Nuclear Factor-κB Activation Leads to Down-regulation of Fatty Acid Oxidation during Cardiac Hypertrophy*

Little is known about the mechanisms responsible for the fall in fatty acid oxidation during the development of cardiac hypertrophy. We focused on the effects of nuclear factor (NF)-κB activation during cardiac hypertrophy on the activity of peroxisome proliferator-activated receptor (PPAR) β/δ, which is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. Phenylephrine-induced cardiac hypertrophy in neonatal rat cardiomyocytes caused a reduction in the expression of pyruvate dehydrogenase kinase 4 (Pdk4), a target gene of PPARβ/δ involved in fatty acid utilization, and a fall in palmitate oxidation that was reversed by NF-κB inhibitors. Lipopolysaccharide stimulation of NF-κB in embryonic rat heart-derived H9c2 myotubes, which only express PPARβ/δ, caused both a reduction in Pdk4 expression and DNA binding activity of PPARβ/δ to its response element, effects that were reversed by NF-κB inhibitors. Coimmunoprecipitation studies demonstrated that lipopolysaccharide strongly stimulated the physical interaction between the p65 subunit of NF-κB and PPARβ/δ, providing an explanation for the reduced activity of PPARβ/δ. Finally, we assessed whether this mechanism was present in vivo in pressure overload-induced cardiac hypertrophy. In hypertrophied hearts of banded rats the reduction in the expression of Pdk4 was accompanied by activation of NF-κB and enhanced interaction between p65 and PPARβ/δ. These results indicate that NF-κB activation during cardiac hypertrophy down-regulates PPARβ/δ activity, leading to a fall in fatty acid oxidation, through a mechanism that involves enhanced protein-protein interaction between the p65 subunit of NF-κB and PPARβ/δ.

congestive heart failure, arrhythmia, and sudden death (1,2). Cardiac hypertrophy is associated with an increase in glucose utilization and a decrease in fatty acid oxidation, which is characteristic of the fetal heart (3,4). It is still a matter of controversy whether changes in intracellular substrate and metabolite levels in cardiomyocytes are a consequence or the reason for cardiac hypertrophy. However, several factors support a role for cardiac metabolism in the development of cardiac hypertrophy. Thus, defects in mitochondrial fatty acid oxidation enzymes cause childhood hypertrophic cardiomyopathy (5), and perturbation of fatty acid oxidation in animal models causes cardiac hypertrophy (6,7), demonstrating that substrate utilization is important in the pathogenesis of hypertrophy. Nonetheless, little is known about the molecular mechanisms linking cardiac hypertrophy and the fall in the expression of genes involved in cardiac fatty acid metabolism.
Peroxisome proliferator-activated receptors (PPARs) 1 are ligand-activated transcription factors that regulate the expression of genes involved in fatty acid uptake and oxidation, lipid metabolism, and inflammation (8). The PPAR subfamily consists of three subtypes, PPAR␣ (NR1C1 according to the unified nomenclature system for the nuclear receptor superfamily), PPAR␤/␦ (NR1C2), and PPAR␥ (NR1C3) (9). PPAR␣ is expressed primarily in tissues that have a high level of fatty acid catabolism such as liver, brown fat, kidney, heart, and skeletal muscle (10). PPAR␤/␦ is ubiquitously expressed, and PPAR␥ has a restricted pattern of expression, mainly in white and brown adipose tissues, whereas other tissues such as skeletal muscle and heart contain limited amounts. To be transcriptionally active, PPARs need to heterodimerize with the 9-cis-retinoic acid receptor (NR2B). PPAR-retinoic acid receptor heterodimers bind to DNA-specific sequences called peroxisome proliferator-response elements (PPREs), consisting of an imperfect direct repeat of the consensus binding site for nuclear hormone receptors (AGGTCA) separated by one nucleotide (DR-1). These sequences have been characterized within the promoter regions of PPAR target genes. However, the regulation of gene transcription by PPARs extends beyond their ability to transactivate specific target genes. PPARs are also able to regulate gene expression independently of binding to DNA through a mechanism termed receptor-dependent trans-repression (11). One of these mechanisms involves a physical interaction of PPAR␣ with nuclear factor (NF)-B, that may lead to suppression activity of the former (12). Interestingly, the NF-B signaling pathway plays a pivotal role in the hypertrophic growth of the myocardium and its inhibition blocks or attenuates the hypertrophic response of cultured cardiac myocytes (13)(14)(15)(16).
Given that Gilde and co-workers (17), using neonatal rat cardiomyocytes as well as the embryonic rat heart-derived H9c2 cells, clearly demonstrated that PPAR␤/␦ is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism, we focused on the effects of NF-B activation during cardiac hypertrophy on PPAR␤/␦ activity. Furthermore, to avoid interference of the other PPAR subtypes we took advantage of the use of the embryonic rat heart-derived H9c2 myotubes, which only express PPAR␤/␦. Our findings indicate that NF-B activation down-regulates PPAR␤/␦ activity, leading to a fall in fatty acid oxidation through a mechanism involving enhanced proteinprotein interaction between the p65 subunit of NF-B and PPAR␤/␦.

MATERIALS AND METHODS
Cell Culture-Neonatal rat ventricular myocytes from 1-to 2-day-old Sprague-Dawley rats were prepared and cultured overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as described previously (18). The media was changed to serum-free Dulbecco's modified Eagle's medium supplemented with transferrin (10 g/ml), insulin (1 g/ml), and bromodeoxyuridine (0.1 mmol/liters) 24 h before treatments. In this study phenylephrine (PE) was used to stimulate neonatal rat cardiomyocytes in the absence or presence of 10 mol/liter parthenolide for the last 6 h. Animal handling and disposal were performed in accordance with law 5/1995, 21st July, of the Generalitat de Catalunya.
The embryonic rat heart-derived H9c2 cells (ATCC) were maintained in growth medium composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. H9c2 cells were plated at a density of 5000 cells/cm 2 and allowed to proliferate in growth medium. Medium was changed every 3 days. To induce differentiation of H9c2 myoblasts into myotubes, growth medium was replaced with differentiation medium (Dulbecco's modified Eagle's medium containing 2% horse serum) when cells had reached near confluence. For mRNA analysis H9c2 cells were stimulated with LPS (10 ng/ml) for 24 h in the presence or absence of 10 mol/liter parthenolide for the last 6 h.

Incorporation of [ 3 H]Leucine-
To examine the effect of PE on protein synthesis, the incorporation of [ 3 H]leucine was measured essentially by the method of Thaik et al. (19). Cultured neonatal rat ventricular myocytes were treated with PE in the presence or absence of pathenolide and coincubated with [ 3 H]leucine (1 Ci/ml) for 24 h. The cells were washed with phosphate-buffered saline and then treated with 10% trichloroacetic acid at 4°C for 30 min to precipitate the protein. The precipitates were then dissolved in NaOH (0.25 N). Aliquots were counted with a scintillation counter.
Pressure Overload-induced Cardiac Hypertrophy-Male Sprague-Dawley rats (225 to 250 g) were maintained under standard conditions of illumination (12-h light/dark cycle) and temperature (21 Ϯ 1°C). They were fed a standard diet (Panlab, Barcelona, Spain) for 5 days before the studies began. The animals were randomly distributed into two groups: sham-operated rats and pressure overloaded rats. Pressure overload was induced by constriction of the abdominal aorta at the suprarenal level with 7-0 nylon strings by ligation with a blunted 25-gauge needle, which was then pulled out. For the age-matched sham operation, the identical procedure was performed except that the suture was not tied around the aorta. Hearts were harvested 15 days after the surgical operation. The heart weight/body weight ratio was calculated and the heart samples were frozen in liquid nitrogen and then stored at Ϫ80°C. Animal handling and disposal were performed in accordance with the law 5/1995, 21st July, from the Generalitat de Catalunya.
Coimmunoprecipitation-Cell nuclear extracts were brought to a final volume of 0.5 ml with buffer containing 10 mM phosphate-buffered saline, 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl 2 , 8.5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, 2% bovine serum albumin, and 1 mg/ml nonfat milk for 6 h at 4°C, and incubated with 4 g of anti-p65. Immunocomplex was captured by incubating the samples with protein A-agarose suspension overnight at 4°C on a rocking platform. Agarose beads were collected by centrifugation and washed three times with phosphate-buffered saline containing protease inhibitors. After microcentrifugation, the pellet was washed with 60 l of SDS-PAGE sample buffer and boiled for 5 min at 100°C. An aliquot of the supernatant was subjected to electrophoresis on 10% SDS-PAGE and immunoblotted with an antibody against PPAR␤/␦.
Statistical Analyses-Results were obtained from at least four independent experiments and presented as mean Ϯ S.D. Significant differences were established by Student's t test or analysis of variance, according to the number of groups compared. When significant variations were found, the Tukey-Kramer multiple comparisons test was performed (GraphPad Software version 2.03) (GraphPad Software Inc., San Diego, CA). Differences were considered significant at p Ͻ 0.05.

Down-regulation of Fatty Acid Oxidation in Hypertrophied
Neonatal Rat Cardiomyocytes Is Reversed by NF-B Inhibitors-Cardiac hypertrophy is characterized by increased protein content (e.g. [ 3 H]leucine uptake) and induction of fetaltype genes (e.g. Anf). PE-induced cardiac hypertrophy enhanced [ 3 H]leucine incorporation (1.6-fold, p Ͻ 0.001) and mRNA levels of Anf in neonatal rat cardiomyocytes (ϳ2-fold induction, p Ͻ 0.01) (Fig. 1, A and B). Next, we evaluated whether induction of cardiac hypertrophy led to a reduction in the expression of cardiac genes involved in lipid metabolism. We examined the expression of the PPAR␤/␦ target gene (22) Pdk4, which suppresses glucose oxidation by its inhibitory effect on the pyruvate dehydrogenase complex leading to an increase in fatty acid utilization (23). Induction of cardiac hypertrophy by PE led to a reduction in the transcript levels of Pdk4 (30%, p Ͻ 0.05). Because it has been previously demonstrated that PE-induced cardiac hypertrophy involves NF-B activation (14), we confirmed NF-B activation by measuring in neonatal rat cardiomyocytes the mRNA levels of Mcp-1, a gene under the transcriptional control of NF-B (24). PE stimulation enhanced 2-fold (p Ͻ 0.01) the expression of this gene (Fig. 1C). These findings indirectly suggested a link between activation of NF-B and reduction in the expression of genes involved in cardiac fatty acid metabolism during the development of cardiac hypertrophy. To clearly demonstrate this relationship we assessed the effects of NF-B inhibitors on the expression of these genes and in palmitate oxidation. The changes in Pdk4 and Mcp-1 mRNA levels in PE-induced cardiac hypertrophy were prevented when cardiomyocytes were incubated with parthenolide, which specifically inhibits activation of NF-B by preventing IB degradation (27) (Fig. 2). Thus, the 33% reduction (p Ͻ 0.05) observed in the transcript levels of Pdk4 after PE stimulation was prevented by parthenolide treatment and even a 5-fold induction was observed compared with control cells (Fig. 2A). Similarly, the 1.8-fold induction (p Ͻ 0.05) in Mcp-1 mRNA levels assessed by PE was prevented when cells were incubated with parthenolide (Fig.  2B). Furthermore, we determined whether these changes affected the palmitate oxidation rate in neonatal rat cardiomyocytes either in the absence or presence of NF-B inhibitors such as parthenolide and SN-50, a cell-permeable peptide highly selective against NF-B (25) that inhibits NF-B by blocking its translocation to the nucleus (26). PE-induced cardiac hypertrophy led to a 44% reduction (p Ͻ 0.001) in palmitate oxidation compared with untreated cardiomyocytes (Fig. 2C). In contrast, when PE-treated cardiomyocytes were coincubated with either parthenolide or SN-50, statistically significant increases were observed in palmitate oxidation compared with cardiomyocytes treated only with PE.
Reduced DNA Binding Activity of PPAR␤/␦ in LPS-stimulated H9c2 Myotubes: Restoration by NF-B Inhibitors-Data obtained in neonatal rat cardiomyocytes clearly establish a link between NF-B activation during cardiac hypertrophy and the fall in fatty acid oxidation. However, because PPAR␣ and PPAR␤/␦ are expressed in neonatal and adult heart (17)  difficult to discard the potential involvement of the former in the changes reported. Thus, to clearly establish the role of PPAR␤/␦ in the changes observed we used H9c2 myotubes, which only express the PPAR␤/␦ subtype (17) (Fig. 3A), whereas PPAR␣ is undetectable. To activate NF-B, H9c2 myotubes were stimulated for 1 h with LPS, which has been reported to activate NF-B in cardiomyocytes (28). LPS-induced activation of NF-B in H9c2 myotubes was confirmed by EMSA and by assessing the expression of Mcp-1. EMSA showed that the NF-B probe formed three complexes with cardiac nuclear proteins (Fig. 3B, complexes I to III). Specificity of the three DNA-binding complexes was assessed in competition experiments by adding an excess of unlabeled NF-B oligonucleotide to incubation mixtures. NF-B binding activity, mainly of specific complex II, increased in cells stimulated with LPS for 1 h. Characterization of NF-B was performed by incubating nuclear extracts with an antibody directed against the p65 subunit of NF-B. Addition of this antibody to incubation mixtures resulted in complete supershift of complex II, demonstrating that this complex contained p65. As expected, a robust induction (7-fold, p Ͻ 0.001) was observed in the mRNA levels of the NF-B target gene Mcp-1 after LPS stimulation (Fig. 3B). This increase was significantly reduced in the presence of the NF-B inhibitor, parthenolide. Furthermore, stimulation of H9c2 myotubes with LPS also caused a 40% reduction in the mRNA levels of Pdk4 that was completely prevented in the presence of parthenolide (Fig. 3C). Therefore, two different stimulus leading to NF-B activation caused a reduction in the expression of Pdk4 in cardiac cells, strengthening the correlation between both processes.
We next sought to determine the molecular mechanism by which NF-B activation leads to reduced expression of PPAR␤/␦ target genes, such as Pdk4. EMSA were performed to examine the interaction of PPARs with its cis-regulatory element using a 32 P-labeled PPRE probe and cardiac nuclear extracts from H9c2 myotubes stimulated with LPS for 1 h. The PPRE probe formed two main complexes with cardiac nuclear proteins (Fig. 3E, complexes I and II). Competition studies performed with a molar excess of unlabeled probe revealed that both complexes represented specific PPREprotein interactions. Supershift studies performed using an antibody against PPAR␦/␤ demonstrated that complex I contained this PPAR subtype. In nuclear extracts from LPSstimulated H9c2 myotubes a significant reduction was observed in the binding activity of complex I compared with nuclear extracts from control cells (Fig. 3F). In contrast, this binding activity was restored when LPS-stimulated H9c2 myotubes were coincubated with two inhibitors of NF-B, pyrrolidine dithiocarbamate and parthenolide. Nuclear extracts from LPS-stimulated H9c2 cells incubated with the selective PPAR␤/␦ activator L-165041 were used as a positive control to demonstrate that enhanced complex I binding activity was because of increased PPAR␤/␦ activity. These results clearly indicate that NF-B activity regulates the binding activity of PPAR␤/␦ to its cis-regulatory element.
NF-B Activation Enhances the Interaction of p65 with PPAR␤/␦-The reduction in the DNA binding activity of PPAR␤/␦ after LPS treatment may result from different molecular mechanisms. First, these changes may be caused by a reduction in the expression of PPAR␤/␦. However, this possibility is unlikely because no significant changes were observed in the protein expression of either PPAR␤/␦ or the p65 subunit of NF-B after LPS stimulation (Fig. 4A). In addition, the p65 subunit of NF-B may interact physically with PPARs. This association has been described for PPAR␣ and prevents this nuclear receptor from binding to its response element and thereby inhibits its ability to induce gene transcription (12). It is not known yet if a similar mechanism may affect PPAR␤/␦ in cardiac cells. To evaluate this possibility we performed coimmunoprecipitation studies with isolated nuclear extracts using antibodies against the p65 subunits of NF-B and PPAR␤/␦. Data shown in Fig. 4B demonstrate that LPS stimulation strongly enhanced the physical interaction between p65 and PPAR␤/␦, suggesting that increased association between these two proteins is the mechanism through which PPAR␤/␦ activity is reduced after LPS stimulation.
Enhanced p65-PPAR␤/␦ Interaction in Pressure Overloadinduced Cardiac Hypertrophy-Finally, we wanted to evaluate whether the mechanism proposed was also observed in vivo in pressure overload-induced cardiac hypertrophy. Cardiac hypertrophy was assessed by measuring the heart weight/body weight ratio. This parameter significantly increased (1.35-fold, p Ͻ 0.001) after aortic constriction compared with sham-operated rats (Fig. 5A). Furthermore, pressure overload also led to ϳ2-fold induction in the mRNA levels of Anf compared with sham-operated rats (data not shown). In the heart of banded rats Pdk4 mRNA levels were down-regulated by 35% (p Ͻ 0.01) compared with sham-operated rats (Fig. 5B), which is in agreement with the reduction in fatty acid oxidation during the development of cardiac hypertrophy (3,4). We also performed EMSA to demonstrate NF-B activation in pressure overloadinduced cardiac hypertrophy. These studies showed that the NF-B probe formed four main specific complexes with cardiac nuclear proteins (Fig. 5C, complexes I to IV), based on competition experiments performed by adding an excess of unlabeled NF-B oligonucleotide to incubation mixtures. NF-B binding activity increased in banded rats, especially complex III, compared with sham-operated rats. Characterization of NF-B was performed by incubating nuclear extracts with an antibody directed against the p65 subunit of this transcription factor (Fig. 5D). No changes were observed in the DNA binding of cardiac nuclear proteins to an Oct-1 probe, indicating that the increase observed for the NF-B probe was specific (Fig. 5E). Finally, we evaluated whether the impairment in the expression of PPAR␤/␦ target gene Pdk4 observed during cardiac hypertrophy may result from enhanced p65-PPAR␤/␦ interaction. Nuclear extracts isolated from hearts were immunoprecipitated using anti-p65 antibody coupled to protein A-agarose beads. Immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-PPAR␤/␦ antibody. Data shown in Fig. 5F demonstrate that pressure overload-induced cardiac hypertrophy enhanced the physical interaction of p65 with PPAR␤/␦, suggesting that increased association between these proteins is a mechanism contributing to the reported reduction in the expression of PPAR␤/␦ target genes involved in fatty acid metabolism. DISCUSSION Fuel generation in the adult myocardium relies on the oxidation of long chain fatty acids by the mitochondria for production of energy. Development of cardiac hypertrophy is associated with a suppression of fatty acid oxidation and metabolic reversion of the heart toward increased glucose utilization, which is characteristic of fetal heart (3,4). It is unclear at present, however, which consequences might result from impaired oxidation of fatty acids in the heart, but several studies have demonstrated that substrate utilization is important in the pathogenesis of cardiac hypertrophy (5)(6)(7). In the present study we demonstrate that the NF-B signaling pathway, which is one of the most important signal transduction pathways involved in the hypertrophic growth of the myocardium, may also be involved in the down-regulation of fatty acid oxidation. Therefore, inhibition of NF-B activation during cardiac hypertrophy may also ameliorate cardiac fatty acid oxidation, achieving a better improvement in the prevention or inhibition of this pathological process. Furthermore, these data may also contribute to explain how, given the complexity of the hypertrophic response, inhibition of NF-B may be sufficient to prevent cardiac hypertrophy (29). Barger et al. (30) demonstrated that during hypertrophic growth cardiac PPAR␣ gene expression fell and its activity was altered at the post-transcriptional level via the extracellular signal-regulated kinase mitogen-activated protein kinase pathway. However, nothing was known until now about the effects of cardiac hypertrophy on PPAR␤/␦ activity. Gilde et al. (17) recently suggested that PPAR␤/␦ plays an important function in the heart. In fact, these authors show that both PPAR␣ and PPAR␤/␦ were expressed in comparable levels in the heart, whereas PPAR␥ was barely detectable. Furthermore, PPAR␤/␦ was fatty acid inducible and activated the expression of PPAR␣ target genes involved in fatty acid utilization in cardiac myocytes. The authors of this study suggested that PPAR␣ and PPAR␤/␦ shared similar functions in cardiac cells regarding cardiac fatty acid metabolism. In agreement with this idea, Muoio et al. (31) have shown that fatty acid oxidation in skeletal muscle of PPAR␣ Ϫ/Ϫ mice was not impaired, probably because of PPAR␤/␦ compensated for the lack of PPAR␣ in these mice.
In the present study stimulation of rat neonatal cardiomyocytes with PE, which leads to NF-B activation (14), caused cardiac hypertrophy that was accompanied by a fall in the expression of Pdk4, a PPAR␤/␦ target gene involved in fatty acid metabolism (22). Furthermore, the fall in fatty acid oxidation observed in PE-stimulated rat neonatal cardiomyocytes was restored by NF-B inhibitors. These data pointed to the involvement of NF-B in the down-regulation of fatty acid oxidation during the development of cardiac hypertrophy. However, given that both PPAR␣ and PPAR␤/␦ are expressed in rat neonatal cardiomyocytes (17), this could lead to a potential interference caused by PPAR␣ in the changes observed. This possibility was avoided by using H9c2 myotubes, which only express the PPAR␤/␦ subtype (17). Stimulation of H9c2 myotubes with LPS, which has been reported to activate NF-B in cardiomyocytes (28), also lead to the reduction in the expression of Pdk4, supporting a link between NF-B activation and the reduced expression of PPAR␤/␦ target genes involved in fatty acid oxidation. All these findings suggest that the impairment of PPAR␤/␦ activity plays an important role in the fall of myocardial fatty acid oxidation during cardiac hypertrophy. In agreement with this idea, a recent study demonstrated that cardiomyocyterestricted PPAR␤/␦ deletion reduced myocardial fatty acid oxidation and the mRNA expression of genes involved in this process, such as Pdk4, and led to cardiomyopathy (32).
The mechanism by which activation of NF-B results in reduced expression of PPAR␤/␦ target genes seems to involve reduced interaction of this PPAR subtype with its cis-regulatory element, because exposure of cardiac cells to LPS caused a dramatic reduction in the binding of PPAR␤/␦ protein to the PPRE probe. This reduction was partially reversed by coincubation of the cells with NF-B inhibitors, confirming the involvement of this transcription factor in the changes observed. Therefore, the reduced binding activity of PPAR␤/␦ seemed to be related to the activation of NF-B in cardiac cells. However, it remained to establish through which mechanism NF-B activation avoided the interaction of PPAR␤/␦ with its response element. NF-B is present in the cytoplasm as an inactive heterodimer, consisting of the p50 and p65 subunits. However, after activation this heterodimer translocates to the nucleus and regulates the expression of genes involved in inflammatory and immune processes. Our results indicate that once the p65 subunit of NF-B reaches the nucleus it interacts with PPAR␤/␦. This association prevents PPAR␤/␦ from binding to its response element and thereby inhibits its ability to induce gene transcription, leading to a reduction in the expression of Pdk4. These findings are in concordance with the results reported by Westergaard et al. (33), who showed that PPAR␤/␦ physically interacts with p65 in psoriatic lesions. Furthermore, they showed a p65-dependent repression of PPAR␤/␦, but not of PPAR␣ or PPAR␥.
In summary, in the present study we show that the NF-B signaling pathway, which plays a pivotal role in the hyper-trophic growth of the myocardium, down-regulates PPAR␤/␦ activity in vitro and in vivo through a mechanism that involves enhanced protein-protein interaction of the p65 subunit of NF-B with this PPAR subtype. These data link NF-B activation during the development of cardiac hypertrophy with the fall in fatty acid oxidation.