Thierry Pedrazzini

Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor.

Adenosine is released from metabolically active cells by facilitated diffusion, and is generated extracellularly by degradation of released ATP. It is a potent biological mediator that modulates the activity of numerous cell types, including various neuronal populations, platelets, neutrophils and mast cells, and smooth muscle cells in bronchi and vasculature. Most of these effects help to protect cells and tissues during stress conditions such as ischaemia. Adenosine mediates its effects through four receptor subtypes: the A1, A2a, A2b and A3 receptors. The A2a receptor (A2aR), is abundant in basal ganglia, vasculature and platelets, and stimulates adenylyl cyclase. It is a major target of caffeine, the most widely used psychoactive drug. Here we investigate the role of the A2a receptor by disrupting the gene in mice. We found that A2aR-knockout (A2aR−/−) mice were viable and bred normally. Their exploratory activity was reduced, whereas caffeine, which normally stimulates exploratory behaviour, became a depressant of exploratory activity. Knockout animals scored higher in anxiety tests, and male mice were much more aggressive towards intruders. The response of A2aR−/−mice to acute pain stimuli was slower. Blood pressure and heart rate were increased, as well as platelet aggregation. The specific A2a agonist CGS 21680 lost its biological activity in all systems tested.

Insulin Resistance, Hyperlipidemia, and Hypertension in Mice Lacking Endothelial Nitric Oxide Synthase

Background—Insulin resistance and arterial hypertension are related, but the underlying mechanism is unknown. Endothelial nitric oxide synthase (eNOS) is expressed in skeletal muscle, where it may govern metabolic processes, and in the vascular endothelium, where it regulates arterial pressure. Methods and Results—To study the role of eNOS in the control of the metabolic action of insulin, we assessed insulin sensitivity in conscious mice with disruption of the gene encoding for eNOS. eNOS−/− mice were hypertensive and had fasting hyperinsulinemia, hyperlipidemia, and a 40% lower insulin-stimulated glucose uptake than control mice. Insulin resistance in eNOS−/− mice was related specifically to impaired NO synthesis, because in equally hypertensive 1-kidney/1-clip mice (a model of renovascular hypertension), insulin-stimulated glucose uptake was normal. Conclusions—These results indicate that eNOS is important for the control not only of arterial pressure but also of glucose and lipid homeostasis. A single gene defect, eNOS deficiency, may represent the link between metabolic and cardiovascular disease.

Neuropeptide Y: the universal soldier.

Abstract. The peptidic neurotransmitter neuropeptide Y (NPY) has received great attention because it has been implicated in the regulation of several organ systems. In particular, NPY is involved in the regulatory loops that control food intake in the hypothalamus and appears also to be important for regulating the activity of neuroendocrine axes under poor metabolic conditions. Furthermore, NPY exerts vasoconstrictive action on the vasculature and potentiates the actions of many other vasoconstrictors. In addition, it was demonstrated to have trophic properties and could therefore contribute to cardiovascular remodeling. These various effects plus a number of others make NPY an attractive target for the potential treatment of human diseases, such as obesity, metabolic disorders, hypertension and heart failure.

Extracellular Superoxide Dismutase Is a Major Determinant of Nitric Oxide Bioavailability In Vivo and Ex Vivo Evidence From ecSOD-Deficient Mice

Abstract— The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O2·−). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O2·− is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II–induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD+/+ and ecSOD−/− mice. After 6 days of angiotensin II–treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD−/− than ecSOD+/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD−/− mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD+/+ mice. Compared with sham-operated ecSOD+/+ mice, sham-operated ecSOD−/− mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O2·−, as measured by lucigenin chemiluminescence, was higher in ecSOD−/− compared with ecSOD+/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD−/−, as well as from clipped ecSOD+/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD−/− mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O2·−, controlling blood pressure and vascular function in angiotensin II–dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Blood Pressure–Independent Cardiac Hypertrophy Induced by Locally Activated Renin-Angiotensin System

Cardiac hypertrophy is frequent in chronic hypertension. The renin-angiotensin system, via its effector angiotensin II (Ang II), regulates blood pressure and participates in sustaining hypertension. In addition, a growing body of evidence indicates that Ang II acts also as a growth factor. However, it is still a matter of debate whether the trophic effect of Ang II can trigger cardiac hypertrophy in the absence of elevated blood pressure. To address this question, transgenic mice overexpressing the rat angiotensinogen gene, specifically in the heart, were generated to increase the local activity of the renin-angiotensin system and therefore Ang II production. These mice develop myocardial hypertrophy without signs of fibrosis independently from the presence of hypertension, demonstrating that local Ang II production is important in mediating the hypertrophic response in vivo.

FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.

Cardiac Sodium Channel Nav1.5 Is Regulated by a Multiprotein Complex Composed of Syntrophins and Dystrophin

The cardiac sodium channel Nav1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Nav1.5 C-terminus. Pull-down experiments were performed using Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx5cv mice to study the role of this protein complex in Nav1.5 function. Western blot experiments revealed a 50% decrease in the Nav1.5 protein levels in mdx5cv hearts, whereas Nav1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx5cv cardiomyocytes. Finally, ECG measurements of the mdx5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Nav1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.

Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.

Induction of Cardiogenesis in Embryonic Stem Cells via Downregulation of Notch1 Signaling

Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain- and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes.