Irène Papageorgiou

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.

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.

Myocardial fatty acid oxidation during ischemia and reperfusion

Inhibition of fatty acid oxidation is an early event in myocardial ischemia that most likely contributes to tissue injury by the accumulation of potentially toxic intermediates such as acylCoA and acylcarnitine. After reperfusion both myocardial oxygen consumption and fatty acid oxidation may rapidly recover to preischemic levels, even when contractile function remains depressed. The mechanisms underlying the apparent dissociation between contractile function and oxidative metabolism early during reperfusion are still controversial. In isolated rat hearts subjected to 60 min of no-flow ischemia myocardial oxygen consumption and oxidation of palmitate were lowered during reperfusion by 3 mM of NiCl2 and by 6 μM of ruthenium red. The results provide indirect evidence for the hypothesis that intracellular calcium transport may be involved in the mechanisms responsible for the high oxidative metabolic rate early after reperfusion. (Mol Cell Biochem 116: 103–109, 1992)

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.

Epinephrine-stimulated contractile and metabolic reserve in postischemic rat myocardium

SummaryRecovery of contractilc function and of fatty acid oxidation may be delayed in viable postischemic myocardium. To determine whether a metabolic reserve is preserved after reperfusion of reversibly injured myocardium, we studied the effect of epinephrine on myocardial fatty acid oxidation in isolated rat hearts perfused retrogradely with crythrocyte enriched buffer containing albumin 0.4 mM, palmitate 0.4 mM, and glucose 11 mM. Hearts were subjected to 60 min of low-flow ischemia (5% of control flow) followed by 60 min of reperfusion. Five minutes following the onset of reperfusion, developed left ventricular pressure (DLVP) and oxidation of palmitate were reduced to 53% (p<0.01) and 46% (p<0.01), respectively, of values measured in nonischemic control hearts. Subsequently, DLVP and oxidation of palmitate gradually recovered to 78% (NS) and 91% (NS) by 60 min of reperfusion. Epinephrine 5·10−4M elicited an immediate stimulation of both contractile function and palmitate oxidation. Early after reperfusion stimulated DLVP and palmitate oxidation were still lower compared to values measured in control hearts exposed to the same concentration of epinephrine. Later than 15 min after the onset of reperfusion the response of DLVP and of palmitate oxidation to epinephrine no longer differed between control and reperfused hearts.These results indicate that viable postischemic myocardium exhibits a remarkable oxidative metabolic reserve. The observation provides further evidence for the view that impairment of myocardial energy production is not responsible for contractilc dysfunction early after reperfusion.

Differential regulation of stimulated glucose transport by free fatty acids and PPARα or δ agonists in cardiac myocytes

Stimulation of glucose transport in response to insulin or metabolic stress is an important determinant of cardiac myocyte function and survival, particularly during ischemia-reperfusion episodes. The impact of dyslipidemia and its consequence PPAR activation on stimulated glucose transport in cardiac myocytes remains unknown. Isolated adult rat cardiac myocytes were chronically exposed to free fatty acids (FFA) or PPAR agonists. Insulin- (ISGT) and oligomycin-stimulated glucose transport (OSGT) and related cell signaling were analyzed. Exposure of cardiac myocytes to FFA reduced both ISGT and OSGT. Exposure to either PPARα or PPARδ agonists, but not to a PPARγ agonist, reduced ISGT but not OSGT and increased fatty acid oxidation (FAO). The reduction in ISGT was associated with impaired insulin signaling and, in the case of PPAR stimulation, overexpression of SOCS-3, a protein known to hinder proximal insulin signaling. In contrast, the reduction of OSGT could not be explained by a reduced activity of the cellular energy-sensing system, as assessed from the maintained phosphorylation state of AMPK. Inhibition of FAO at the level of mitochondrial acylcarnitine uptake restored OSGT but not ISGT. Seemingly paradoxically, further stimulation of FAO with PPARα or PPARδ agonists also restored OSGT but not ISGT. Together, these results suggest that inhibition of OSGT occurs downstream of energy gauging and is caused by some intermediate(s) of fatty acid oxidation, which does not appear to be acylcarnitines. The results indicate that the mechanisms underlying FFA-mediated inhibition of ISGT and OSGT differ remarkably.

Retinoic acids increase expression of GLUT4 in dedifferentiated and hypertrophied cardiac myocytes.

Sufficient expression of the insulin–sensitive glucose transporter GLUT4 may be crucial for the survival of cardiac myocytes in situations of stress. Expression of GLUT4 in cardiac myocytes correlates with cell differentiation and is reduced in the hypertrophied and failing myocardium. Adult rat cardiomyocytes (ARC) in primary culture undergo dedifferentiation and reduction of GLUT4 expression. Depending on the culture condition partial redifferentiation and/or hypertrophy follows. All–trans (at) and 9–cis retinoic acids (RA) are morphogenetic agents important for cell differentiation. Both atRA and 9–cisRA restored GLUT4 expression in dedifferentiated ARC, while only 9–cisRA could increase GLUT4 expression in hypertrophic ARC. The effects of RA were associated with improved differentiation of the cardiac myocytes, as assessed from the expression of atrial natriuretic factor and the morphology of the contractile apparatus. In neonatal rat cardiomyocytes, 9–cisRA, but not atRA, stimulated transcription from the glut4 promoter. In conclusion, treatment with RA can restore the down–regulated expression of GLUT4 in cardiomyocytes in association with a partial improvement of the differentiated phenotype.

Dual effect of the heart-targeting cytokine cardiotrophin-1 on glucose transport in cardiomyocytes

Cardiotrophin-1 (CT-1) is a heart-targeting cytokine that is increased in the metabolic syndrome due to overexpression in the adipocytes. The effects of CT-1 on cardiomyocyte substrate metabolism remain unknown. We therefore determined the effects of CT-1 on basal and stimulated glucose transport in cardiomyocytes exposed to a low dose (1nM) or a high dose (10nM). Dose-response curves for insulin showed that 1nM CT-1 reduced insulin responsiveness, while 10nM CT-1 increased insulin responsiveness. In either condition insulin sensitivity was unaffected. Similarly 1nM CT-1 reduced the stimulation of glucose transport in response to metabolic stress, induced by the mitochondrial poison oligomycin, while 10nM CT-1 increased this response. Reduction of stimulated glucose transport by 1nM CT-1 was associated with overexpression of SOCS-3, a protein known to hinder proximal insulin signaling, and increased phosphorylation of STAT5. In cardiomyocytes exposed to 1nM CT-1 there was also reduced phosphorylation of Akt and AS160 in response to insulin, and of AMPK in response to oligomycin. Insulin-stimulated glucose transport and signaling were restored by inhibition of STAT5 activity. On the other hand in cardiomyocytes exposed to 10nM CT-1 there was increased phosphorylation of the AS160 and Akt in response to insulin. Most importantly, basal and oligomycin-stimulated phosphorylation of AMPK was markedly increased in cardiomyocytes exposed to 10nM CT-1. The enhancement of basal and stimulated-glucose transport was abolished in cardiomyocytes treated with the calmodulin-dependent kinase II (CaMKII) inhibitor KN93, and so was AMPK phosphorylation. This suggests that activation of CaMKII mediates activation of AMPK by a high dose of CT-1 independently of metabolic stress. Our results point to a role for CT-1 in the regulation of myocardial glucose metabolism and implicate entirely separate mechanisms in the inhibitory or stimulatory effects of CT-1 on glucose transport at low or high concentrations respectively.

Differential effects of high-fat diet on myocardial lipid metabolism in failing and nonfailing hearts with angiotensin II-mediated cardiac remodeling in mice

Normal myocardium adapts to increase of nutritional fatty acid supply by upregulation of regulatory proteins of the fatty acid oxidation pathway. Because advanced heart failure is associated with reduction of regulatory proteins of fatty acid oxidation, we hypothesized that failing myocardium may not be able to adapt to increased fatty acid intake and therefore undergo lipid accumulation, potentially aggravating myocardial dysfunction. We determined the effect of high-fat diet in transgenic mice with overexpression of angiotensinogen in the myocardium (TG1306/R1). TG1306/R1 mice develop ANG II-mediated left ventricular hypertrophy, and at one year of age approximately half of the mice present heart failure associated with reduced expression of regulatory proteins of fatty acid oxidation and reduced palmitate oxidation during ex vivo working heart perfusion. Hypertrophied hearts from TG1306/R1 mice without heart failure adapted to high-fat feeding, similarly to hearts from wild-type mice, with upregulation of regulatory proteins of fatty acid oxidation and enhancement of palmitate oxidation. There was no myocardial lipid accumulation or contractile dysfunction. In contrast, hearts from TG1306/R1 mice presenting heart failure were unable to respond to high-fat feeding by upregulation of fatty acid oxidation proteins and enhancement of palmitate oxidation. This resulted in accumulation of triglycerides and ceramide in the myocardium, and aggravation of contractile dysfunction. In conclusion, hearts with ANG II-induced contractile failure have lost the ability to enhance fatty acid oxidation in response to increased fatty acid supply. The ensuing accumulation of lipid compounds may play a role in the observed aggravation of contractile dysfunction.

Role of ERK1/2 activation in microtubule stabilization and glucose transport in cardiomyocytes

We previously demonstrated that microtubule disruption impairs stimulation of glucose uptake in cardiomyocytes and that 9-cis retinoic acid (9cRA) treatment preserved both microtubule integrity and stimulated glucose transport. Herein we investigated whether 1) activation of the extracellular signal-regulated kinases (ERK1/2) is responsible for microtubule destabilization and 2) ERK1/2 inactivation may explain the positive effects of 9cRA on glucose uptake and microtubule stabilization. Adult rat cardiomyocytes in primary culture showed increased basal ERK1/2 phosphorylation. Cardiomyocytes exposed to inhibitors of the ERK1/2 kinase mitogen/extracellular signal-regulated kinase (MEK) 1/2 had preserved microtubular scaffold, including microtubule-organizing centers (MTOC), together with increased insulin and metabolic stress-stimulated glucose transport as well as signaling, thus replicating the effects of 9cRA treatment. Although 9cRA treatment did not significantly reduce global ERK1/2 activation, it markedly reduced perinuclear-activated ERK1/2 at the location of MTOC. 9cRA also triggered relocation of the ERK1/2 phosphatase mitogen-activated protein kinase phosphatase-3 from the cytosol to the nucleus. These results indicate that, in cardiomyocytes, microtubule destabilization, leading to impaired stimulation of glucose transport, is mediated by ERK1/2 activation, impacting on the MTOC. 9cRA acid restores stimulated glucose transport indirectly through compartmentalized inactivation of ERK1/2.