Roselle Gélinas

A-769662 potentiates the effect of other AMP-activated protein kinase activators on cardiac glucose uptake.

AMP-activated protein kinase (AMPK), a key cellular sensor of energy, regulates metabolic homeostasis and plays a protective role in the ischemic or diabetic heart. Stimulation of cardiac glucose uptake contributes to this AMPK-mediated protection. The small-molecule AMPK activator A-769662, which binds and directly activates AMPK, has recently been characterized. A-769662-dependent AMPK activation protects the heart against an ischemia-reperfusion episode but is unable to stimulate skeletal muscle glucose uptake. Here, we tried to reconcile these conflicting findings by investigating the impact of A-769662 on cardiac AMPK signaling and glucose uptake. We showed that A-769662 promoted AMPK activation, resulting in the phosphorylation of several downstream targets, but was incapable of stimulating glucose uptake in cultured cardiomyocytes and the perfused heart. The lack of glucose uptake stimulation can be explained by A-769662s narrow specificity, since it selectively activates cardiac AMPK heterotrimeric complexes containing α2/β1-subunits, the others being presumably required for this metabolic outcome. However, when combined with classical AMPK activators, such as metformin, phenformin, oligomycin, or hypoxia, which impact AMPK heterotrimers more broadly via elevation of cellular AMP levels, A-769662 induced more profound AMPK phosphorylation and subsequent glucose uptake stimulation. The synergistic effect of A-769662 under such ischemia-mimetic conditions protected cardiomyocytes against ROS production and cell death. In conclusion, despite the fact that A-769662 activates AMPK, it alone does not significantly stimulate glucose uptake. However, strikingly, its ability of potentiating the action on other AMPK activators makes it a potentially useful participant in the protective role of AMPK in the heart.

TECRL, a new life‐threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole‐exome sequencing (WES) was carried out on patients from three different families that presented with life‐threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans‐2,3‐enoyl‐CoA reductase‐like protein. Both patients had cardiac arrest, stress‐induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL. Analysis of intracellular calcium ([Ca2+]i) dynamics in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) generated from this individual (TECRLHom‐hiPSCs), his heterozygous but clinically asymptomatic father (TECRLHet‐hiPSCs), and a healthy individual (CTRL‐hiPSCs) from the same Sudanese family, revealed smaller [Ca2+]i transient amplitudes as well as elevated diastolic [Ca2+]i in TECRLHom‐hiPSC‐CMs compared with CTRL‐hiPSC‐CMs. The [Ca2+]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine‐induced [Ca2+]i transients. In addition, the decay phase of the [Ca2+]i transient was slower in TECRLHom‐hiPSC‐CMs due to decreased SERCA and NCX activities. Furthermore, TECRLHom‐hiPSC‐CMs showed prolonged action potentials (APs) compared with CTRL‐hiPSC‐CMs. TECRL knockdown in control human embryonic stem cell‐derived CMs (hESC‐CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom‐hiPSC‐CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT. Patient‐specific hiPSC‐CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias.

O-GlcNAcylation, enemy or ally during cardiac hypertrophy development?

O-linked attachment of the monosaccharide β-N-acetyl-glucosamine (O-GlcNAcylation) is a post-translational modification occurring on serine and threonine residues, which is evolving as an important mechanism for the regulation of various cellular processes. The present review will, first, provide a general background on the molecular regulation of protein O-GlcNAcylation and will summarize the role of this post-translational modification in various acute cardiac pathologies including ischemia-reperfusion. Then, we will focus on research studies examining protein O-GlcNAcylation in the context of cardiac hypertrophy. A particular emphasis will be laid on the convergent but also divergent actions of O-GlcNAcylation according to the type of hypertrophy investigated, including physiological, pressure overload-induced and diabetes-linked cardiac hypertrophy. In an attempt to distinguish whether O-GlcNAcylation is detrimental or beneficial, this review will present the different O-GlcNAcylated targets involved in hypertrophy development. We will finally argue on potential interest to target O-GlcNAc processes to treat cardiac hypertrophy. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation.

AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.AMPK activation inhibits cardiac hypertrophy. Here the authors show that this occurs independently of previously proposed mechanisms and that AMPK controls the phosphorylation of the aminotransferase GFAT, thereby preventing cardiac hypertrophy through the reduction of protein O-GlcNAcylation.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucines action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucines inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucines effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucines inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucines action, suggesting acetylation involvement in this phenomenon.Listen to this articles corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

AMP-Activated Protein Kinase and O-GlcNAcylation, Two Partners Tightly Connected to Regulate Key Cellular Processes

The AMP-activated protein kinase (AMPK) is an important cellular energy sensor. Its activation under energetic stress is known to activate energy-producing pathways and to inactivate energy-consuming pathways, promoting ATP preservation and cell survival. AMPK has been shown to play protective role in many pathophysiological processes including cardiovascular diseases, diabetes, and cancer. Its action is multi-faceted and comprises short-term regulation of enzymes by direct phosphorylation as well as long-term adaptation via control of transcription factors and cellular events such as autophagy. During the last decade, several studies underline the particular importance of the interaction between AMPK and the post-translational modification called O-GlcNAcylation. O-GlcNAcylation means the O-linked attachment of a single N-acetylglucosamine moiety on serine or threonine residues. O-GlcNAcylation plays a role in multiple physiological cellular processes but is also associated with the development of various diseases. The first goal of the present review is to present the tight molecular relationship between AMPK and enzymes regulating O-GlcNAcylation. We then draw the attention of the reader on the putative importance of this interaction in different pathophysiological events.

PO14 Cytosquelette d’actine et protéine G Rac1, cibles potentielles pour le traitement de l’insulinorésistance

Introduction Nous avons precedemment montre que lactivation de la proteine kinase activee par lAMP (AMPK) par les biguanides (metformine ou phenformine) augmente la sensibilite a linsuline et le transport de glucose cardiaque. Nous avons tente didentifier les mecanismes moleculaires impliques dans cet effet insulino-sensibilisateur. Nous nous sommes interesses a la reorganisation du cytosquelette dactine et a Rac1, la proteine G regulant cette reorganisation. La voir Rac1/actine est, en effet, connue pour etre impliquee dans la translocation insulino-dependante des transporteurs de glucose Glut4 a la membrane. Materiels et methodes Des cardiomyocytes de rat en culture primaire insulinosensibles et insulinoresistants ont ete soumis a differents traitements. Le transport de glucose a ete evalue par mesure de la detritiation du 2-3H-glucose. Les divers elements de signalisation ont ete evalues par western-blot et/ou mesure dactivite. Resultats Dans des cardiomyocytes insulinosensibles, lactivation de lAMPK par les biguanides stimule le transport de glucose de maniere similaire a linsuline (respectivement 5 et 6 fois, p p Conclusion La voie Rac1/Actine est impliquee dans laction insulino-sensibilisatrice de lAMPK sur le transport de glucose cardiaque. Cette voie pourrait etre une cible therapeutique potentielle pour traiter linsulinoresistance cardiaque. Declaration d’interet Les auteurs declarent ne pas avoir dinteret direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

0325 : Catabolism of leucine in the heart inhibits glucose transport

Branched-chain amino acids like leucine induce insulin resistance in muscle and adipose tissues. The mechanism explaining leucine action involves mTOR/p70S6K signaling. This pathway is activated by leucine and is implicated in the stimulation of an insulin negative feedback loop. Knowing that insulin-resistance participates in diabetic cardiomyopathy, we were interested in studying leucine action in cardiomyocytes. Primary cultured adult rat cardiomyocytes were pretreated with different concentrations of leucine (1 to 10mM) during different periods of time (up to 20h) before being exposed to insulin (3x10-9M, 30min). Insulin increased glucose transport. This correlated with the increase of PKB and AS160 phosphorylation, both known to regulate GLUT4 translocation to the plasma membrane allowing glucose uptake. 1h pre-incubation with leucine stimulated mTOR/p70S6K pathway. This is accompanied by a decrease in PKB and AS160 phosphorylation but, surprisingly, insulin-stimulated glucose uptake was preserved. On the other hand, a longer incubation (14h) with leucine induced a drastic decrease in glucose transport. The mTOR/p70S6K inhibitor rapamycin did not prevent this inhibition. The non-metabolized leucine analog BCH had no effect on the insulin-induced glucose uptake. By contrast, intermediates of leucine catabolism, alpha-ketoisocaproate and ketone bodies inhibited glucose uptake similarly to leucine. This inhibition is clearly independent of insulin signaling because leucine also inhibited basal glucose transport and glucose uptake stimulated by the insulin-unrelated pathway involving AMPK. The leucine-mediated inhibition of glucose transport resulted from the inhibition of GLUT4 translocation. The exact molecular mechanism downstream leucine’s metabolites and responsible for the inhibition of GLUT4 translocation and glucose uptake is under investigation. Leucine catabolism reduces cardiac glucose transport independently of insulin signaling by an undefined mechanism.

0296 : Regulating O-GlcNAcylation by AMP-activated protein kinase, a new way to prevent hypertrophy development

Background We have previously shown that the AMPK specific activator, A769662, is able to block phenylephrine (PE)-induced hypertrophy without affecting the previously identified AMPK-related key regulators of cardiac hypertrophy, namely protein synthesis, NFAT and MAP kinase signaling. Hence, this study was undertaken to identify a novel mechanism by which AMPK can regulate cardiac hypertrophy. Methods Hypertrophy was induced in neonatal rat cardiomyocytes (NRVMs) using PE for 24h and AMPK was chronically activated with A769662. Hypertrophy was evaluated by immunostaining and cell surface area measurement. In vivo cardiac hypertrophy was induced by Angiotensin II (AngII) infusion by osmotic mini-pumps and AMPK was activated by metformin in drinking water. Protein expression, AMPK phosphorylation and OGlcNAc levels were evaluated by QPCR and western blot. Results First, we showed that PE-dependent cardiomyocyte hypertrophy was accompanied by an increase in O-GlcNAc levels. This hypertrophy was prevented by A769662 and this was associated with a decrease in O-GlcNAc levels. Then, using hexosamine biosynthesis pathway activators, i. e PUGNAc or glucosamine, we showed that increase in O-GlcNAc levels was able to reverse the anti-hypertrophic effect of AMPK activation in NRVMs. Similar results were obtained in adult rat cardiomyocytes. On the other hand, inhibition of hexosamine biosynthesis pathway by Azaserine or DON inhibited PE-induced cardiomyocyte hypertrophy and this effect was reversed by PUGNAc or glucosamine. In vivo experiments confirmed these in vitro data. Indeed, metformin was able to reduce cardiac hypertrophy and this correlated with a decrease in O-GlcNAc levels in WT mice but not in AMPKα2 knockout mice. Conclusion Collectively, our results suggest that AMPK regulates cardiac hypertrophy mainly by inhibiting O-GlcNAcylation signaling. AMPK and OGlcNAcylation signaling could be putative new therapeutic targets for treatment of cardiac hypertrophy.

P152 La leucine, un puissant inhibiteur du captage de glucose cardiaque

Introduction La leucine est un acide amine branche capable dinduire une resistance a linsuline dans le muscle et le tissu adipeux. Le mecanisme propose pour expliquer laction inhibitrice de la leucine implique la voie mTOR/p70S6K/IRS-1. Cette voie peut etre activee par la leucine et est impliquee dans lactivation dune boucle de retrocontrole negatif de linsuline. Sachant que linsulino-resistance participe a letablissement dune cardiomyopathie diabetique, nous avons etudie leffet inhibiteur de la leucine dans le cardiomyocyte. Materiels et methodes Des cardiomyocytes de rats adultes en culture primaire ont ete pretraites avec differentes concentrations de leucine (entre 1 et 10mM) pendant differentes periodes dincubation avant detre exposes a linsuline (3×10 −9 M, 30min). Resultats En absence de leucine, linsuline induit une augmentation du captage de glucose (0,31±0,04 vs. 0,05±0,01μmoles/mg. h). Celle-ci correle avec laugmentation de la phosphorylation de PKB et AS160, connus pour reguler le captage de glucose en aval de linsuline. Une pre-incubation de 1h en leucine active la voie mTOR/p70S6K resultant en linhibition dIRS-1 situe en amont dans la voie de signalisation insulinique. Ceci saccompagne dune diminution significative de la phosphorylation de PKB et AS160. Etonnamment, le captage de glucose stimule par linsuline est preserve malgre cette inhibition (0,31±0,05μmoles/mg. h). Dautre part, une pre-incubation plus longue (14h) en leucine induit une diminution drastique du captage de glucose (0,056±0,01μmoles/mg. h). La rapamycine, un inhibiteur de mTOR/p70S6K, nempeche pas cette inhibition. En outre, un analogue non metabolisable de la leucine (BCH) stimule la voie mTOR/p70S6K sans avoir deffet sur le captage de glucose. En revanche, les intermediaires du catabolisme de la leucine, lα- cetoisocaproate, lacetoacetate, le β-hydroxybutyrate, inhibent le transport de glucose de maniere similaire a la leucine. Conclusion Le catabolisme de la leucine reduit le transport de glucose independamment de la signalisation insulinique.