Magali Balteau

NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1

AIMS Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase in cardiomyocytes, but the underlying mechanism remains elusive. In this study, we have dissected the link between glucose transport and metabolism and NADPH oxidase activation under hyperglycaemic conditions. METHODS AND RESULTS Primary cultures of adult rat cardiomyocytes were exposed to HG concentration (HG, 21 mM) and compared with the normal glucose level (LG, 5 mM). HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane, resulting in NADPH oxidase (NOX2) activation, increased ROS production, insulin resistance, and eventually cell death. Comparison of the level of O-linked N-acetylglucosamine (O-GlcNAc) residues in LG- and HG-treated cells did not reveal any significant difference. Inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide counteracted ROS production in response to HG but did not prevent Rac-1 upregulation and p47phox translocation leading to NOX2 activation. Modulation of glucose uptake barely affected oxidative stress and toxicity induced by HG. More interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and α-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of the sodium/glucose cotransporter SGLT1 by phlorizin counteracted HG-induced NOX2 activation and ROS production. CONCLUSION Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.

AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-β2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.

Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1.

Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-β1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.

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.

Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells

AIMS Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulation.

Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart.

Hyperglycemia (HG) stimulates the production of reactive oxygen species in the heart through activation of NADPH oxidase 2 (NOX2). This production is independent of glucose metabolism but requires sodium/glucose cotransporters (SGLT). Seven SGLT isoforms (SGLT1 to 6 and sodium-myoinositol cotransporter-1, SMIT1) are known, although their expression and function in the heart remain elusive. We investigated these 7 isoforms and found that only SGLT1 and SMIT1 were expressed in mouse, rat and human hearts. In cardiomyocytes, galactose (transported through SGLT1) did not activate NOX2. Accordingly, SGLT1 deficiency did not prevent HG-induced NOX2 activation, ruling it out in the cellular response to HG. In contrast, myo-inositol (transported through SMIT1) reproduced the toxic effects of HG. SMIT1 overexpression exacerbated glucotoxicity and sensitized cardiomyocytes to HG, whereas its deletion prevented HG-induced NOX2 activation. In conclusion, our results show that heart SMIT1 senses HG and triggers NOX2 activation. This could participate in the redox signaling in hyperglycemic heart and contribute to the pathophysiology of diabetic cardiomyopathy.

0335: Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1

Objective Cardiac fibroblasts (CF) are crucial in left ventricular (LV) remodelling after myocardial infarction (MI). They predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptive heart responses remains unexplored. In this study, we aim at further substantiating the role of this AMPK isoform in the pathogenesis of post-MI LV remodelling and more particularly in the regulation of fibrotic properties of CF. Methods AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic MI. Cardiac fibrosis was monitored using qRT-PCR analysis, histology and immunohistofluorescent staining. LV function and remodelling was assessed by echocardiography. Results In the absence of AMPKα1, the CF proliferative response was increased in infarcted myocardia. It resulted in elevated levels of fibrotic factors but did not lead to excessive matrix deposition or degradation in KO infarcts. While CF proliferation was increased, expression of the myodifferentiation marker a-smooth muscle actin was decreased. This faulty maturation of myofibroblasts might derive from down-regulation of the transforming growth factor-β1/p38 mitogen-activated protein kinase pathway in KO infarcts. Although infarct size was similar in KO and WT hearts subjected to MI, these changes resulted in defective scar collagen maturation. This was associated with an exacerbated adverse remodelling as indicated by increased LV diastolic dimension 30 days after MI. Conclusion Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology

0311: Role of AMP-activated protein kinase in regulating proliferation of mesenchymal stem cells

Background Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischemic heart disease. Mechanisms underlying control of their proliferation capacity, which is known to be inversely correlated to their differentiation abilities, have not been fully characterized. The AMP-activated protein kinase (AMPK) controls several cell functions including cell proliferation. Objective We hypothesized that AMPK plays a crucial role in the control of MSC proliferation. Methods MSCs isolated from WT or AMPKα1 KO adult mouse bone marrow were stimulated with the specific AMPK activator, A769662. Protein phosphorylation state was determined by immunoblotting, cell viability was quantified by FACS after AnnexinV and Propidium Iodide staining, cell proliferation was quantified by EdU incorporation and gene expression was analyzed by real-time PCR. Results MSCs isolated from murine bone marrow exclusively expressed the AMPKa1 catalytic subunit. A769662 was able to induce a robust and sustained AMPK activation in WT MSCs but not in AMPKa1 KO MSCs. This A769662-dependent AMPK activation blocks WT MSC proliferation but not in KO MSCs without affecting their viability. Moreover, AMPKa1 deletion induces an increase of basal MSC proliferation. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression prevented the A-769662- dependent inhibition of MSC proliferation. Conclusions These results suggest that activating AMPK promotes a principal prerequisite for cell differentiation, the arrest of MSC proliferation via p27 regulation.

P2028 Identification d’un senseur au glucose dans le tissu cardiaque : mise en évidence de l’isoforme SGLT3

Introduction L’hyperglycemie exerce des effets deleteres sur le myocarde suite a une activation de la NADPH oxydase (NOX2). L’activation de NOX2 ne necessite pas la metabolisation du glucose mais est le resultat du transport de glucose par un cotransporteur sodium-glucose ou SGLT. L’expression des isoformes de SGLT dans le coeur et plus particulierement dans le cardiomyocyte n’est pas decrite a ce jour. Le but de ce travail est de detecter l’expression des isoformes principales de SGLT dans le coeur et les cardiomyocytes isoles. Materiels et methodes L’expression genique et proteique des isoformes 1, 2 et 3 de SGLT a ete etudiee par RT-PCR et immunodetection sur des extraits de coeurs entiers et de cardiomyocytes isoles de rat adulte en culture primaire. Resultats L’ARNm de SGLT1 est detecte dans le coeur et dans les cardiomyocytes. Par immunodetection, la proteine SGLT1 est mise en evidence dans le coeur entier et les cardiomyocytes mais le niveau d’expression est beaucoup plus faible que dans des cellules intestinales. SGLT2 est exprime dans un extrait de cœur entier. Par contre, cette isoforme n’est pas detectee dans le cardiomyocyte isole. De maniere plus interessante, l’isoforme SGLT3b est detectee par RT-PCR dans le coeur entier et de maniere plus evidente encore, dans les cardiomyocytes. Une proteine de 55 kDa est detectee par l’anticorps antiSGLT3 humain dans les extraits de coeur et de cardiomyocytes. Conclusion Nous montrons pour la premiere fois l’expression de l’isoforme SGLT3b dans le coeur et surtout dans le cardiomyocyte. Nous confirmons la presence de SGLT1 dans le coeur. Par contre et contrairement a SGLT1 et SGLT3b, SGLT2 n’est pas exprime dans le cardiomyocyte. SGLT3b est considere comme un senseur au glucose et pourrait etre responsable d’une activation de la signalisation intracellulaire menant a l’activation de NOX2 en reponse a une hyperglycemie.