Christophe Montessuit

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.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism

OBJECTIVES Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Chemokine CCL5/RANTES inhibition reduces myocardial reperfusion injury in atherosclerotic mice

Although beneficial for cardiomyocyte salvage and to limit myocardial damage and cardiac dysfunction, restoration of blood flow after prolonged ischemia exacerbates myocardial injuries. Several deleterious processes that contribute to cardiomyocyte death have been proposed, including massive release of reactive oxygen species, calcium overload and hypercontracture development or leukocyte infiltration within the damaged myocardium. Chemokines are known to enhance leukocyte diapedesis at inflammatory sites. The aim of the present study was to investigate the effect of chemokine CCL5/RANTES antagonism in an in vivo mouse model of ischemia and reperfusion. ApoE(-/-) mice were submitted to 30 min ischemia, by ligature of the left coronary artery, followed by 24 h reperfusion. Intraperitoneal injection of 10 mug of CCL5/RANTES antagonist [(44)AANA(47)]-RANTES, 5 min prior to reperfusion, reduced infarct size as well as Troponin I serum levels compared to PBS-treated mice. This beneficial effect of [(44)AANA(47)]-RANTES treatment was associated with reduced leukocyte infiltration into the reperfused myocardium, as well as decreased chemokines Ccl2/Mcp-1 and Ccl3/Mip-1alpha expression, oxidative stress, and apoptosis. However, mice deficient for the CCL5/RANTES receptor Ccr5 did not exhibit myocardium salvage in our model of ischemia-reperfusion. Furthermore, [(44)AANA(47)]-RANTES did not mediate cardioprotection in these ApoE(-/-) Ccr5(-/-) deficient mice, probably due to enhanced expression of compensatory chemokines. This study provides the first evidence that inhibition of CCL5/RANTES exerts cardioprotective effects during early myocardial reperfusion, through its anti-inflammatory properties. Our findings indicate that blocking chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes.

Forkhead Transcription Factors Coordinate Expression of Myocardial KATP Channel Subunits and Energy Metabolism

Coordinate adaptation of myocyte metabolism and function is fundamental to survival of the stressed heart, but the mechanisms for this coordination remain unclear. Bioinformatics led us to discover that Foxs are key transcription factors involved. We performed experiments on the mouse atrial cell line HL-1, neonate rat heart myocytes, and an adult rat model of myocardial infarction. In electrophoretic mobility-shift assays, FoxO1 binds to the FoxO concensus site of the KATP channel subunit KIR6.1 promoter. In primary atrial culture, targeting FoxO1 and FoxO3 with siRNA specifically reduces mRNA expression of FoxO1 and -O3 and KIR6.1. Western blots, confocal immunofluorescence, and quantitative RT-PCR was applied for measuring expression of 10 Fox, 6 KATP channel subunits, and 12 metabolic genes. FoxF2, -O1, and -O3 strongly associate with expression of KATP channel subunits (in particular, KIR6.1, SUR1A and SUR2B) in different heart tissues and in the periinfarct zone of the left ventricle. Patch-clamp recordings demonstrate that molecular plasticity of these channels is matched by pharmacological plasticity and increased sensitivity to a metabolic challenge mimicked by the protonophore CCCP. A balance of FoxF2 and FoxO also regulates expression of at least 9 metabolic genes involved in setting the balance of glycolysis and &bgr;-oxidation. Bioinformatics shows that the transcriptional mechanisms are highly conserved among chicken, mouse, rat, and human, and Fox are intimately linked to other metabolic sensors. Thus, FoxF2 and -O are key transcription factors coordinating expression of KATP channels and energy metabolism.

Role of NADPH oxidase isoforms NOX1, NOX2 and NOX4 in myocardial ischemia/reperfusion injury.

Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia-reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease.

Inhibition of Nicotinamide Phosphoribosyltransferase Reduces Neutrophil-Mediated Injury in Myocardial Infarction

AIMS Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. RESULTS Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD(+)-dependent deacetylase in the production of this chemokine. INNOVATION The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. CONCLUSIONS Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice.

Single Administration of the CXC Chemokine-Binding Protein Evasin-3 During Ischemia Prevents Myocardial Reperfusion Injury in Mice

Objective—Evasins (chemokine-binding proteins) have been shown to selectively neutralize chemokine bioactivity. We investigated the potential benefits of Evasin-3 on mouse myocardial ischemia/reperfusion injury. Methods and Results—In vivo and ex vivo (Langendorff model) left coronary artery ligature was performed in C57Bl/6 mice. Coronary occlusion was maintained for 30 minutes, followed by different times (up to 24 hours) of reperfusion. Five minutes after coronary occlusion, mice received 1 intraperitoneal injection of Evasin-3 or vehicle. Infarct size was assessed histologically and by serum cardiac troponin I ELISA. In vitro neutrophil chemotaxis, immunohistology, oxidative stress quantification, real-time RT-PCR analysis of leukocyte chemoattractants, and Western blots for cardioprotective intracellular pathway activation were performed. Evasin-3 reduced infarct size and cardiac troponin I levels compared with vehicle. This effect was associated with the reduction of neutrophil infiltration and reactive oxygen species production within the infarcted myocardium. Evasin-3 did not reduce infarct size in the absence of circulating neutrophils (Langendorff model). Evasin-3 did not influence the activation of intracellular cardioprotective pathways or the expression of leukocyte chemoattractants during early phases of reperfusion. Conclusion—Single administration of Evasin-3 during myocardial ischemia significantly reduced infarct size by preventing CXC chemokine-induced neutrophil recruitment and reactive oxygen species production in myocardial ischemia/reperfusion.

Angiotensin II downregulates the fatty acid oxidation pathway in adult rat cardiomyocytes via release of tumour necrosis factor-α

AIMS Advanced heart failure is often associated with reduced myocardial fatty acid oxidation capacity. We have previously observed that failing hearts of mice with overexpression of angiotensinogen in the myocardium exhibit marked reduction of key regulatory proteins of fatty acid oxidation. In the present study, we determined whether exposure of adult rat cardiac (ARC) myocytes to angiotensin II (Ang II) influences expression of fatty acid translocase, muscle-type carnitine palmitoyl transferase-I, and medium-chain acyl-CoA dehydrogenase. METHODS AND RESULTS Ang II reduced mRNA expression of the three regulatory proteins in ARC myocytes during the entire 14-days culture period. However, protein expression and palmitate oxidation rate remained unaltered for 7 days, but subsequently markedly decreased. The decrease of protein expression and of fatty acid oxidation coincided with the onset of increased protein expression of tumour necrosis factor-alpha (TNF-alpha). The effect of Ang II was completely abolished by either blocking TNF-alpha formation through inhibition of reactive oxygen species-mediated activation of nuclear factor-kappaB or by neutralizing TNF-alpha with a specific antibody. Activation of peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARbeta/delta counteracted Ang II-mediated reduction of the fatty acid oxidation pathway. CONCLUSION Prolonged exposure of cardiac myocytes to Ang II elicits downregulation of the fatty acid oxidation pathway mediated by enhanced synthesis of TNF-alpha.

Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation

Myocardium undergoing remodeling in vivo exhibits insulin resistance that has been attributed to a shift from the insulin‐sensitive glucose transporter GLUT4 to the fetal, less insulin‐sensitive, isoform GLUT1. To elucidate the role of altered GLUT4 expression in myocardial insulin resistance, glucose uptake and the expression of the glucose transporter isoforms GLUT4 and GLUT1 were measured in adult rat cardiomyocytes (ARC). ARC in culture spontaneously undergo dedifferentiation, hypertrophy‐like spreading, and return to a fetal‐like gene expression pattern. Insulin stimulation of 2‐deoxy‐D‐glucose uptake was completely abolished on day 2 and 3 of culture and recovered thereafter. Although GLUT4 protein level was reduced, the time‐ course of unresponsiveness to insulin did not correlate with altered expression of GLUT1 and GLUT4. However, translocation of GLUT4 to the sarcolemma in response to insulin was completely abolished during transient insulin resistance. Insulin‐mediated phosphorylation of Akt was not reduced, indicating that activation of phosphatidylinositol 3‐kinase (PI3K) was preserved. On the other hand, total and phosphorylated Cbl was reduced during insulin resistance, suggesting that activation of Cbl/CAP is essential for insulin‐mediated GLUT4 translocation, in addition to activation of PI3K. Pharmacological inhibition of contraction in insulin‐sensitive ARC reduced insulin sensitivity and lowered phosphorylated Cbl. The results suggest that transient insulin resistance in ARC is related to impairment of GLUT4 translocation. A defect in the PI3K‐independent insulin signaling pathway involving Cbl seems to contribute to reduced insulin responsiveness and may be related to contractile arrest.

Regulation and dysregulation of glucose transport in cardiomyocytes

The ability of the heart muscle to derive energy from a wide variety of substrates provides the myocardium with remarkable capacity to adapt to the ever-changing metabolic environment depending on factors including nutritional state and physical activity. There is increasing evidence that loss of metabolic flexibility of the myocardium contributes to cardiac dysfunction in disease conditions such as diabetes, ischemic heart disease and heart failure. At the level of glucose metabolism reduced metabolic adaptation in most cases is characterized by impaired stimulation of transarcolemmal glucose transport in the cardiomyocytes in response to insulin, referred to as insulin resistance, or to other stimuli such as energy deficiency. This review discusses cellular mechanisms involved in the regulation of glucose uptake in cardiomyocytes and their potential implication in impairment of stimulation of glucose transport under disease conditions. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.