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Circulation Research. 2003;92:518-524
Published online before print February 6, 2003, doi: 10.1161/01.RES.0000060700.55247.7C
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(Circulation Research. 2003;92:518.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Peroxisome Proliferator-Activated Receptor (PPAR) {alpha} and PPARß/{delta}, but not PPAR{gamma}, Modulate the Expression of Genes Involved in Cardiac Lipid Metabolism

Andries J. Gilde*, Karin A.J.M. van der Lee*, Peter H.M. Willemsen, Giulia Chinetti, Feike R. van der Leij, Ger J. van der Vusse, Bart Staels, Marc van Bilsen

From the Department of Physiology (A.J.G., K.A.J.v.d.L., P.H.M.W., G.J.v.d.V., M.v.B.), Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands; U 545 INSERM, Département d’Athérosclerose (G.C., B.S.), Institut Pasteur de Lille and Faculté de Pharmacie, Université de Lille, France; and Department of Pediatrics (F.R.v.d.L.), Beatrix Children’s Hospital, University of Groningen, the Netherlands.

Correspondence to Dr M. van Bilsen, Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands. E-mail Marc.vanBilsen{at}FYS.Unimaas.NL


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Long-chain fatty acids (FA) coordinately induce the expression of a panel of genes involved in cellular FA metabolism in cardiac muscle cells, thereby promoting their own metabolism. These effects are likely to be mediated by peroxisome proliferator-activated receptors (PPARs). Whereas the significance of PPAR{alpha} in FA-mediated expression has been demonstrated, the role of the PPARß/{delta} and PPAR{gamma} isoforms in cardiac lipid metabolism is unknown. To explore the involvement of each of the PPAR isoforms, neonatal rat cardiomyocytes were exposed to FA or to ligands specific for either PPAR{alpha} (Wy-14,643), PPARß/{delta} (L-165041, GW501516), or PPAR{gamma} (ciglitazone and rosiglitazone). Their effect on FA oxidation rate, expression of metabolic genes, and muscle-type carnitine palmitoyltransferase-1 (MCPT-1) promoter activity was determined. Consistent with the PPAR isoform expression pattern, the FA oxidation rate increased in cardiomyocytes exposed to PPAR{alpha} and PPARß/{delta} ligands, but not to PPAR{gamma} ligands. Likewise, the FA-mediated expression of FA-handling proteins was mimicked by PPAR{alpha} and PPARß/{delta}, but not by PPAR{gamma} ligands. As expected, in embryonic rat heart-derived H9c2 cells, which only express PPARß/{delta}, the FA-induced expression of genes was mimicked by the PPARß/{delta} ligand only, indicating that FA also act as ligands for the PPARß/{delta} isoform. In cardiomyocytes, MCPT-1 promoter activity was unresponsive to PPAR{gamma} ligands. However, addition of PPAR{alpha} and PPARß/{delta} ligands dose-dependently induced promoter activity. Collectively, the present findings demonstrate that, next to PPAR{alpha}, PPARß/{delta}, but not PPAR{gamma}, plays a prominent role in the regulation of cardiac lipid metabolism, thereby warranting further research into the role of PPARß/{delta} in cardiac disease.


Key Words: cardiomyocytes • H9c2 cells • fatty acids • gene expression • uncoupling protein


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
It is generally acknowledged that dietary factors, among which are major energy-providing substrates such as fatty acids (FA), act as signaling molecules that modulate gene expression in a variety of tissues, including the heart (for review see Van Bilsen et al1 and Taegtmeyer2). Using primary cultures of neonatal rat cardiomyocytes, it was recently demonstrated that exposure of these cells to long-chain FA markedly enhances the expression of genes encoding proteins involved in FA transport and metabolism.3,4 In addition, evidence was provided that the modulating effect of FA is mediated by peroxisome proliferator-activated receptors (PPARs).3,4 Of the three PPAR isoforms known to date, the significance of PPAR{alpha} and PPAR{gamma} in the regulation of whole-body and cellular lipid metabolism has been firmly established (reviewed in Schoonjans et al5). The function of the ubiquitously expressed PPARß/{delta} isoform, however, remains largely elusive.

Through the development of specific synthetic ligands for PPAR{alpha},6 PPAR{gamma},7 and more recently for PPARß/{delta},8,9 powerful tools have become available to delineate the biological roles of each of the PPAR isoforms in more detail. This prompted us to investigate the specific roles of PPAR{alpha}, PPARß/{delta}, and PPAR{gamma} in the cardiac muscle cell with respect to the regulation of genes encoding proteins involved in energy metabolism. Thereto, neonatal rat cardiomyocytes as well as the embryonic rat heart-derived H9c2 cells were cultured in defined medium in the absence or presence of FA. To fully appreciate the role of each of the three PPAR isoforms, their expression pattern was assessed in cardiac cells and H9c2 myoblasts and myotubes. Next, we examined whether specific ligands for PPAR{alpha} (Wy-14,643), PPARß/{delta} (L-165041, GW501516), and PPAR{gamma} (ciglitazone and rosiglitazone) were able to mimic the modulating effect of FA on metabolic gene expression in these cell models. Accordingly, their effects on the expression of various proteins involved in cardiac FA metabolism, such as acyl-coenzyme A synthetase (ACS), muscle-type carnitine palmitoyltransferase-1 (MCPT-1), long-chain acyl-coenzyme A dehydrogenase (LCAD), and the uncoupling proteins 2 and 3 (UCP-2/-3) were determined at the mRNA level. To assess the functional consequences of PPAR-mediated transcription on cellular lipid metabolism, FA oxidation rates were measured. For comparison, mRNA levels of proteins involved in glucose transport (GLUT4) and metabolism (hexokinase II [HKII]) were determined. Finally, the effect of the isoform-specific PPAR ligands was explored further in transient transfection studies using the PPAR-responsive human MCPT-1 promoter.3

The collective data indicate that, besides PPAR{alpha}, the PPARß/{delta} isoform is involved in regulation of cardiac lipid metabolism. In contrast, the role of PPAR{gamma} in this process appears to be insignificant.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
Neonatal rat ventricular cardiomyocytes were prepared as described previously.10 Experiments were approved by the Institutional Animal Care and Use Committee of the Maastricht University.

H9c2 cells (ATCC, Manassas, Va; No. CRL-1446; passage Nos. 18 to 22) were maintained in growth medium composed of DMEM supplemented with 10% fetal bovine serum (Life Technologies). H9c2 cells were plated at a density of 2000 cells/cm2 and allowed to proliferate in growth medium. Medium was changed twice a week. To induce differentiation of H9c2 myoblasts into myotubes, growth medium was replaced with differentiation medium (DMEM containing 2% horse serum) when cells had reached near confluence. The extent of differentiation of the H9c2 cells was evaluated on the basis of morphological criteria and by measuring cellular creatine kinase activity. Cells were considered differentiated after formation of multinucleated myotubes and a >10-fold increase in creatine kinase activity.

At the start of the experiments, H9c2 myoblasts (at near confluence), H9c2 myotubes, and neonatal cardiomyocytes were incubated in serum-free medium consisting of a 4:1 mixture of DMEM/M199, gentamicin (50 mg/L), and glucose (10 mmol/L) as the sole substrate, for 24 hours. Subsequently, the cells were rinsed once with serum-free medium, whereupon experimental medium was applied to the cells. The experimental medium consisted of serum-free medium enriched with 0.25 mmol/L L-carnitine, 0.25 mU/mL insulin, and 0.15 mmol/L BSA (A-7906, Sigma). When indicated, the medium was further supplemented with FA (palmitic and oleic acids 0.25 mmol/L, each complexed to 0.15 mmol/L BSA, as described elsewhere10). To test the effects of PPAR-specific ligands, cells receiving glucose as the only substrate were incubated with the PPAR{alpha}-ligand Wy-14,643 (reported EC50=0.63 µmol/L7, Biomol), the PPARß/{delta} ligands L-165041 (a gift from Dr J. Berger, Merck Research Laboratories, Rahway, NJ) and GW501516 (EC50=3.8 and 0.024 µmol/L, respectively),7,9 or the PPAR{gamma}-ligands ciglitazone (Biomol) and rosiglitazone (BRL49653, kindly provided by Glaxo SmithKline) (EC50=3.0 and 0.076 µmol/L, respectively7), at the indicated concentrations (see Results). For detailed information regarding the potency and selectivity of the various ligands for the three PPAR isoforms, we refer to Willson et al7 and Oliver et al.9 PPAR ligands were dissolved in DMSO, which was also added as vehicle (0.1% vol/vol) to control cells.

Tissues and Cells
Hearts and epididymal white adipose tissue were dissected from neonatal and adult Lewis rats. To determine the presence of PPAR isoforms in cardiac myocytes, the myocyte fraction was isolated from neonatal and adult hearts after collagenase treatment by Percoll-gradient centrifugation10 and sedimentation,11 respectively. Tissues and cell fractions were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

RNA Analysis
After 48 hours of incubation, RNA was isolated with TRIzol reagent (Life Technologies). Total RNA (10 µg) was size-fractionated, transferred to nylon membranes, and probed with cDNA fragments of GLUT4, HKII, ACS, LCAD, MCPT-1, UCP-2, and UCP-3 as described previously.4,12,13 A 0.9-kb PstI fragment of mouse PPAR{alpha}, a 1.5-kb BamHI fragment of rat PPARß/{delta} (a gift of Dr P.A. Grimaldi, University of Nice, France), and a 0.6-kb BglII fragment of hamster PPAR{gamma} were also used for hybridization. The cDNA probes were labeled with [32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech) by random priming (Radprime, Life Technologies) to a specific activity of >0.5x109 cpm/µg DNA. To correct for possible differences in transfer and loading, the membranes were also hybridized with a 32P-labeled ribosomal 18S probe. After hybridization, membranes were washed at the appropriate stringency to remove nonspecific binding. The membranes were exposed to imaging screens and subsequently scanned on the Personal FX Imager (Bio-Rad Laboratories). Signals were quantified using Quantity One software (Bio-Rad).

Protein Extraction and Western Blot
Tissues and cells were homogenized in lysis buffer (in mmol/L, Tris-HCl 10, NaCl 150, and EDTA 1; 0.1% NP-40; 0.5% sodium deoxycholate; 1% SDS; and a protease inhibitor cocktail), and proteins were collected by centrifugation at 4°C. Proteins (100 µg) were loaded onto 10% SDS-PAGE gels. Ponceau’s red staining was performed to verify equal loading. Western blot analysis was performed using a monoclonal antibody against PPAR{gamma} (Santa Cruz Biotechnology, Inc) or against {alpha}-actinin (Sigma). Specific signals were visualized using the enhanced chemiluminescence system (Amersham).

Transient Transfection
For transient transfection, neonatal cardiomyocytes were seeded on 6-well plates at a density of 2.5x105 cells in 2 mL of medium per well. Cells were transfected 16 hours before addition of experimental medium using the transfection reagent FuGENE 6 (Roche). Cells were transfected with 0.5 µg of promoter/reporter vector, ie, a 1.3-kb fragment of the human MCPT-1 promoter generated by high-fidelity polymerase chain reaction (PCR; Expand, Roche) of genomic DNA (corresponding to positions 61203 to 62543; GenBank accession No. U6231714) and cloned into the pGL3 luciferase vector (MCPT-1-luc), or a construct containing three copies of the PPRE site of the human apolipoprotein A-II promoter (PPRE3-TK-luc).15 The cytomegalovirus ß-galactosidase–containing vector pON249 (0.25 µg) was cotransfected to control for transfection efficiency.16 In a subseries of experiments, 0.5 µg of the pSG5 expression vector containing the cDNA of PPAR{gamma}17 was cotransfected. The total amount of plasmid DNA per well was always kept constant by adding empty pSG5 vector (Promega).

Cells were harvested 24 hours after addition of experimental medium (see above) and immediately processed for the determination of reporter activity. Luciferase activity was determined using a commercial firefly luciferase assay according to the supplier’s instructions (Steady Glo, Promega) in white 96-well plates (Nalge Nunc International) and FluorS imager (Bio-Rad) for measuring luminescence. ß-Galactosidase activity was determined spectrophotometrically (Titertek Multiskan Plus MKII, Thermo LabSystems) as described previously.16

FA Oxidation
Experiments to determine the oxidation capacity of [1-14C]palmitic acid (Amersham) in neonatal rat ventricular cardiomyocytes were performed according to van der Lee et al.4

Statistics
Results are obtained from at least three independent experiments and presented as sample mean±SD. Comparison between groups was performed with one-way ANOVA. In cases in which the F ratio obtained indicated that significant differences between groups were present, a two-tailed Student’s t test for unpaired data was carried out, applying a Bonferroni adjustment for multiple comparison.18 Differences were considered significant at P<0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
FA Supplementation and Gene Expression
To assess the effects of FA on gene expression, neonatal rat cardiomyocytes and myoblasts and myotubes of the embryonic rat heart-derived H9c2 cell line were cultured in the presence of either glucose or a combination of glucose and FA (palmitic and oleic acid) as substrates.

Cardiomyocytes
Consistent with previous studies,4 48 hours after exposure to FA, mRNA levels of the glucose transporter GLUT4 and the glycolytic enzyme HKII were lower (Figure 1A). In contrast, the mRNA levels of ACS, LCAD, and the uncoupling protein UCP-2 were markedly increased.



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  		Figure 1. Effects of FA on mRNA levels of genes involved in glucose and FA uptake and metabolism in neonatal rat cardiomyocytes (A), H9c2 myoblasts (B), and H9c2 myotubes (C). Cells were exposed to either 10 mmol/L glucose (open bars) or a combination of glucose and 0.5 mmol/L FA (hatched bars) for 48 hours. The mRNA levels of GLUT4, HKII, ACS, LCAD, and UCP2 are expressed relative to the control group, the level of which was set at 1.0 arbitrarily. Data are mean±SD (n=5 to 7). *Significantly different (P<0.05) from glucose group.

H9c2 Cells
The differentiation of H9c2 myoblasts into myotubes was associated with a rise in the mRNA levels of proteins involved in both glucose (GLUT4 and HKII) and FA (ACS and LCAD) metabolism, but still the mRNA levels were substantially lower than those in cardiomyocytes exposed to identical conditions (data not shown). Likewise, the FA-induced changes in gene expression were less pronounced in the H9c2 cells, reaching statistical significance for HKII, LCAD, and UCP-2 in the myoblasts (Figure 1B), and for GLUT4, HKII, ACS, LCAD as well as UCP-2 in the myotubes, respectively (Figure 1C).

PPAR Isoform Expression
As shown in Figure 1, the effects of FA on gene expression are more pronounced in cardiac myocytes than in H9c2 cells. As the effects of FA on cardiac myocytes are considered to be PPAR mediated,3,4 we first looked for possible differences in PPAR isoform expression or abundance. Thereto, mRNA levels of the PPAR isoforms were determined in the different cell types and in ventricular tissue of neonatal and adult rats (Figure 2). In H9c2 myoblasts and myotubes, as well as in cardiac myocytes and ventricular tissue, PPARß/{delta} was abundantly expressed. Interestingly, PPAR{alpha} mRNA was not detectable in H9c2 cells but was clearly present in isolated cardiomyocytes and cardiac tissue. In contrast to its preponderance in white adipose tissue, a clear PPAR{gamma} mRNA signal was not detected either in H9c2 cells, in isolated cardiomyocytes, or in intact ventricular tissue. As the presence of PPAR{gamma} in cardiac myocytes is controversial,19,20 we also applied Western blotting to further check for the possible presence of this isoform in neonatal and adult cardiac myocytes. PPAR{gamma} immunoreactivity was readily observed in white adipose tissue, whereas substantially longer exposure times were required to detect signals in cardiac tissue and cardiomyocytes (Figure 3). Collectively these findings demonstrate that the PPAR isoforms are all present in cardiac muscle cells, albeit that {gamma}-isoform is expressed at relatively low levels.



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  		Figure 2. Northern blots showing the distribution of PPAR{alpha}, PPARß/{delta}, and PPAR{gamma} mRNA in isolated H9c2 myoblasts, H9c2 myotubes, neonatal and adult rat cardiomyocytes (CM), and neonatal and adult rat heart tissue (representative of 3 experiments). White adipose tissue (WAT) is included as positive control for PPAR{gamma}. 18S rRNA signal demonstrates equal loading of gel.



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  		Figure 3. Western blot showing PPAR{gamma} expression in neonatal and adult rat ventricular tissue and freshly isolated cardiomyocytes (CM) and white adipose tissue (WAT; representative of 2 isolations). Note that for the detection of PPAR{gamma} immunoreactivity in cardiac fractions, substantially longer exposure times (60 minutes instead of 15 minutes) were required. Sarcomeric {alpha}-actinin immunoreactivity is included as control.

Effects of Isoform-Specific PPAR Agonists
Next, the involvement of each of the PPAR isoforms in the FA-mediated changes in gene expression was assessed functionally. Thereto, cardiomyocytes and H9c2 cells were treated with Wy-14,643, L-165041, and ciglitazone, specific agonists for PPAR{alpha}, PPARß/{delta}, and PPAR{gamma}, respectively. At concentrations of 10 µmol/L, these PPAR agonists were previously shown to be effective and isoform specific in other cell types.6–8

Cardiomyocytes
As shown in Figure 4, none of the PPAR ligands significantly affected the expression of genes involved in glucose uptake (GLUT4) and metabolism (HKII). In contrast, the PPAR{alpha}-specific ligand Wy-14,643 was as effective as FA in inducing the expression of the FA-handling proteins ACS, MCPT-1, LCAD, and UCP-2 in cardiomyocytes. The addition of the PPARß/{delta} agonist L-165041 also led to an increase, albeit less pronounced, in the mRNA levels of ACS, MCPT-1, and UCP-2.



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  		Figure 4. Effects of FA and isoform-specific PPAR ligands on cardiomyocyte gene expression. Cells were exposed to glucose (control group, open bars), either in a combination with FA (FA, hatched bars) or with one of the isoform-specific PPAR ligands (10 µmol/L) using 0.1% DMSO as vehicle (black bars) for 48 hours. The mRNA levels of GLUT4, HKII, ACS, MCPT-1, LCAD, and UCP2 are expressed relative to the control group, the level of which was set at 1.0 arbitrarily. Representative Northern blot signals of the mRNAs investigated are included. Data are mean±SD (n=5 to 7). *Significantly different (P<0.05) from control group.

H9c2 Cells
In accordance with the expression pattern of the PPAR isoforms in H9c2 myoblasts and myotubes, the PPAR{alpha}- and PPAR{gamma}-specific ligands were ineffective (Figure 5). In contrast, the PPARß/{delta} ligand L-165041 increased mRNA levels of both ACS and LCAD in H9c2 cells to the same degree as FA. Likewise, UCP-2 mRNA levels were clearly upregulated by both FA and L-165041 in H9c2 myoblasts. Conversely, although mRNA of UCP-3 could not be detected in H9c2 myoblasts, in H9c2 myotubes UCP-3 expression was markedly induced by both FA and the PPARß/{delta} agonist. Interestingly, the PPAR ligands did not affect GLUT4 and HKII mRNA levels in myotubes, but exposure to FA was associated with a decline, which suggests that FA also exert PPAR-independent effects.



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  		Figure 5. Northern blots displaying the effects of isoform-specific PPAR ligands on the expression of metabolic genes in H9c2 cells. RNA levels of the glucose transporter GLUT4, HKII, ACS, LCAD, and the uncoupling proteins UCP-2 and UCP-3 are shown (representative of 3 experiments). H9c2 myoblasts and myotubes were incubated in the absence or presence of either Wy-14,643, L-165041, ciglitazone (ligands specific for PPAR{alpha}, PPARß/-{delta}, and PPAR{gamma}, respectively, at 10 µmol/L), or with 0.5 mmol/L FA for 48 hours. Medium containing glucose and 0.1% DMSO served as vehicle control. 18S rRNA signal shows equal loading.

PPAR Isoform-Specific Transcriptional Activation
To demonstrate the PPAR isoform-specific response of neonatal cardiomyocytes, transient transfection was applied. Thereto, the dose response to the various isoform-specific ligands was investigated using the human muscle-type CPT-1 promoter containing a functional PPAR-responsive element3 (Figure 6). The PPAR{alpha} ligand Wy-14,643 as well as the PPARß/{delta} ligands L-165041 and GW501516 dose-dependently induced promoter activity. For each ligand at {approx}5 µmol/L, the response reached maximal values, but the level of induction was still lower than that with FA (3.5±1.0-fold). The PPARß/{delta} ligand GW501516 proved to be the most potent ligand used and significantly increased MCPT-1 promoter activity at 10 nmol/L already. The specific PPAR{gamma} ligands ciglitazone and rosiglitazone, however, were unable to induce MCPT-1 promoter activity, irrespective of the dose applied. The forced overexpression of PPAR{gamma} substantially increased basal and rosiglitazone-activated transcription activity of both the MCPT-1 promoter (Figure 7A) and a minimal promoter construct containing three copies of the apolipoprotein A-II PPRE (PPRE3-TK-Luc) (Figure 7B), thereby demonstrating the potential of PPAR{gamma} to regulate transcription in the cardiomyocyte context, provided that PPAR{gamma} is expressed at high levels.



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  		Figure 6. Dose response of the human MCPT-1 promoter in neonatal cardiomyocytes exposed to the PPAR{alpha} ligand Wy-14,643 ({bullet}), PPARß/{delta} ligands L-165041 ({blacksquare}) and GW501516 ({square}), and PPAR{gamma} ligands ciglitazone ({blacktriangledown}) and rosiglitazone ({triangledown}), respectively. Cells were transiently transfected with the MCPT-1 promoter/luciferase reporter vector and were subsequently treated with the different ligands. Results are expressed as fold induction compared with control (no ligand), which was arbitrarily set 1.0.



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  		Figure 7. Effects of PPAR{gamma} overexpression on the activity of the MCPT-1 promoter (A) and a 3x PPRE containing minimal promoter construct PPRE3-TK-Luc (B) in the absence or presence of the selective PPAR{gamma} ligand rosiglitazone (10 µmol/L) in cardiomyocytes. Induction of promoter activity is shown relative to vehicle (0.1% DMSO)–treated control cells (open bars), the luciferase activity of which was arbitrarily set at 1.0. Data are mean±SD from 3 independent experiments. *Significantly different (P<0.05) from control.

PPAR Isoforms and FA Oxidation
To test whether the PPAR-mediated changes at the mRNA level were associated with functional alterations in FA metabolism, the effects of the isoform-specific ligands on palmitate oxidation rate were determined (Figure 8). In line with the mRNA data, 14CO2 production from palmitate increased in cardiomyocytes pretreated with the PPAR{alpha} ligand Wy-14,643 and the PPARß/{delta} ligands L-165041 and GW501516, the latter being more potent. In contrast, the PPAR{gamma} ligands ciglitazone and rosiglitazone were both ineffective.



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  		Figure 8. Effect of the PPAR isoform-specific ligands on FA oxidation. The oxidation of radiolabeled palmitate was measured as the production of 14CO2 for 30 minutes in cells pretreated for 48 hours with 10 µmol/L of either the PPAR{alpha} ligand Wy-14,643, the PPARß/{delta} ligands L-165041 and GW501516, or the PPAR{gamma} ligands ciglitazone and rosiglitazone. Results are expressed as fold induction compared with control (no ligand), which was arbitrarily set at 1.0 and corresponds with 0.15±0.04 nmol palmitate/min per mg protein. Data are mean±SD (n=9 of 3 different cultures). *Significantly different (P<0.05) from vehicle-treated cells.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present findings demonstrate for the first time that the long-chain FA-induced increase in the expression of genes in cardiac muscle cells is mediated not only by PPAR{alpha}, but also by PPARß/{delta}. With the use of a panel of PPAR isoform-specific ligands, it is demonstrated that in neonatal cardiomyocytes PPAR{alpha} and PPARß/{delta}, but not PPAR{gamma}, selectively increase the expression of genes involved in cardiac FA metabolism. The transient transfection studies with the MCPT-1 promoter indicate that these effects are mediated at the transcriptional level. As a consequence, the FA oxidation rate in cardiomyocytes was shown to be enhanced. The fact that in H9c2 cells, which only express the PPARß/{delta} isoform, the effects of FA can only be reproduced by the PPAR ß/{delta}-specific ligand further substantiates the notion that FA act as endogenous ligands for PPARß/{delta} in the cardiac muscle context.

PPAR{gamma} and Cardiomyocyte Gene Expression
Whereas the presence of PPAR{alpha} and PPARß/{delta} in cardiac tissue has been firmly established,21,22 the presence of PPAR{gamma} is controversial. PPAR{gamma} mRNA either was detected at low levels17,22–24 or could not be detected at all in the intact heart.19,25 Recently, however, Takano et al20 demonstrated the presence of PPAR{gamma} mRNA and immunoreactivity in primary cultures of neonatal rat cardiomyocytes. In the present study, PPAR{gamma} mRNA appeared to be undetectable by Northern blotting but was detectable using a more sensitive method such as RT-PCR (data not shown) in freshly isolated neonatal and adult cardiomyocytes. Moreover, PPAR{gamma} immunoreactivity was detectable, albeit weak, revealing small amounts of PPAR{gamma} protein in cardiac myocytes and tissue. Our results corroborate the findings of Escher et al,24 who showed by quantitative RT-PCR that in the adult heart the mRNA content of PPAR{gamma} was low as compared with that of PPAR{alpha} and PPARß/{delta}.

The absence of significant effects of the PPAR{gamma} ligands ciglitazone and rosiglitazone on mRNA levels of all genes investigated, MCPT-1 promoter activity, and cellular FA oxidation rate argues against a role for PPAR{gamma} in the regulation of cardiac lipid metabolism. Even FAT/CD36, a gene that has convincingly been shown to behave as a PPAR{gamma} target gene in macrophages,26 did not respond to PPAR{gamma} ligands in neonatal cardiomyocytes (authors’ unpublished observation, 2001). As the forced overexpression of PPAR{gamma} in cardiomyocytes is accompanied by basal and ligand-activated transcription of the MCPT-1 promoter, it is conceivable that the virtual absence of PPAR{gamma}-mediated effects on FA metabolism resides in its low abundance in this cell type.

Other studies, however, revealed effects of PPAR{gamma} ligands on cardiomyocyte and skeletal muscle cell function and phenotype. In skeletal muscle the stimulatory effects of the glitazones on mitochondrial fuel oxidation were demonstrated to be independent of PPAR{gamma}-mediated gene expression, suggesting that the drugs exerted PPAR{gamma}-independent effects.27 Indeed, other signaling pathways, among which is the nuclear factor-{kappa}B pathway, have been shown to be modulated by PPAR{gamma} ligands, involving both PPAR{gamma}-dependent and PPAR{gamma}-independent mechanisms.28,29 Collectively, these observations suggest that, notwithstanding the limited relevance of PPAR{gamma} in cardiac lipid metabolism, a biological role for PPAR{gamma} in other processes in the heart cannot be dismissed.

Role of PPAR{alpha} and PPARß/{delta}
Both in vitro and in vivo studies demonstrated PPAR{alpha} to be instrumental in the regulation of the expression of genes coding for proteins involved in cardiac FA metabolism.4,30,31 The present findings with respect to the PPAR isoform expression pattern and the effect of the PPAR{alpha} ligand Wy-14,643 on gene expression and FA oxidation corroborate these earlier observations.

Along with PPAR{alpha}, the PPARß/{delta} isoform was shown to be present in cardiomyocytes. However, in myoblasts and myotubes of the embryonic heart-derived cell line H9c2, PPARß/{delta} turned out to be the only isoform present. In contrast to the other isoforms, the biological role of PPARß/{delta} has remained largely enigmatic up to now. Interestingly, in H9c2 cells the effects of FA on gene expression was mimicked by the PPARß/{delta} ligand only. This effect was most obvious for the uncoupling proteins. In H9c2 myoblasts, FA and L-165041 induced UCP-2 mRNA levels to a similar extent, whereas in myotubes UCP-3 responded in a similar way. These findings support the notion that FA function as ligands for PPARß/{delta} in these cells and, by inference, in the cardiac muscle cell as well. Indeed, several recent studies are indicative of a role of PPARß/{delta} in cellular lipid metabolism. In skeletal muscle cells, PPARß/{delta} was shown to be the PPAR subtype responsible for the induction of UCP-3 and UCP-2 mRNA.32,33 In cultured brain cells and keratinocytes, the PPARß/{delta}-specific ligand L-165041 increased expression of the ACS isoform ACS2 and FAT/CD36, respectively.34,35 Furthermore, Oliver et al9 demonstrated increased transcription of the cholesterol transport protein ABCA1 by the highly specific PPARß/{delta} ligand GW501516 in macrophages.

The current findings indicate that activation of PPARß/{delta} also modulates neonatal cardiomyocyte gene expression. The mRNA and promoter/reporter studies reveal that the human MCPT-1 gene, previously shown to be responsive to PPAR{alpha}, is equally responsive to the PPARß/{delta}-specific ligands L-165041 and GW501516. On the other hand, differences in response to {alpha}- and ß/{delta}-ligands have to be appreciated too, as the rise in endogenous ACS and LCAD mRNA levels is less pronounced with L-165041 than with Wy-14,643 when applied at equimolar doses (see Figures 4 and 5). Whether the differences in response of individual genes to PPAR{alpha} and PPARß/{delta} ligands are related to the potency of the respective ligands on the one hand or to subtle differences within PPAR-response elements or flanking sequences on the other awaits further investigation. In this respect it is worth mentioning that differences in the consensus PPAR{alpha} and PPARß/{delta} binding sites have indeed been reported.36

The present findings strongly suggest that PPAR{alpha} and PPARß/{delta} share similar functions. Indeed, administration of PPARß/{delta} agonists to PPAR{alpha}-null mice induces peroxisome proliferation in liver,37 a phenomenon formerly exclusively ascribed to PPAR{alpha}. Similarly, in PPAR{alpha}-/- mice, the fasting-induced upregulation of several PPAR-responsive genes in liver and heart is attenuated but not completely blunted.30 The latter findings suggest a residual effect, or partial compensation, involving PPARß/{delta}. Furthermore, the recent observation by Muoio et al38 that FA oxidation in skeletal muscle of PPAR{alpha}-/- mice is not impaired also led these investigators to conclude that PPARß/{delta} compensates for the lack of PPAR{alpha} in these mice.

Taken together, the present findings argue against a significant role for PPAR{gamma} in the regulation of metabolic gene expression in heart muscle cells. Furthermore, unequivocal evidence is presented that, next to PPAR{alpha}, PPARß/{delta} is an important regulator of the expression of genes involved in cardiac lipid metabolism. Accordingly, further investigation into the role of PPARß/{delta} isoform in the regulation of cardiac FA homeostasis during cardiac disease is warranted.


*    Acknowledgments
 
This research was funded by grants from the Netherlands Heart Foundation (NHS 1998T015 to M.v.B. and NHS 1997B093 to F.R.v.d.L.), the European Union (QLG1-1999-01007 to G.C.), the Leducq Foundation, and the FEDER/Conseil Régional Nord/Pas-de-Calais (Genopole 01360124 to B.S.). We thank E.M.H. Cuijpers and R.L.M. Lotz for technical support.


*    Footnotes
 
*Both authors contributed equally to this study. Back

Received June 19, 2002; revision received December 13, 2002; accepted January 29, 2003.


*    References
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