80 T 0.05 mg/g,
P < 0.05), was partially reduced by FE (6.53 T 0.15 mg/g,
P < 0.05 vs. JVS mice) and CA (6.01 T 0.08 mg/g, P < 0.05
vs. JVS mice), and was even more reduced by CA + FE
treatment (5.16 T 0.08 mg/g, P < 0.05 vs. CA-treated JVS
mice). Thus, CA + FE treatment markedly attenuated the
cardiac hypertrophy in JVS mice.
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
8 weeks 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 16 weeks 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
Table 1
Hemodynamics and cardiac function in WT and JVS mice
WT mice
JVS mice
Control
FE
CA
CA + FE
Control
FE
CA
CA + FE
8 weeks
SBP (mm Hg)
HR (bpm)
FS (%)
LVEDD (mm)
110 T 3
633 T 24
54.5 T 1.8
2.46 T 0.08
106 T 2
621 T 41
52.6 T 0.7
2.35 T 0.03
108 T 3
538 T 28
51.2 T 1.1
2.29 T 0.04
102 T 4
604 T 25
53.1 T 0.9
2.24 T 0.03
88 T 4*
502 T 35
39.2 T 0.8*
2.56 T 0.23
88 T 2*
473 T 49
40.7 T 0.6*
2.62 T 0.07
93 T 2*
553 T 34
46.0 T 0.3*,.
2.58 T 0.04
98 T 2
636 T 36
50.4 T 0.8.,2.36 T 0.05
16 weeks
SBP (mm Hg)
HR (bpm)
FS (%)
LVEDD (mm)
107 T 2
647 T 25
53.4 T 1.0
2.56 T 0.11
103 T 4
633 T 35
52.0 T 0.9
2.53 T 0.09
108 T 3
546 T 23
52.8 T 0.7
2.61 T 0.04
105 T 4
624 T 23
50.9 T 0.6
2.56 T 0.08
85 T 3*
520 T 37
23.5 T 2.9*
4.14 T 0.26*
89 T 3*
529 T 64
23.6 T 1.6*
3.77 T 0.19*
92 T 2*
543 T 37
38.3 T 0.7*,.
2.89 T 0.10.
98 T 2
637 T 36
48.4 T 0.8.,2.55 T 0.06.
SBP, systolic blood pressure; HR, heart rate; FS, fractional shortening; LVEDD, left ventricular end-diastolic dimension.
Values are mean T S.E.M. n = 8 each group.
* P < 0.05 vs. WT-control.
.
P < 0.05 vs. JVS-control.
P < 0.05 vs. JVS-CA.
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T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
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 (400) from WT and JVS mice. The bar indicates 10Am. (f – o) Representative hematoxylin –
eosin-stained cross sections at the level of papillary muscles (10) from WT and JVS mice at 8 and 16 weeks of age. The bar indicates 400 Am.
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.
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 8 weeks 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
Table 2
Morphological analysis of the hearts in WT and JVS mice at 8weeks of age
WT mice
Myocyte width
Septum
Posterior
RV
JVS mice
Control
FE
CA
CA + FE
Control
FE
CA
CA + FE
(lm)
6.40 T 0.30
6.12 T 0.15
5.93 T 0.07
6.60 T 0.19
6.35 T 0.41
6.03 T 0.19
6.72 T 0.13
6.51 T 0.15
6.31 T 0.24
6.65 T 0.15
6.64 T 0.20
6.30 T 0.22
9.71 T 0.35*
9.95 T 0.22*
9.28 T 0.62*
9.23 T 0.30*
9.79 T 0.28*
9.22 T 0.27*
9.27 T 0.29*
8.70 T 0.35*
8.54 T 0.33*
6.92 T 0.23.,7.63 T 0.26.
6.91 T 0.20.
207 T 3
202 T 3
123 T 9
203 T 9
224 T 8
123 T 6
192 T 7
220 T 12
123 T 4
304 T 7*
350 T 18*
187 T 3*
302 T 10*
309 T 7*
186 T 7*
292 T 9*
310 T 8*
169 T 6*
232 T 11.,262 T 15.
144 T 3.
Wall thickness (lm)
Septum
196 T 9
Posterior
225 T 2
RV
120 T 6
RV, right ventricle.
Values are mean T S.E.M. n = 4 each group.
* P < 0.01 vs. all WT mice groups.
.
P < 0.05 vs. JVS-control.
P < 0.01 vs. JVS-CA.
T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
571
Table 3
Biochemical characteristics of blood in WT and JVS mice at 8weeks of age
WT mice
PG (mg/dL)
TG (mg/dL)
FFA (mEq/L)
JVS mice
Control
FE
CA
CA + FE
Control
FE
CA
CA + FE
160.8 T 10.0
46.8 T 2.8
0.81 T 0.05
113.9 T 5.2
24.3 T 2.9*
0.78 T 0.05
153.1 T 21.5
44.8 T 3.0
0.80 T 0.09
121.6 T 8.5
29.1 T 4.4*
0.75 T 0.05
38.4 T 2.3*
437.6 T 55.8*
3.11 T 0.13*
60.9 T 2.8*
15.5 T 1.0*,.,1.05 T 0.06.,-
116.4 T 7.8.
40.9 T 4.5.
1.44 T 0.04*,.
108.8 T 10.7.
23.0 T 2.3*,.,0.95 T 0.04.,-
PG, plasma glucose; TG, triglycerides; FFA, free fatty acids.
Values are mean T S.E.M. n = 8 each group.
* P < 0.05 vs. WT-control.
.
P < 0.05 vs. JVS-control.
P < 0.05 vs. JVS-CA.
[20]. These increases observed in JVS mice were significantly reduced by CA + FE treatment.
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.
3.5. Real-time RT-PCR
As shown in Fig. 4, the expression of PPARa, 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 PPARa 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.
3.6. Myocardial ATP content
To assess the effects of treatment on energetic status, we
determined the myocardial ATP content (Fig. 5). At 8 weeks
of age, the myocardial ATP content in JVS mice was
markedly lower than that in WT mice (1.74 T 0.16 vs.
2.91 T 0.24 mol/g, P < 0.01). CA + FE treatment increased
the myocardial ATP content to nearly WT levels in JVS
mice (2.76 T 0.11 mol/g in CA + FE-treated JVS mice,
P < 0.01 vs. JVS mice) [14].
Fig. 4. Relative expression levels of PPARa (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 T S.E.M. n = 5 each group. *P < 0.01, .P < 0.05 vs. all WT mice groups, -P < 0.05 vs. JVS-control.
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T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
3.7. Myocardial lipid contents
Fig. 5. Myocardial ATP levels in WT and JVS mice at 8 weeks of age.
Values are mean T S.E.M. n = 6 each group. *P < 0.01 vs. WT mice,
.
P < 0.01 vs. JVS-control, -P < 0.05 vs. JVS-CA.
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 8 weeks 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 T 7.1 vs. 6.2 T 0.4 Ag/mg dry weight,
P < 0.01) (Fig. 6B). Thus, substantial cardiac steatosis was
evident at 8 weeks in JVS mice. The increased triglyceride
levels in JVS mice were reduced in CA-treated JVS mice
(48.3 T 2.6 Ag/mg dry weight, P < 0.01 vs. JVS mice).
CA + FE treatment dramatically reduced the accumulation
of triglycerides in JVS mice (15.3 T 0.8 Ag/mg dry weight,
P < 0.01 vs. CA-treated JVS mice). In fact, neutral lipid
Fig. 6. (A) Representative oil red O-stained myocardial sections (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 T S.E.M. n = 8 each group. *P < 0.01 vs. WT-control, .P < 0.01 vs.
JVS-control, -P < 0.01 vs. JVS-CA.
T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
573
Fig. 7. Fatty acid composition of myocardial 1,2-diacylglycerol in WT and JVS mice at 8 weeks of age. Values are mean T S.E.M. n = 8 each group. *P < 0.01 vs.
WT-control, .P < 0.01, -P < 0.05 vs. JVS-control.
accumulation within cardiomyocytes in JVS mice and CAtreated 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.
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.
3.8. Fatty acid composition of myocardial DAG
Fig. 8 shows representative immunoblots and calculated
membrane-to-cytosol fraction ratios of cardiac PKCs h2 and
( at 8 weeks of age. There were no significant differences
among groups in terms of the membrane-to-cytosol fraction
ratio of PKC(. The membrane-to-cytosol ratios of PKCh2 in
both JVS and CA-treated JVS mice were higher than those
in WT mice, and were significantly reduced by CA + FE
As shown in Fig. 7, the fatty acid composition of DAG in
JVS mice was significantly different from that in WT mice
at 8 weeks 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.
3.9. Western blot analysis of PKC isoforms
Fig. 8. Representative immunoblots of cardiac PKCh2 (A) and PKC( (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 T S.E.M. n = 6 each group. *P < 0.01 vs. WT mice, .P < 0.01 vs. JVS-control, -P < 0.01 vs. JVS-CA.
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T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
treatment. Thus, the activation of PKCh2 shown by
membrane translocation in JVS mice was significantly
inhibited by CA + FE treatment.
4. Discussion
To our knowledge, this is the first study to demonstrate
the efficacy of PPARa 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 PPARa 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 (100 mg/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 highdose 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 PPARa 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 PPARa, 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 PPARa, mCPT-1 and MCAD may be exerted
only in energetically compromised hearts of JVS mice. The
upregulation of PPARa 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 longchain 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
PKCh2. 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
PKCh was increased in failed human heart [44] and that
T. Asai et al. / Cardiovascular Research 70 (2006) 566 – 577
cardiac-specific overexpression of the PKCh2 isoform
caused pathological cardiac hypertrophy [45,46]. We
therefore suggest that the alteration of the molecular species
composition of DAG and consequent inhibition of PKCh2
redistribution, at least in part, lead to the attenuation of
pathological cardiac hypertrophy in JVS mice.
The role of PPARa in the pathogenesis of various heart
diseases has not been well defined. In PPARa-null mice,
altered expression of PPARa-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 PPARa exhibited signs of diabetic cardiomyopathy
including ventricular hypertrophy and dysfunction [36]. In
a murine model of pressure overload-induced cardiac
hypertrophy, reactivation of PPARa 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 PPARa 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 PPARa in the heart
suggests that the function of this transcription factor differs
in various pathological states and that the activation of
PPARa 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 PPARa activation can be
obtained only when combined with l-carnitine supplementation in lipotoxic cardiomyopathy due to SCD.
Previous studies have assessed the effects of PPARa
activation on the heart in normal animals. Although WY14,643, a PPARa agonist, increased cardiac mRNA levels
of PPARa-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
575
include the experimental group of WT mice treated with
fenofibrate treatment alone for further investigation. Taken
together, the net effects of PPARa 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-weekold 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 lcarnitine attenuated the pathological cardiac hypertrophy at
8 weeks of age and completely prevented the LV remodeling
at 16 weeks of age. Therefore, we consider that analysis at
8 weeks of age provides key findings for elucidating the
pathophysiology of cardiomyopathy in JVS mice and that
intervention to inhibit the pathological hypertrophy at
8 weeks of age leads to the prevention of detrimental LV
remodeling.
In conclusion, PPARa activation, in addition to lcarnitine 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
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.
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