P. H. M. Willemsen

Peroxisome Proliferator-Activated Receptor (PPAR) α and PPARβ/δ, but not PPARγ, Modulate the Expression of Genes Involved in Cardiac Lipid Metabolism

Abstract— 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&agr; in FA-mediated expression has been demonstrated, the role of the PPAR&bgr;/&dgr; and PPAR&ggr; 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&agr; (Wy-14,643), PPAR&bgr;/&dgr; (L-165041, GW501516), or PPAR&ggr; (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&agr; and PPAR&bgr;/&dgr; ligands, but not to PPAR&ggr; ligands. Likewise, the FA-mediated expression of FA-handling proteins was mimicked by PPAR&agr; and PPAR&bgr;/&dgr;, but not by PPAR&ggr; ligands. As expected, in embryonic rat heart-derived H9c2 cells, which only express PPAR&bgr;/&dgr;, the FA-induced expression of genes was mimicked by the PPAR&bgr;/&dgr; ligand only, indicating that FA also act as ligands for the PPAR&bgr;/&dgr; isoform. In cardiomyocytes, MCPT-1 promoter activity was unresponsive to PPAR&ggr; ligands. However, addition of PPAR&agr; and PPAR&bgr;/&dgr; ligands dose-dependently induced promoter activity. Collectively, the present findings demonstrate that, next to PPAR&agr;, PPAR&bgr;/&dgr;, but not PPAR&ggr;, plays a prominent role in the regulation of cardiac lipid metabolism, thereby warranting further research into the role of PPAR&bgr;/&dgr; in cardiac disease.

Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage.

Disturbances in lipid metabolism may play an important role in the onset of irreversible myocardial damage. To investigate the effect of ischemia and reperfusion on lipid homeostasis and to delineate its possible consequences for myocardial damage, Krebs-Henseleit-perfused, working rat hearts were subjected to various periods of no-flow ischemia (10 to 90 minutes) with or without 30 minutes of reperfusion. During ischemia, the rise in nonesterified fatty acids (NEFAs) was preceded by the accumulation of substantial amounts of glycerol, indicating the presence of an active triacylglycerol-NEFA cycle. The subsequent rise in NEFAs (from 0.25 to 1.64 μmol/g dry residue wt after 90 minutes [means]) coincided with the reduction of ATP to values lower than 10 μmol/g dry wt and the rise of AMP, a potent inhibitor of acyl-coenzyme A synthetase, to values exceeding 2 μmol/g dry wt, making the latter compound a good candidate to hamper the turnover of endogenous lipids during prolonged ischemia. Reperfusion resulted in an additional rise in NEFAs (up to 4.1 μmol/g dry residue wt after 60 minutes of ischemia). Neither ischemia nor reperfusion resulted in significant decreases in the tissue content of triacylglycerols and the various phospholipids. During reperfusion recovery of stroke volume was still adequate at tissue NEFA levels thought to be incompatible with normal mitochondrial function.37 A positive correlation (r=0.81) was found between NEFA content of reperfused hearts and cumulative release of lactate dehydrogenase during reperfusion. Accordingly it is concluded that 1) reperfusion results in additional changes in myocardial lipid homeostasis, 2) the accumulating NEFAs are compartmentalized, possibly at the cellular level, and 3) the accumulation of NEFAs is a sensitive marker for myocardial cell damage.

Inflammatory Pathways Are Activated during Cardiomyocyte Hypertrophy and Attenuated by Peroxisome Proliferator-activated Receptors PPARα and PPARδ

Accumulating evidence indicates an important role for inflammation in cardiac hypertrophy and failure. Peroxisome proliferator-activated receptors (PPARs) have been reported to attenuate inflammatory signaling pathways and, as such, may interfere with cardiac remodeling. Accordingly, the objectives of the present study were to explore the relationship between cardiomyocyte hypertrophy and inflammation and to investigate whether PPARα and PPARδ are able to inhibit NF-κB activation and, consequently, the hypertrophic growth response of neonatal rat cardiomyocytes (NCM). mRNA levels of markers of both hypertrophy and inflammation were increased following treatment with the pro-hypertrophic factor phenylephrine (PE) or the chemokine TNF-α. Induction of inflammatory genes was found to be fast (within 2 h after stimulation) and transient, while induction of hypertrophic marker genes was more gradual (peaking at 24–48 h). Inflammatory and hypertrophic pathways appeared to converge on NF-κB as both PE and TNF-α increased NF-κB binding activity as measured by electrophoretic mobility shift assay. Following transient transfection, the p65-induced transcriptional activation of a NF-κB reporter construct was significantly blunted after co-transfection of PPARα or PPARδ in the presence of their respective ligands. Finally, adenoviral overexpression of PPARα and PPARδ markedly attenuated cell enlargement and the expression of hypertrophic marker genes in PE-stimulated NCM. The collective findings reveal a close relationship between hypertrophic and inflammatory signaling pathways in the cardiomyocyte. It was shown that both PPARα and PPARδ are able to mitigate cardiomyocyte hypertrophy in vitro by inhibiting NF-κB activation.

Cardiac hypertrophy is enhanced in PPARα−/− mice in response to chronic pressure overload

AIMS Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPARalpha modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPARalpha aggravates the cardiac hypertrophic response to pressure overload. METHODS AND RESULTS Male PPARalpha-/- and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPARalpha-/- than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPARalpha-/- mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, alpha-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-alpha, cyclo-oxygenase-2) marker genes were higher in PPARalpha-/- than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPARalpha-/- mice, but were not further compromised by TAC. CONCLUSION The present findings show that the absence of PPARalpha results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPARalpha exerts salutary effects during cardiac hypertrophy.

Effects of fatty acids on uncoupling protein-2 expression in the rat heart

Fatty acids are thought to play a role in the activity of uncoupling proteins (UCP) and have been shown to regulate the expression of genes encoding proteins involved in fatty acid handling. Therefore, we investigated whether fatty acids, which are the main substrates for the heart, affect rat cardiac UCP‐2 expression in vivo and in vitro. After birth, when the contribution of fatty acid oxidation to cardiac energy conversion increases, UCP‐2 expression enhanced rapidly. In the adult heart, however, UCP‐2 mRNA levels did not alter during conditions associated with either enhanced (fasting, diabetes) or decreased (hypertrophy) fatty acid utilization. Exposure of neonatal cardiomyocytes and embryonic rat heart‐derived H9c2 cells to fatty acids (palmitic and oleic acid) for 48 h strongly induced UCP‐2 expression. Stimulation of neonatal cardio‐myocytes with triiodothyronine also increased UCP‐2 mRNA levels, though only in the presence of fatty acids. Ligands specific to the fatty acid‐activated transcription factor PPARa, but not to PPAR7, acted as inducers of cardiomyocyte UCP‐2 expression. It is concluded that fatty acids promote UCP‐2 expression in neonatal cardiomyocytes, which might explain the rapid increase in UCP‐2 mRNA in the postnatal heart. However, UCP‐2 mRNA levels in the adult heart appear to be insensitive to changes in cardiac fatty acid handling under various pathological conditions.—van der Lee, K. A. J. M., Willemsen, P. H. M., van der Vusse, G. J., van Bilsen, M. Effects of fatty acids on uncoupling protein‐2 expression in the rat heart. FASEB J. 14, 495–502 (2000)

Cardiac fatty acid metabolism is preserved in the compensated hypertrophic rat heart

Abstract Cardiac hypertrophy and failure are associated with alterations in cardiac substrate metabolism. It remains to be established, however, whether genomically driven changes in cardiac glucose and fatty acid (FA) metabolism represent a key event of the hypertrophic remodeling process. Accordingly, we investigated metabolic gene expression and substrate metabolism during compensatory hypertrophy, in relation to other cardiac remodeling processes.Thereto, cardiac hypertrophy was induced in rats by supra–renal aortic constriction to various degrees, resulting in increased heart/body weight ratios of 22% (Aob–1), 24% (Aob–2) and 32% (Aob–3) (p < 0.005) after 4 weeks. The unaltered ejection fraction in all groups indicated that the hypertrophy was still compensatory in nature. β–Myosin Heavy Chain protein and ANF mRNA levels were increased in all groups. Only in Aob–3 rats were SERCA2a mRNA levels markedly reduced. In this group, glycolytic capacity was modestly elevated (+ 25%; p < 0.01). Notwithstanding these phenotypical changes, the expression of genes involved in FA metabolism and FA oxidation rate in cardiac homogenates was completely preserved, irrespective of the degree of hypertrophy. These findings indicate that cardiac FA oxidative capacity is preserved during compensatory hypertrophy, and that a decline in metabolic gene expression does not represent a hallmark of the development of hypertrophy.

Co-expression in rat heart and skeletal muscle of four genes coding for proteins implicated in long-chain fatty acid uptake.

It has been suggested that specific membrane-associated and cytoplasmic proteins cooperate in the uptake of long-chain fatty acids by cardiac and skeletal muscle cells. A prerequisite for this hypothesis would be the co-occurrence of these proteins in muscle. Thus, we studied the possible co-expression in rat muscles of the genes coding for the integral membrane proteins fatty acid transport protein (FATP) and fatty acid translocase (FAT), the membrane-associated plasmalemmal fatty acid-binding protein (FABPpm) and the cytoplasmic heart-type fatty acid-binding protein (H-FABPc). The transcripts of the four proteins were assessed in heart and skeletal muscles of adult Wistar rats, in isolated cells and cell lines from rat heart and also in rat heart during development and upon streptozotocin-induced diabetes. All four genes showed high expression levels in heart, somewhat lower in red skeletal muscle (soleus) and appreciably lower in white skeletal muscle (extensor digitorum longus). FATP, FAT and H-FABPc showed a 3- to 5-fold increase in mRNA expression during maturational growth of the heart, while the FABPpm expression remained virtually constant. In the heart, streptozotocin-diabetes induced a slight, but statistically not significant, increase in the expression of all four genes. In conclusion, this study shows the co-expression of FATP, FAT, FABPpm and H-FABPc in rat muscles. This finding supports the possible cooperation of these proteins in the uptake of long-chain fatty acids by muscle cells.

Transcriptomic analysis of PPARalpha-dependent alterations during cardiac hypertrophy.

Peroxisome proliferator-activated receptor (PPAR)alpha regulates lipid metabolism at the transcriptional level and modulates the expression of genes involved in inflammation, cell proliferation, and differentiation. Although PPARalpha has been shown to mitigate cardiac hypertrophy, knowledge about underlying mechanisms and the nature of signaling pathways involved is fragmentary and incomplete. The aim of this study was to identify the processes and signaling pathways regulated by PPARalpha in hearts challenged by a chronic pressure overload by means of whole genome transcriptomic analysis. PPARalpha-/- and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days, and left ventricular gene expression profile was determined with Affymetrix GeneChip Mouse Genome 430 2.0 arrays containing >45,000 probe sets. In unchallenged hearts, the mere lack of PPARalpha resulted in 821 differentially expressed genes, many of which are related to lipid metabolism and immune response. TAC resulted in a more pronounced cardiac hypertrophy and more extensive changes in gene expression (1,910 and 312 differentially expressed genes, respectively) in PPARalpha-/- mice than in wild-type mice. Many of the hypertrophy-related genes were related to development, signal transduction, actin filament organization, and collagen synthesis. Compared with wild-type hypertrophied hearts, PPARalpha-/- hypertrophied hearts revealed enrichment of gene clusters related to extracellular matrix remodeling, immune response, oxidative stress, and inflammatory signaling pathways. The present study therefore demonstrates that, in addition to lipid metabolism, PPARalpha is an important modulator of immune and inflammatory response in cardiac muscle.

Stretch-induced hypertrophy of isolated adult rabbit cardiomyocytes

Both mechanical and humoral triggers have been put forward to explain the hypertrophic response of the challenged cardiomyocyte. The aim of the present study was to investigate whether cyclic equibiaxial stretch is a direct stimulus for isolated adult rabbit cardiomyocytes to develop hypertrophy and to explore the potential involvement of the autocrine/paracrine factors ANG II, transforming growth factor (TGF)-beta(1), and IGF-I in this process. Isolated cardiomyocytes were exposed to 10% cyclic equibiaxial stretch (1 Hz) for up to 48 h or treated with ANG II (100 nM), TGF-beta(1) (5 ng/ml), IGF-I (100 ng/ml), ANG II type 1 (AT(1)) receptor blockers, or conditioned medium of stretched fibroblasts. Cyclic stretch significantly increased cell surface area (+3.1%), protein synthesis (+21%), and brain natriuretic peptide (BNP) mRNA expression (6-fold) in cardiomyocytes. TGF-beta(1) expression increased (+42%) transiently at 4 h, whereas cardiomyocyte IGF-I expression was not detectable under all experimental conditions. The AT(1) receptor blockers candesartan and irbesartan (100 nM) did not prevent the stretch-induced hypertrophic response. Direct exposure to ANG II, TGF-beta(1), or IGF-I did not enhance cardiomyocyte BNP expression. In cardiac fibroblasts, stretch elicited a significant approximately twofold increase in TGF-beta(1) and IGF-I expression. Conditioned medium of stretched fibroblasts increased BNP expression in cardiomyocytes ( approximately 2-fold, P = 0.07). This study clearly indicates that cyclic stretch is a strong, direct trigger to induce hypertrophy in fully differentiated rabbit cardiomyocytes. The present findings do not support the notion that stretch-mediated hypertrophy of adult rabbit cardiomyocytes involves autocrine/paracrine actions of ANG II, TGF-beta(1), or IGF-I.

Lactate-induced stimulation of myocardial triacylglycerol turnover

The addition of lactate (5.6 mM) to a perfusion medium containing glucose (11 mM) stimulated the turnover of the cardiac triacylglycerol pool throughout the perfusion period as indicated by increased glycerol release in association with maintained levels of triacylglycerols. Attenuation of feedback inhibition of triacylglycerol lipase by fatty acids as a possible cause of the elevated triacylglycerol turnover rate should be ruled out, since tissue fatty acid levels were 3-times higher in glucose plus lactate perfused hearts than in hearts perfused with glucose as the sole substrate. The present findings are in favor of the notion that lactate enhances triacylglycerol turnover through increased glycerol 3-phosphate levels.