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Inflammation causing oxidative stress in endothelial cells contributes to heart failure development. Sodium/glucose cotransporter 2 inhibitors (SGLT2i's) were shown to reduce heart failure hospitalization and oxidative stress. However, how inflammation causes oxidative stress in endothelial cells, and how SGLT2i's can reduce this is unknown. Here we hypothesized that 1) TNF-α activates the Na + /H + exchanger (NHE) and raises cytoplasmatic Na + ([Na + ] c ), 2) increased [Na + ] c causes reactive oxygen species (ROS) production, and 3) empagliflozin (EMPA) reduces inflammation-induced ROS through NHE inhibition and lowering of [Na + ] c in human endothelial cells. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10 ng/ml TNF-α, 1 µM EMPA or the NHE inhibitor Cariporide (CARI, 10 µM) and NHE activity, intracellular [Na + ] c and ROS were analyzed. TNF-α enhanced NHE activity in HCAECs and HUVECs by 92% (p < 0.01) and 51% (p < 0.05), respectively, and increased [Na + ] c from 8.2 ± 1.6 to 11.2 ± 0.1 mM (p < 0.05) in HCAECs. Increasing [Na + ] c by ouabain elevated ROS generation in both HCAECs and HUVECs. EMPA inhibited NHE activity in HCAECs and in HUVECs. EMPA concomitantly lowered [Na + ] c in both cell types. In both cell types, TNF α-induced ROS was lowered by EMPA or CARI, with no further ROS lowering by EMPA in the presence of CARI, indicating EMPA attenuated ROS through NHE inhibition. In conclusion, inflammation induces oxidative stress in human endothelial cells through NHE activation causing elevations in [Na + ] c , a process that is inhibited by EMPA through NHE inhibition., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aims: Sodium glucose cotransporter 2 (SGLT2) inhibitors have sodium-hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes, but it is unknown whether these properties extend to the intact heart during ischaemia-reperfusion (IR) conditions. NHE inhibitors as Cariporide delay time to onset of contracture (TOC) during ischaemia and reduce IR injury. We hypothesized that, in the ex vivo heart, Empagliflozin (Empa) mimics Cariporide during IR by delaying TOC and reducing IR injury. To facilitate translation to in vivo conditions with insulin present, effects were examined in the absence and presence of insulin., Methods and Results: Isolated C57Bl/6NCrl mouse hearts were subjected to 25 min I and 120 min R without and with 50 mU/L insulin. Without insulin, Empa and Cari delayed TOC by 100 and 129 s, respectively, yet only Cariporide reduced IR injury [infarct size (mean ± SEM in %) from 51 ± 6 to 34 ± 5]. Empa did not delay TOC in the presence of the NHE1 inhibitor Eniporide. Insulin perfusion increased tissue glycogen content at baseline (from 2 ± 2 µmol to 42 ± 1 µmol glycosyl units/g heart dry weight), amplified G6P and lactate accumulation at end-ischaemia, thereby decreased mtHKII and exacerbated IR injury. Under these conditions, Empa (1 µM) and Cariporide (10 µM) were without effect on TOC and IR injury. Empa and Cariporide both inhibited NHE activity, in isolated cardiomyocytes, independent of insulin., Conclusions: In the absence of insulin, Empa and Cariporide strongly delayed the time to onset of contracture during ischaemia. In the presence of insulin, both Empa and Cari were without effect on IR, possibly because of severe ischaemic acidification. Insulin exacerbates IR injury through increased glycogen depletion during ischaemia and consequently mtHKII dissociation. The data suggest that also in the ex vivo intact heart Empa exerts direct cardiac effects by inhibiting NHE during ischaemia, but not during reperfusion., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background/aims: Heart failure is characterized by chronic low-grade vascular inflammation, which in itself can lead to endothelial dysfunction. Clinical trials showed reductions in heart failure-related hospitalizations of type 2 diabetic patients using sodium glucose co-transporter 2 inhibitors (SGLT2i's). Whether and how SGLT2i's directly affect the endothelium under inflammatory conditions is not completely understood. The aim of the study was to investigate whether the SGLT2i Empagliflozin (EMPA) and Dapagliflozin (DAPA) reduce tumor necrosis factor α (TNFα) induced endothelial inflammation in vitro., Methods: Human coronary arterial endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were (pre-)incubated with 1 µM EMPA or DAPA and subsequently exposed to 10 ng/ml TNFα. ROS and NO were measured using live cell imaging. Target proteins were either determined by infrared western blotting or fluorescence activated cell sorting (FACS). The connection between Cav-1 and eNOS was determined by co-immunoprecipitation., Results: Nitric oxide (NO) bioavailability was reduced by TNFα and both EMPA and DAPA restored NO levels in TNFα-stimulated HCAECs. Intracellular ROS was increased by TNFα, and this increase was completely abolished by EMPA and DAPA in HCAECs by means of live cell imaging. eNOS signaling was significantly disturbed after 24 h when cells were exposed to TNFα for 24h, yet the presence of both SGLT2is did not prevent this disruption. TNFα-induced enhanced permeability at t=24h was unaffected in HUVECs by EMPA. Similarly, adhesion molecule expression (VCAM-1 and ICAM-1) was elevated after 4h TNFα (1.5-5.5 fold increase of VCAM-1 and 4-12 fold increase of ICAM-1) but were unaffected by EMPA and DAPA in both cell types. Although we detected expression of SGLT2 protein levels, the fact that we could not silence this expression by means of siRNA and the mRNA levels of SGLT2 were not detectable in HCAECs, suggests aspecificity or our SGLT2 antibody and absence of SGLT2 in our cells., Conclusion: These data suggest that EMPA and DAPA rather restore NO bioavailability by inhibiting ROS generation than by affecting eNOS expression or signaling, barrier function and adhesion molecules expression in TNFα-induced endothelial cells. Furthermore, the observed effects cannot be ascribed to the inhibition of SGLT2 in endothelial cells., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)