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      Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na +/H + exchanger, lowering of cytosolic Na + and vasodilation

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          Abstract

          Aims/hypothesis

          Sodium–glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na + ([Na +] c) and cytosolic Ca 2+ ([Ca 2+] c) concentrations through inhibition of Na +/H + exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na +] c; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice.

          Methods

          Cardiac NHE activity and [Na +] c in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 μmol/l), DAPA (1 μmol/l), CANA (3 μmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O 2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined.

          Results

          EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH 4 + pulse: EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p < 0.001 for all three comparisons) and reduced [Na +] c (in mmol/l: EMPA 10.0 ± 0.5, DAPA 10.7 ± 0.7 and CANA 11.0 ± 0.9 vs vehicle 12.7 ± 0.7; p < 0.001). Docking studies provided high binding affinity of all three SGLT2i with the extracellular Na +-binding site of NHE. EMPA and CANA, but not DAPA, induced coronary vasodilation of the intact heart. PCr/ATP remained unaffected.

          Conclusions/interpretation

          EMPA, DAPA and CANA directly inhibit cardiac NHE flux and reduce [Na +] c, possibly by binding with the Na +-binding site of NHE-1. Furthermore, EMPA and CANA affect the healthy heart by inducing vasodilation. The [Na +] c-lowering class effect of SGLT2i is a potential approach to combat elevated [Na +] c that is known to occur in heart failure and diabetes.

          Electronic supplementary material

          The online version of this article (10.1007/s00125-017-4509-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

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          Most cited references14

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          Empagliflozin decreases myocardial cytoplasmic Na + through inhibition of the cardiac Na + /H + exchanger in rats and rabbits

          Aims/hypothesis Empagliflozin (EMPA), an inhibitor of the renal sodium–glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na+ ([Na+]c) and Ca2+ ([Ca2+]c) concentrations and decreased mitochondrial Ca2+ concentration ([Ca2+]m) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na+]c, [Ca2+]c and [Ca2+]m in cardiomyocytes. Methods [Na+]c, [Ca2+]c, [Ca 2+]m and Na+/H+ exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats. Results An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na+]c and [Ca2+]c levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na+]c and [Ca2+]c and increased [Ca2+]m. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na+]c and NHE flux in the absence of extracellular glucose. Conclusions/interpretation The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [Na+]c and [Ca2+]c and enhancing [Ca2+]m, through impairment of myocardial NHE flux, independent of SGLT2 activity. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-4134-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
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            Intracellular Na(+) concentration is elevated in heart failure but Na/K pump function is unchanged.

            Intracellular sodium concentration ([Na(+)](i)) modulates cardiac contractile and electrical activity through Na/Ca exchange (NCX). Upregulation of NCX in heart failure (HF) may magnify the functional impact of altered [Na(+)](i). We measured [Na(+)](i) by using sodium binding benzofuran isophthalate in control and HF rabbit ventricular myocytes (HF induced by aortic insufficiency and constriction). Resting [Na(+)](i) was 9.7+/-0.7 versus 6.6+/-0.5 mmol/L in HF versus control. In both cases, [Na(+)](i) increased by approximately 2 mmol/L when myocytes were stimulated (0.5 to 3 Hz). To identify the mechanisms responsible for [Na(+)](i) elevation in HF, we measured the [Na(+)](i) dependence of Na/K pump-mediated Na(+) extrusion. There was no difference in V(max) (8.3+/-0.7 versus 8.0+/-0.8 mmol/L/min) or K(m) (9.2+/-1.0 versus 9.9+/-0.8 mmol/L in HF and control, respectively). Therefore, at measured [Na(+)](i) levels, the Na/K pump rate is actually higher in HF. However, resting Na(+) influx was twice as high in HF versus control (2.3+/-0.3 versus 1.1+/-0.2 mmol/L/min), primarily the result of a tetrodotoxin-sensitive pathway. Myocyte [Na(+)](i) is elevated in HF as a result of higher diastolic Na(+) influx (with unaltered Na/K-ATPase characteristics). In HF, the combined increased [Na(+)](i), decreased Ca(2+) transient, and prolonged action potential all profoundly affect cellular Ca(2+) regulation, promoting greater Ca(2+) influx through NCX during action potentials. Notably, the elevated [Na(+)](i) may be critical in limiting the contractile dysfunction observed in HF.
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              Glycemic Control with Ipragliflozin, a Novel Selective SGLT2 Inhibitor, Ameliorated Endothelial Dysfunction in Streptozotocin-Induced Diabetic Mouse

              Background Endothelial dysfunction caused by increased oxidative stress is a critical initiator of macro- and micro-vascular disease development in diabetic patients. Ipragliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, offers a novel approach for the treatment of diabetes by enhancing urinary glucose excretion. The aim of this study was to examine whether ipragliflozin attenuates endothelial dysfunction in diabetic mice. Methods Eight-week-old male C57BL/6 mice were treated with streptozotocin (150 mg/kg) by a single intraperitoneal injection to induce diabetes mellitus. At 3 days of injection, ipragliflozin (3 mg/kg/day) was administered via gavage for 3 weeks. Vascular function was assessed by isometric tension recording. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RNA and protein expression were examined by quantitative RT-PCR (qPCR) and western blot, respectively. Oxidative stress was determined by measuring urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) level. Results Ipragliflozin administration significantly reduced blood glucose level (P < 0.001) and attenuated the impairment of endothelial function in diabetic mice, as determined by acetylcholine-dependent vasodilation (P < 0.001). Ipragliflozin did not alter metabolic parameters, such as body weight and food intake. Ipragliflozin administration ameliorated impaired phosphorylation of Akt and eNOSSer1177 in the abdominal aorta and reduced reactive oxygen species generation as determined by urinary excretion of 8-OHdG in diabetic mice. Furthermore, qPCR analyses demonstrated that ipragliflozin decreased the expression of inflammatory molecules [e.g., monocyte chemoattractant protein-1 (MCP-1) vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule (ICAM)-1] in the abdominal aorta (P < 0.05). In in vitro studies, incubation with methylglyoxal, one of the advanced glycation end products, significantly impaired phosphorylation of Akt and eNOSSer1177 (P < 0.01) and increased the expression of MCP-1, VCAM-1, and ICAM-1 in HUVEC. Conclusion Ipragliflozin improved hyperglycemia and prevented the development of endothelial dysfunction under a hyperglycemic state, at least partially by attenuation of oxidative stress.
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                Author and article information

                Contributors
                c.j.zuurbier@amc.uva.nl
                Journal
                Diabetologia
                Diabetologia
                Diabetologia
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0012-186X
                1432-0428
                2 December 2017
                2 December 2017
                2018
                : 61
                : 3
                : 722-726
                Affiliations
                [1 ]ISNI 0000000084992262, GRID grid.7177.6, Laboratory of Experimental Intensive Care and Anaesthesiology, Department of Anaesthesiology, Academic Medical Center, , University of Amsterdam, ; Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
                [2 ]ISNI 0000000084992262, GRID grid.7177.6, Department of Clinical and Experimental Cardiology, Academic Medical Center, , University of Amsterdam, ; Amsterdam, the Netherlands
                [3 ]ISNI 0000000084992262, GRID grid.7177.6, Department of Medical Biochemistry, Academic Medical Center, , University of Amsterdam, ; Amsterdam, the Netherlands
                Article
                4509
                10.1007/s00125-017-4509-7
                6448958
                29197997
                18453c48-8884-4b06-9bc5-d1ed8835a5ab
                © The Author(s) 2017

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 5 October 2017
                : 1 November 2017
                Funding
                Funded by: Academic Medical Center (AMC)
                Categories
                Short Communication
                Custom metadata
                © Springer-Verlag GmbH Germany, part of Springer Nature 2018

                Endocrinology & Diabetes
                cardiac,diabetes,heart failure,na+/h+ exchanger,sglt2i,sodium,vasodilation
                Endocrinology & Diabetes
                cardiac, diabetes, heart failure, na+/h+ exchanger, sglt2i, sodium, vasodilation

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