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Background Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Purpose Our study sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions by decreasing phospholipid content. Methods We generated a new Cre transgenic mouse strain that allows megakaryocyte/platelet specific ACC1 deletion (GpIbCre+/− x ACC1 flx/flx mouse). In vitro, platelet functions were assessed by aggregometry and flow cytometry. In vivo, hemostasis was assessed via the measurement of bleeding time. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Results As expected, ACC1 deletion was restricted to the megakaryocytic lineage. Hematological parameters in platelet-specific ACC1 knockout mice showed a decrease in platelet count by 30% and an increase in platelet volume by 31%, compared to ACC1 flx/flx platelets. In vitro, platelets from platelet-specific ACC1 knockout mice displayed a decrease in thrombin and CRP-induced platelet aggregation, associated with impaired dense granules secretion. In contrast, ADP-induced platelet aggregation was higher in the absence of ACC1. In vivo, platelet-specific ACC1 knockout mice showed a normal bleeding time. In agreement with our hypothesis, lipidomics analyses showed that ACC1 deletion in platelets was associated with a significant decrease in arachidonic acid-contaning phosphatidylethanolamine plasmalogen, and subsequently with a reduced production of thromboxane A2 upon thrombin or CRP stimulation. Conclusion Platelet-specific ACC1 deletion led to a decrease in phospholipid content which, in turn, decreased platelet thromboxane A2 generation, dense granules secretion and aggregation upon thrombin and CRP, but not ADP stimulation. Further studies are needed to elucidate the impact of ADP on platelet functions Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Background Septic shock generates an important inflammatory reaction, endothelial activation and a procoagulant state leading to microvascular thrombosis and subsequent organ impairment [1]. Similarly, a severe inflammatory reaction and a coagulopathy with pulmonary micro-thrombosis eventually leading to acute lung injury, is a typical feature of critical form of Coronavirus disease 2019 (Covid-19) [2]. Our aim was to compare coagulation, platelet activation and platelets-neutrophils interplay between control, septic shock and critical Covid-19 patients. Methods/Materials A total of 118 patients were included in our prospective, monocentric, observational study between February 2019 and June 2020. Septic shock (n=48) and Covid-19 (n=22) patients were consecutively included at admission in our ICU department. Control patients (n=48) with matched gender and co-morbidities were recruited at central lab consultation. Results Septic shock patients had worse severity scores due to multiple organ failure (assessed by APACHE II and SOFA score) whereas Covid-19 patients had more severe respiratory failure and a longer ICU length-of-stay (Table 1). At the time of inclusion, CRP and lymphocyte count were comparable between septic shock and Covid-19 patients. White cell count ad neutrophil count was higher for septic shock patients. Analysis of coagulation showed a prolonged INR, TT and aPTT in septic shock although only INR was prolonged in Covid-19. Thrombin antithrombin complex (TATc) formation was similar in both pathologies, whereas consumption of antithrombin III (ATIII) and D-dimers formation was more pronounced in septic shock. Platelet count was lower in septic shock and platelet activation, assessed via plasmatic levels of soluble P-selectin (sCD62P) and Trem-like transcript 1 (sTLT-1), was more important in septic shock. Neutrophil activation and NETosis, evaluated by levels of circulating myeloperoxidase (MPO) and citrullinated histone 3 (H3-Cit), was similarly increased in both groups (Figure 1). Conclusions This study confirmed an activation of coagulation cascade, platelet activation and NETosis in both septic shock and critical Covid-19, compared with control patients. Importantly, the extent of these changes was similar or less pronounced in critical COVID-19 compared with septic shock. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondation Saint Luc Figure 1

Summary: Adenosine monophosphate–activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK–induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK–induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function. Key Words: AMPK, acetyl-CoA carboxylase, coronary artery disease, lipidomics, platelet

We have previously demonstrated that systemic AMP-activated protein kinase α1 (AMPKα1) invalidation enhanced adverse LV remodelling by increasing fibroblast proliferation, while myodifferentiation and scar maturation were impaired. We thus hypothesised that fibroblastic AMPKα1 was a key signalling element in regulating fibrosis in the infarcted myocardium and an attractive target for therapeutic intervention. The present study investigates the effects of myofibroblast (MF)-specific deletion of AMPKα1 on left ventricular (LV) adaptation following myocardial infarction (MI), and the underlying molecular mechanisms. MF-restricted AMPKα1 conditional knockout (cKO) mice were subjected to permanent ligation of the left anterior descending coronary artery. cKO hearts exhibit exacerbated post-MI adverse LV remodelling and are characterised by exaggerated fibrotic response, compared to wild-type (WT) hearts. Cardiac fibroblast proliferation and MF content significantly increase in cKO infarcted hearts, coincident with a significant reduction of connexin 43 (Cx43) expression in MFs. Mechanistically, AMPKα1 influences Cx43 expression by both a transcriptional and a post-transcriptional mechanism involving miR-125b-5p. Collectively, our data demonstrate that MF-AMPKα1 functions as a master regulator of cardiac fibrosis and remodelling and might constitute a novel potential target for pharmacological anti-fibrotic applications. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00846-y.

Aims Besides regulating calcium‐phosphate metabolism, fibroblast growth factor 23 (FGF‐23) has been associated with incident heart failure (HF) and left ventricular hypertrophy. However, data about FGF‐23 in HF and preserved ejection fraction (HFpEF) remain limited. The aim of this study was to assess the association between FGF‐23 levels, clinical and imaging characteristics, particularly diffuse myocardial fibrosis, and prognosis in HFpEF patients. Methods and results We prospectively included 143 consecutive HFpEF patients (78 ± 8 years, 61% female patients) and 31 controls of similar age and gender (75 ± 6 years, 61% female patients). All subjects underwent a complete two‐dimensional echocardiography and cardiac magnetic resonance with extracellular volume (ECV) assessment by T1 mapping. FGF‐23 was measured at baseline. Among the patients, differences in clinical and imaging characteristics across tertiles of FGF‐23 levels were analysed with a trend test across the ordered groups. Patients were followed over time for a primary endpoint of all‐cause mortality and first HF hospitalization and a secondary endpoint of all‐cause mortality. Median FGF‐23 was significantly higher in HFpEF patients compared with controls of similar age and gender (247 [115; 548] RU/mL vs. 61 [51; 68] RU/mL, P

Introduction Host immune response to the coronavirus disease 2019 (COVID-19) is variable and can induce a dysregulated inflammatory response associated with venous and arterial thrombosis called COVID-19 associated coagulopathy (CAC). During septic shock, inflammatory reaction generates endothelial activation and procoagulant state with microvascular thrombi inducing disseminated intravascular coagulation (DIC). Although CAC and DIC induce altered coagulation responses, their clinical outcomes are different. Objective We investigated and compared coagulopathy between septic shock and critical COVID-19 patients. Method Septic shock patients were diagnosed following the Survival Sepsis Campaign guidelines. COVID-19 patients were admitted in intensive care unit (ICU) for severe acute respiratory distress syndrome. Both were included in the study within 2 days after admission. Biomarkers were measured by ELISA from patient's plasma. Results We observed an increase in vWF and TFPI in both septic and COVID-19 patients compared to controls, highlighting endothelial damage. Interestingly, circulating TF was only elevated in COVID-19 patients. Platelet activation differed between the two cohorts of patients. P-selectin and TLT-1 were specifically heightened in septic shock whereas CD40L was only augmented in COVID-19. Coagulation markers were increased in a disease-dependent way, with PAI-1, tPA and D-Dimers higher in septic shock and fibrinogen level, higher in COVID-19. Discussion COVID-19 patients had longer length-of-stay with more pronounced respiratory failure. This strong lung disruption overtime induced plasmatic TF release with sustained inflammatory response characterized by sCD40L and fibrinogen secretion. Given the similarities between COVID-19 and septic shock regarding fibrinolysis and coagulation, but not platelet activation, endothelium seems to play a central role in COVID-19 and might explain the differences between CAC and DIC.

Introduction Vascular hyperpermeability is a major aspect of sepsis pathophysiology. We previously demonstrated that AMPK protects against sepsis induced vascular hyperpermeability by mechanisms involving interendothelial junctions (IEJs) regulation. The SGLT2 inhibitor Canagliflozin, prescribed in diabetes and known for its cardiovascular protective properties, has recently been shown to activate AMPK in different cell types. Objectives Here, we show that Canagliflozin might be considered as a new therapeutic approach against sepsis induced vascular hyperpermeability. We also decipher molecular mechanisms underlying this protective effect. Methods In vivo experiments were performed on endothelial-specific α1AMPK knockout (e-AMPK KO) mice. Canagliflozin administration was achieved by oral gavage (100 mg/kg) and validated by plasmatic dosages. LPS was administered by intraperitoneal injection (10 mg/kg). Myocardial edema was assessed by Evans Blue Dye extravasation measurement. In vitro experiments were performed in Human Dermal Blood Endothelial Cells (HDBEC). α1AMPK was invalidated with a specific siRNA or activated by Canagliflozin (10 μM). Results We show that Canagliflozin activates AMPK in HDBEC at physiological concentrations. Canagliflozin administration strongly protects against LPS induced vascular leakage in WT but not in e-AMPK KO mice. Accordingly, albuminemia is preserved by Canagliflozin treatment in septic WT mice. More interestingly, Canagliflozin treatment increases survival in response to sepsis injury. In vitro, AMPK activation by Canagliflozin preserves the integrity of IEJs (Cx43, VeCad, ZO1). Conclusion Our work demonstrates the protective effects of Canagliflozin on sepsis induced vascular hyperpermeability and mortality, and highlights key molecular mechanisms involved in this protection. This should open the field to consider Canagliflozin as a new therapeutic support of sepsis induced vascular hyperpermeability.

Background Cardiac fibroblasts (CFs) are crucial components of the fibrotic response following myocardial infarction (MI). We have previously shown that AMPK is a key regulator of CF properties and fibrosis. Our data suggest that AMPK exerts anti-fibrotic effects by regulating connexin 43 (Cx43) expression, a protein known to modulate CF activity and cardiac fibrosis. Objectives The present study sought to decipher the molecular mechanisms underlying Cx43 regulation by AMPK in CFs. Methods Cultured human CFs (HCFs) were transfected with a specific siRNA targeting AMPK. Transcriptional and post-transcriptional regulation of Cx43 was assessed by luciferase reporter assay, actinomycin and proteasome inhibitor experiments. Its post-transcriptional regulation was further investigated using microRNAs (miRs) array, mimics and antimiRs. Results In cultured HCFs, AMPK deletion is associated with a drastic reduction of Cx43 protein expression. We demonstrated that proteasome is not involved in this regulation. Using luciferase reporter assay, we showed that AMPK deficiency decreases Cx43 promoter activity by 40%, indicating that AMPK contributes to the transcriptional regulation of Cx43. In addition, we provided evidence that AMPK can also affect Cx43 mRNA stability, suggesting the potential implication of miRs in the control of CX43 expression. Accordingly, the analysis of miRs profiling with microarrays revealed that several miRs are dysregulated in AMPK-depleted HCFs. Among them, in silico analysis identified miR-125b as a Cx43 regulator. We then confirmed that transfection of miR-125b mimic down-regulates Cx43 in HCFs. Importantly, inhibition of miR-125b expression using antimiR prevents Cx43 loss in absence of AMPK. Conclusions Collectively, our data demonstrate that AMPK regulates Cx43 in CFs, transcriptionally and post-transcriptionally, by a mechanism involving miR-125b. Further investigations are needed to define whether miR-125b might be a pro-fibrotic biomarker.

Introduction Acetyl-CoA carboxylase (ACC) is the first committed enzyme regulating lipids synthesis. We have recently demonstrated that the overexpression of a constitutively active form of ACC in platelets leads to an increase in the phospholipid content which stimulates thrombus formation. Based on these data, we believe that ACC inhibition might be a counter-regulatory mechanism limiting endogenous lipogenesis and platelet reactivity in certain pathological circumstances. Aim Our study aims to assess the impact of pharmacological ACC1 inhibition on platelet functions, de novo lipogenesis and lipid content. Methods Platelets were treated with the long-chain fatty acid (FA) analog TOFA (30 μM) or the active site-directed CP640.186 (CP, 60 μM), two ACC inhibitors, for 2H before thrombin stimulation. Lipogenesis was measured via 14C-acetate incorporation into FA. Platelet functions were assessed by aggregometry. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Results Both ACC inhibitors decreased lipogenesis and thrombin-induced platelet aggregation. TOFA, but not CP, drastically decreased mitochondrial oxygen consumption rate, an effect associated with an increased reactive oxygen species production and AMPK activation. Surprisingly, the quantitative lipidomics analyses showed that a preincubation with CP did not affect global platelet lipid content. However, the short-term ACC inhibition (and potential acetyl-CoA accumulation) was sufficient to modify the level of acetylated proteins such as tubulin, a key player in the regulation of platelet shape change and aggregation. Conclusion Given the effect of TOFA on mitochondrial respiration, oxidative stress and AMPK activation, CP seems more appropriate to investigate the impact of ACC inhibition in platelets. A short-term acute treatment with CP inhibits thrombin-induced aggregation by a mechanism which doesn’t depend on lipid content but might involve changes in protein acetylation.

Objectives Activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) leads to phosphorylation of acetyl-CoA carboxylase (ACC) on serine 79 (phosphoACC) and its subsequent inhibition. Given the role of AMPK-ACC signaling in platelet lipid metabolism and function, this pathway could be affected in the platelets of patients with coronary artery disease (CAD), where the atherogenic environment has an impact on platelet biology. We hypothesized that platelet AMPKACC signaling is activated by atherogenic lipids and could be considered as a metabolic signature in high-risk CAD patients. We also explored the consequences of platelet phosphoACC on platelet lipid metabolic profile. Approach and results Blood samples from 188 consecutive patients admitted for coronary angiography were processed. Lipid extracts from the platelets of 31 patients were subjected to lipidomic analysis. PhosphoACC level in circulating platelets of CAD patients were significantly increased and was linked to the severity of coronary artery calcification as well as acute coronary syndrome (OR: 6.71, 95% CI: 2.06–21.91; P = 0.002). The triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) ratio, a well-known atherogenic marker, was strongly associated with increased phosphoACC in our CAD cohort. Hence, oxLDL activated AMPK-ACC signaling through a CD36-dependent pathway. Lipidomic analysis revealed that increased phosphoACC led to a down-regulation of intraplatelet TG, particularly of those containing C14:0 fatty acid chains. Conclusion Platelet phosphoACC is a marker for risk stratification in suspected CAD patients and reveals an interaction between platelets and lipids. Inhibitory phosphoACC impacts platelet lipid content by down-regulating TG, which in turn may affect platelet function (ACCTHEROMA, NCT03034148).

Introduction Sodium glucose co-transporters (SGLT) are well known for their role in glucose transport in the intestin or in the kidney. In a systematic evaluation of SGLT transporters, our group recently confirmed the expression of SGLT1 but discovered that the heart also express a second isoform, SMIT1 (sodium myo-inositol co transporter 1). The absence of SMIT1 does not change heart function and structure under baseline condition. However, the role of SMIT1 or myo-inositol transport in diseased heart remains unexplored. Objective In this study, we aimed to determine the human and murine cardiac expression pattern of SMIT1 in health and diseases. Methods Myocardial biopsies from 23 patients at end stage heart failure were obtained during Left Ventricle Assist Device (LVAD) implantation. Control biopsies corresponded to 11 patients with mitral regurgitation or stenosis without evidence of LV remodeling and were taken during valvular surgery. Mice model of heart failure were generated by a permanent ligation of left anterior descending artery (LAD). Results Compared to control hearts, human failing hearts exhibited higher SMIT1 expression. Increase in SMIT1 was more pronounced in ischemic versus non-ischemic cardiomyopathy. Interestingly, SMIT1 expression correlated with pro-fibroblastic markers in biopsies (αSmooth Muscle Actin and Collagen Ia), suggesting that SMIT1 was tightly associated with fibrosis. In mice model of myocardial infarction, SMIT1 expression was 2.5-fold higher in the scar compared to remote myocardium. As in human heart, SMIT1 expression correlated with pro-fibroblastic markers. Conclusion SMIT1 expression is increased in ischemic failing heart, reflecting increased cardiac fibrosis. The physiological and physiopathological role of SMIT1 in the heart remains to be determined.

Introduction Sodium-glucose transporter 1 (SGLT1) is the major SGLT isoform expressed in the heart. Under baseline condition, its absence does not affect heart function and structure. Using the pharmacological SGLT1 inhibitor, phlorizin, several data suggested that SGLT1 could contribute to overall glucose transport under physiological as well as under pathological conditions, i.e. in the ischemic heart. However, SGL1 role in glucose transport has never been formerly demonstrated. Purpose This study aimed to evaluate, in physiological and pathological conditions, the consequence of SGLT1 transporter invalidation on cardiomyocyte glucose uptake. Methods Glucose uptake was measured in isolated cardiomyocytes using 2,3[H]-glucose and 3[H]-2-deoxyglucose. SGLT1 contribution was evaluated by pharmacologic (phlorizin) and genetic means (KO mice). GLUT transporters expression was examined by RT-qPCR and Western Blot. Results Under basal and hyperglycemic conditions as well as under insulin or oligomycin stimulation, glucose uptake was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. We verified that SGLT1 absence was not compensated by an increase in GLUT1 or GLUT4 expression. Phlorizin inhibited insulin-stimulated glucose transport in a dose dependent manner, leading to its complete inhibition at high concentration (1 mM). However, this effect was independent of SGLT1 as phlorizin impact on glucose transport was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. Furthermore, the phlorizin-induced glucose uptake inhibition was not explained by any change on insulin signaling or GLUT4 translocation, suggesting that phlorizin directly affected the GLUTs facilitated glucose transporters. Conclusion Phlorizin effect on glucose transport does not involve SGLT1 but rather a direct effect on GLUTs. We also demonstrated that SGLT1 does not significantly contribute to glucose uptake in the cardiomyocyte in physiological and pathological conditions.

Introduction At rest, the heart favors fatty acids to produce its energy switching to glucose in response to insulin at postprandial state. Diabetic heart features metabolic adaptation defaults and is no longer able to switch, producing its energy exclusively through fatty acid oxidation. This metabolic inflexibility is a major factor of cardiac dysfunction. Observations made in high-fat diet mouse model lead to an increase in cardiac protein acetylation. We hypothesized that modulation of acetylation impacts the ability of the heart to uptake glucose with a special focus on tubulin. Objectives Microtubules playing a key role in GLUT4 translocation to the plasma membrane, we evaluated the impact of tubulin acetylation on cardiac glucose uptake. Methods The model is primary cultured rat cardiomyocytes. Acetylation levels and signaling pathways are evaluated by western blotting. The impact of long term incubation (20 h) with different pharmacological reducers of acetylation (garcinol 10 μM and anacardic acid 25 μM) on basal and insulin (3 nM, 30 min)-stimulated glucose uptake are measured following the detritiation rate of 2-3H-glucose. Tubacin, the inhibitor of tubulin deacetylase and an adenovirus overexpressing a non-acetylable form of tubulin are used to modulate tubulin acetylation. Results Insulin induces a 6-fold increase in glucose transport. Pharmacologically inhibiting global acetylation increases basal glucose transport similarly to insulin stimulation. By contrast, increasing specifically tubulin acetylation using tubacin inhibits 40% of basal and insulin-induced glucose transport. Accordingly, the overexpression of a dominant negative form and non-acetylable form of tubulin decreases endogenous tubulin acetylation while increasing basal glucose transport. Finally, targeting tubulin acetylation partially restores fatty acid-mediated inhibition of glucose transport. Conclusion There is a clear correlation between tubulin acetylation and glucose uptake level.

Introduction The sodium-glucose transporter 1 (SGLT1) has been identified as the major SGLT isoform expressed in the heart. The exact role of this transporter in the heart is still unknown. Several authors have postulated a role on glucose transport using phlorizin as a specific inhibitor of SGLT transporters. However, high concentrations of phlorizin have been used in these studies and questions remain towards its potential effects on facilitated glucose transporters (GLUTs). Indeed, phlorizin has a similar structure to phloretin, GLUT-specific inhibitor and it could be hydrolyzed into phloretin acting on glucose transport independently of SGLT transporters. Objective The aim of this study is to determine the contribution of SGLT1 transporter on glucose uptake in hearts and the impact of phlorizin on this uptake. Method We studied glucose uptake in vitro (cardiomyocytes), ex vivo (whole working hearts) and in vivo with SGLT1 WT and KO mice using 2-3[H]-glucose. Different phlorizin concentrations were tested to determine its impact on glucose uptake. Results We showed that glucose uptake was similar in SGLT1 WT and KO mice in basal, hyperglycemic and insulin-stimulated conditions. Moreover, we observed that high concentrations of phlorizin (10−3 M) completely inhibit glucose uptake in cardiomyocytes subjected to insulin stimulation and that this inhibition was similar in WT and KO SGLT1 mice. An inhibition was also found at lower concentration of phlorizin (10−4 M) to a lesser extent. Conclusion The contribution of SGLT1 in glucose uptake in the heart is marginal compared to facilitate glucose uptake. At least at high concentrations, phlorizin inhibits glucose uptake independently of SGLT1 questioning the use of this compound to study SGLT-dependent glucose uptake.

Ischemic preconditioning confers powerful protection against myocardial infarction through pre-emptive activation of survival signaling pathways, but it remains difficult to apply to patients with ischemic heart disease, and its effects are transient. Promoting a sustained activation of preconditioning mechanisms in vivo would represent a novel approach of cardioprotection. We tested the role of the protein H11 kinase (H11K), which accumulates by 4- to 6-fold in myocardium of patients with chronic ischemic heart disease and in experimental models of ischemia. This increased expression was quantitatively reproduced in cardiac myocytes using a transgenic (TG) mouse model. After 45 minutes of coronary artery occlusion and reperfusion, hearts from TG mice showed an 82+/-5% reduction in infarct size compared with wild-type (WT), which was similar to the 84+/-4% reduction of infarct size observed in WT after a protocol of ischemic preconditioning. Hearts from TG mice showed significant activation of survival kinases participating in preconditioning, including Akt and the 5'AMP-activated protein kinase (AMPK). H11K directly binds to both Akt and AMPK and promotes their nuclear translocation and their association in a multiprotein complex, which results in a stimulation of survival mechanisms in cytosol and nucleus, including inhibition of proapoptotic effectors (glycogen synthase kinase-3beta, Bad, and Foxo), activation of antiapoptotic effectors (protein kinase Cepsilon, endothelial and inducible NO synthase isoforms, and heat shock protein 70), increased expression of the hypoxia-inducible factor-1alpha, and genomic switch to glucose utilization. Therefore, activation of survival pathways by H11K preemptively triggers the antiapoptotic and metabolic response to ischemia and is sufficient to confer cardioprotection in vivo equally potent to preconditioning.

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.

Purpose The AMP-activated protein kinase (AMPK) regulates metabolic homeostasis and plays a protective role in the ischemic and diabetic hearts. The A-769662 compound, which directly binds and activates AMPK, has recently been characterized. Here, we studied the impact of A-769662 on cardiac AMPK signaling and glucose uptake which is known to participate in the protective action of AMPK. Methods Insulin and/or A-769662 were used to stimulate adult cardiomyocytes. The effect of A-769662 on the action of other AMPK activators has been also tested. Glucose uptake was measured by the detritriation rate of [2-3H] glucose. The phosphorylation state of signaling elements was measured by immunoblot. AMPK activity was assessed by the incorporation of radioactive phosphates on a specific synthetic peptide. Cell death and ROS production were evaluated using propidium iodide or by monitoring oxidation of a fluorescent probe, respectively. Results Glucose uptake was increased by both insulin and oligomycin. In the presence of insulin, oligomycin leads to an overstimulation of glucose uptake that correlated with an Akt overphopsphorylation. In contrast, A-769662 had no insulin sensitizing effects. Indeed, A-769662 promoted AMPK phosphorylation but did not stimulate glucose uptake and did not induce an overphosphorylation of Akt or an overstimulation of glucose uptake in the presence of insulin. Otherwise, A-769662 potentiates the effects of other AMPK activators. We showed an overphosphorylation of AMPK and of its substrate ACC, when A-769662 was added to oligomycin or hypoxia. This potentiation effect was accompanied by an overstimulation of glucose uptake and by a decrease in cell mortality and ROS production. Conclusions A-769662, by itself, is unable to stimulate glucose uptake or to increase insulin sensitivity. However, 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.

Background It was demonstrated that branched-chain amino acids like leucine induce insulin resistance in muscle and adipose tissues. The mechanism proposed to explain leucine action involves mTOR/p70S6K signaling. This pathway can be 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 effect in cardiomyocytes. Methods Primary cultured adult rat cardiomyocytes were pretreated with different concentrations of leucine (from 1 to 10 mM) during different periods of time (up to 20h) before being exposed to insulin (3x10 −9 M, 30 min). Results In absence of leucine, insulin induced a 6-fold increase in glucose uptake (0.31+/−0.04 vs. 0.05+/−0.01 μmoles/mg.h). This correlated with the increase in phosphorylation state of PKB and AS160, both known to regulate glucose transport downstream of insulin. Pre-incubation with leucine for 1 h stimulated mTOR/p70S6K pathway resulting in the inhibiting phosphorylation of IRS-1 located in the proximal insulin signaling pathway. This is accompanied by a significant decrease in PKB and AS160 phosphorylation but, surprisingly, insulin-stimulated glucose uptake was preserved (0.31+/–0.05 μmoles/mg.h). On the other hand, a longer incubation (14h) with leucine induced a drastic decrease in glucose transport (0.056+/–0.01 μmoles/mg.h). The mTOR/p70S6K inhibitor rapamycin did not prevent this inhibition. Moreover, the non-metabolized leucine analog BCH was able to stimulate mTOR/p70S6K pathway but had no effect on the insulin-mediated stimulation of glucose uptake. By contrast, intermediates of leucine catabolism, alpha-ketoisocaproate, acetoacetate and betahydroxybutyrate, inhibited glucose uptake similarly to leucine. Conclusion Leucine catabolism reduces insulin-dependent glucose transport independently of insulin signaling.

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.

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.

BackgroundAMPK activators, like resveratrol or metformin, inhibit pathological cardiac hypertrophy. However, despite evidence for their anti-hypertrophic effect, it seems that this phenomenon is mainly circumstantial. Indeed, those agents induce a rather non-specific AMPK activation by increasing the AMP/ATP ratio or by mimicking AMP. Hence, the aim of this study was to test the ability of a more specific AMPK activator, called A-769662, to prevent phenylephrine (PE)-induced hypertrophy in cultured neonatal rat ventricular myocytes (NRVM) and in adult rat ventricular myocytes (ARVM).MethodAlpha-actinin immunostaining, radio labelled amino acid incorporation, nuclear factor of activated T-cells (NFAT) activity, hypertrophy-linked gene expression and protein phosphorylation were analysed to determine NRVM hypertrophy. Cell surface area and protein phosphorylation were analysed to define ARVM hypertrophy.ResultsUsing dose–response experiments and genetic AMPK silencing, we show here that A-769662 is able to prevent the development of PE-induced NRVM hypertrophy by an AMPK-dependant mechanism. This hypertrophy prevention correlates with the modification of AMPK-related key regulators of cardiac hypertrophy including ~50% lower protein synthesis (p