Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart.

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.

INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown. OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P. METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury. CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

Ticagrelor Prevents Endothelial Cell Apoptosis through the Adenosine Signalling Pathway in the Early Stages of Hypoxia.

BACKGROUND: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia-reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. METHOD: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. RESULTS: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. CONCLUSION: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.

Simultaneous quantification of 8 nucleotides and adenosine in cells and their medium using UHPLC-HRMS.

Purinergic signalling is involved in physiological processes, particularly during ischemia-reperfusion injuries for which it has a protective effect. The purpose of this work was to develop a method for simultaneous quantification of eight nucleotides and adenosine in biological matrices by liquid chromatography coupled with high-resolution mass spectrometry. A method was developed that was sufficiently robust to quantify the targeted analytes in 20 min with good sensitivity. Analysis of extracellular media from cultured endothelial cells detected the release of nucleotides and adenosine during 2 h of hypoxia. The quantification of cylic adenosine monophosphate (cAMP) allowed to establish a dose-response curve after receptor stimulation. Therefore, our method allows us to study the involvement of nucleotides in various processes in both the intracellular and extracellular compartment.

Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs.

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor.

BACKGROUND AND PURPOSE: Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH: The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS: Pretreatment with 1µM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3ß (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS: Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.