Marc van Bilsen

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.

Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) involves liver lipid accumulation (steatosis) combined with hepatic inflammation. The transition towards hepatic inflammation represents a key step in pathogenesis, because it will set the stage for further liver damage, culminating in hepatic fibrosis, cirrhosis, and liver cancer. The actual risk factors that drive hepatic inflammation during the progression to NASH remain largely unknown. The role of steatosis and dietary cholesterol in the etiology of diet‐induced NASH was investigated using hyperlipidemic mouse models fed a Western diet. Livers of male and female hyperlipidemic (low‐density lipoprotein receptor–deficient [ldlr−/−] and apolipoprotein E2 knock‐in [APOE2ki]) mouse models were compared with livers of normolipidemic wild‐type (WT) C57BL/6J mice after short‐term feeding with a high‐fat diet with cholesterol (HFC) and without cholesterol. Whereas WT mice displayed only steatosis after a short‐term HFC diet, female ldlr−/− and APOE2ki mice showed steatosis with severe inflammation characterized by infiltration of macrophages and increased nuclear factor κB (NF‐κB) signaling. Remarkably, male ldlr−/− and APOE2ki mice developed severe hepatic inflammation in the absence of steatosis after 7 days on an HFC diet compared with WT animals. An HFC diet induced bloated, “foamy” Kupffer cells in male and female ldlr−/− and APOE2ki mice. Hepatic inflammation was found to be linked to increased plasma very low‐density lipoprotein (VLDL) cholesterol levels. Omitting cholesterol from the HFC diet lowered plasma VLDL cholesterol and prevented the development of inflammation and hepatic foam cells. Conclusion: These findings indicate that dietary cholesterol, possibly in the form of modified plasma lipoproteins, is an important risk factor for the progression to hepatic inflammation in diet‐induced NASH. (HEPATOLOGY 2008;48:474–486.)

Inflammation as a therapeutic target in heart failure? A scientific statement from the Translational Research Committee of the Heart Failure Association of the European Society of Cardiology

The increasing prevalence of heart failure poses enormous challenges for health care systems worldwide. Despite effective medical interventions that target neurohumoral activation, mortality and morbidity remain substantial. Evidence for inflammatory activation as an important pathway in disease progression in chronic heart failure has emerged in the last two decades. However, clinical trials of ‘anti‐inflammatory’ therapies (such as anti‐tumor necrosis factor‐α approaches) have to date failed to show benefit in heart failure patients. The Heart Failure Association of the European Society of Cardiology recently organized an expert workshop to address the issue of inflammation in heart failure from a basic science, translational and clinical perspective, and to assess whether specific inflammatory pathways may yet serve as novel therapeutic targets for this condition. This consensus document represents the outcome of the workshop and defines key research questions that still need to be addressed as well as considering the requirements for future clinical trials in this area.

Cardiac fatty acid uptake and transport in health and disease.

Fatty acids are important energy donors for the healthy heart. These substrates are supplied to the myocardium bound to albumin to overcome their low solubility in aqueous solutions such as blood plasma. Transport from the microvascular compartment to the mitochondria inside the cardiomyocytes is most likely a combination of passive and protein-mediated diffusion. Alterations in tissue content of fatty acid-transport proteins may contribute to myocardial diseases such as the diabetic heart, and cardiac hypertrophy and failure.

Metabolic remodelling of the failing heart: beneficial or detrimental?

The failing heart is characterized by alterations in energy metabolism, including mitochondrial dysfunction and a reduction in fatty acid (FA) oxidation rate, which is partially compensated by an increase in glucose utilization. Together, these changes lead to an impaired capacity to convert chemical energy into mechanical work. This has led to the concept that supporting cardiac energy conversion through metabolic interventions provides an important adjuvant therapy for heart failure. The potential success of such a therapy depends on whether the shift from FA towards glucose utilization should be considered beneficial or detrimental, a question still incompletely resolved. In this review, the current status of the literature is evaluated and possible causes of observed discrepancies are discussed. It is cautiously concluded that for the failing heart, from a therapeutic point of view, it is preferable to further stimulate glucose oxidation rather than to normalize substrate metabolism by stimulating FA utilization. Whether this also applies to the pre-stages of cardiac failure remains to be established.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Uncoupling protein 3 as a mitochondrial fatty acid anion exporter

In contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is related to cellular long‐chain fatty acid homeostasis. If long‐chain fatty acids enter the mitochondrial matrix in their non‐esterified form, they cannot be metabolized and may exert deleterious effects. To test the feasibility that UCP3 exports fatty acid anions, we systematically interfered at distinct steps in the fatty acid metabolism pathway, thereby creating conditions in which the entry of (non‐esterified) fatty acids into the mitochondrial matrix is enhanced. First, reducing the cellular fatty acid binding capacity, known to increase cytosolic concentrations of non‐esterified fatty acids, up‐regulated UCP3 5.3‐fold. Second, inhibition of mitochondrial entry of esterified long‐chain fatty acids up‐ regulated UCP3 by 1.9‐fold. Third, high‐fat diets, to increase mitochondrial supply of non‐ esterified long‐chain fatty acids exceeding oxidative capacity, up‐regulated UCP3 twofold. However, feeding a similar amount of medium‐chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore do not need to be exported from the matrix, did not affect UCP3 protein levels. These data are compatible with a physiological function of UCP3 in facilitating outward transport of long‐chain fatty acid anions, which cannot be oxidized, from the mitochondrial matrix.

MCIP1 overexpression suppresses left ventricular remodeling and sustains cardiac function after myocardial infarction

Pathological remodeling of the left ventricle (LV) after myocardial infarction (MI) is a major cause of heart failure. Although cardiac hypertrophy after increased loading conditions has been recognized as a clinical risk factor for human heart failure, it is unknown whether post-MI hypertrophic remodeling of the myocardium is beneficial for cardiac function over time, nor which regulatory pathways play a crucial role in this process. To address these questions, transgenic (TG) mice engineered to overexpress modulatory calcineurin-interacting protein-1 (MCIP1) in the myocardium were used to achieve cardiac-specific inhibition of calcineurin activation. MCIP1-TG mice and their wild-type (WT) littermates, were subjected to MI and analyzed 4 weeks later. At 4 weeks after MI, calcineurin was activated in the LV of WT mice, which was significantly reduced in MCIP1-TG mice. WT mice displayed a 78% increase in LV mass after MI, which was reduced by 38% in MCIP1-TG mice. Echocardiography indicated marked LV dilation and loss of systolic function in WT-MI mice, whereas TG-MI mice displayed a remarkable preservation of LV geometry and contractility, a pronounced reduction in myofiber hypertrophy, collagen deposition, and &bgr;-MHC expression compared with WT-MI mice. Together, these results reveal a protective role for MCIP1 in the post-MI heart and suggest that calcineurin is a crucial regulator of postinfarction-induced pathological LV remodeling. The improvement in functional, structural, and molecular abnormalities in MCIP1-TG mice challenges the adaptive value of post-MI hypertrophy of the remote myocardium. The full text of this article is available online at http://circres.ahajournals.org.

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism; intervention; and long-term consequences.

Background Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

17β-Estradiol Antagonizes Cardiomyocyte Hypertrophy by Autocrine/Paracrine Stimulation of a Guanylyl Cyclase A Receptor-Cyclic Guanosine Monophosphate-Dependent Protein Kinase Pathway

Background—Significant gender-related differences exist in the development of left ventricular hypertrophy (LVH). In addition, administration of 17&bgr;-estradiol (E2) to ovariectomized female mice attenuates the development of LVH, demonstrating an antagonistic role for E2 in this process, although no molecular mechanism has been proposed for this phenomenon. Methods and Results—E2 attenuated phenylephrine and endothelin-1 induced hypertrophy in neonatal cardiomyocytes, and E2 directly induced atrial natriuretic factor (ANF) expression as assessed by Northern blot, immunocytochemical analyses, and transient transfection assays using ANF promoter deletion fragments. Both the antihypertrophic effects and ANF induction could be blocked by the estrogen receptor antagonist ICI 182,780, which demonstrates a genomic, estrogen receptor-dependent pathway. To mimic E2-induced autocrine/paracrine effects through stimulation of the guanylyl cyclase A receptor (ANF receptor), cardiomyocytes were stimulated with phenylephrine or endothelin-1 in the presence of exogenous ANF or 8-bromo-cyclic guanosine monophosphate (cGMP), both of which attenuated agonist-induced hypertrophy. Both estrogen and ANF increased cGMP activity. The antihypertrophic effect of ANF could be reduced with extracellular ANF antibodies in a dose-dependent manner. cGMP-dependent protein kinase mediates the antihypertrophic effects of E2, so cardiomyocytes were agonist stimulated in the presence of the cGMP-dependent protein kinase blocker KT-5823. KT-5823 not only reversed the antihypertrophic properties of E2, ANF, or 8-bromo-cGMP, but also evoked potentiation of hypertrophy. Conclusions—E2-mediated induction of ANF in cardiac hypertrophy contributes to its antagonistic effects in LVH.