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Background: SGLT2i directly inhibit the cardiac sodium-hydrogen exchanger-1 (NHE1) in isolated ventricular cardiomyocytes (CMs). However, other studies with SGLT2i have yielded conflicting results. This may be explained by methodological factors including cell isolation techniques, cell types and ambient pH. In this study, we tested whether the use of protease XIV (PXIV) may abrogate inhibition of SGLT2i on cardiac NHE1 activity in isolated rabbit CMs or rat cardiomyoblast cells (H9c2), in a pH dependent manner. Methods: Rabbit ventricular CMs were enzymatically isolated from Langendorff-perfused hearts during a 30-min perfusion period followed by a 25-min after-dissociation period, using a collagenase mixture without or with a low dose PXIV (0.009 mg/mL) present for different periods. Empagliflozin (EMPA) inhibition on NHE activity was then assessed at pH of 7.0, 7.2 and 7.4. In addition, effects of 10 min PXIV treatment were also evaluated in H9c2 cells for EMPA and cariporide NHE inhibition. Results: EMPA reduced NHE activity in rabbit CMs that were not exposed to PXIV treatment or undergoing a 35-min PXIV treatment, independent of pH levels. However, when exposure time to PXIV was extended to 55 min, NHE inhibition by Empa was completely abolished at all three pH levels. In H9c2 cells, NHE inhibition by EMPA was evident in non-treated cells but lost after 10-min incubation with PXIV. NHE inhibition by cariporide was unaffected by PXIV. Conclusion: The use of protease XIV in cardiac cell isolation procedures obliterates the inhibitory effects of SGLT2i on NHE1 activity in isolated cardiac cells, independent of pH. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are the first antidiabetic compounds that effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetics. Being explicitly designed to inhibit SGLT2 in the kidney, SGLT2i have lately been investigated for their off-target cardiac actions. Here, we review the direct effects of SGLT2i Empagliflozin (Empa), Dapagliflozin (Dapa), and Canagliflozin (Cana) on various cardiac cell types and cardiac function, and how these may contribute to the cardiovascular benefits observed in large clinical trials. SGLT2i impaired the Na+/H+ exchanger 1 (NHE-1), reduced cytosolic [Ca2+] and [Na+] and increased mitochondrial [Ca2+] in healthy cardiomyocytes. Empa, one of the best studied SGLT2i, maintained cell viability and ATP content following hypoxia/reoxygenation in cardiomyocytes and endothelial cells. SGLT2i recovered vasoreactivity of hyperglycemic and TNF-α-stimulated aortic rings and of hyperglycemic endothelial cells. Anti-inflammatory actions of Cana in IL-1β-treated HUVEC and of Dapa in LPS-treated cardiofibroblast were mediated by AMPK activation. In isolated mouse hearts, Empa and Cana, but not Dapa, induced vasodilation. In ischemia-reperfusion studies of the isolated heart, Empa delayed contracture development during ischemia and increased mitochondrial respiration post-ischemia. Direct cardiac effects of SGLT2i target well-known drivers of diabetes and heart failure (elevated cardiac cytosolic [Ca2+] and [Na+], activated NHE-1, elevated inflammation, impaired vasorelaxation, and reduced AMPK activity). These cardiac effects may contribute to the large beneficial clinical effects of these antidiabetic drugs. [ABSTRACT FROM AUTHOR]

The cardiac autonomic nervous system (ANS) controls normal atrial electrical function. The cardiac ANS produces various neuropeptides, among which the neurokinins, whose actions on atrial electrophysiology are largely unknown. We here demonstrate that the neurokinin substance-P (Sub-P) activates a neurokinin-3 receptor (NK-3R) in rabbit, prolonging action potential (AP) duration through inhibition of a background potassium current. In contrast, ventricular AP duration was unaffected by NK-3R activation. NK-3R stimulation lengthened atrial repolarization in intact rabbit hearts and consequently suppressed arrhythmia duration and occurrence in a rabbit isolated heart model of atrial fibrillation (AF). In human atrial appendages, the phenomenon of NK-3R mediated lengthening of atrial repolarization was also observed. Our findings thus uncover a pathway to selectively modulate atrial AP duration by activation of a hitherto unidentified neurokinin-3 receptor in the membrane of atrial myocytes. NK-3R stimulation may therefore represent an anti-arrhythmic concept to suppress re-entry-based atrial tachyarrhythmias, including AF. [ABSTRACT FROM AUTHOR]

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 Ca2+ ([Ca2+]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, O2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined.Results: EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4+ 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. [ABSTRACT FROM AUTHOR]

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]) and Ca ([Ca]) concentrations and decreased mitochondrial Ca concentration ([Ca]) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na], [Ca] and [Ca] in cardiomyocytes. Methods: [Na] [Ca], [Ca ] 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] and [Ca] levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na] and [Ca] and increased [Ca]. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na] 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] and [Ca] and enhancing [Ca], through impairment of myocardial NHE flux, independent of SGLT2 activity. [ABSTRACT FROM AUTHOR]

Background: Hypercholesterolemia protects against ventricular fibrillation in patients with myocardial infarction. We hypothesize that hypercholesterolemia protects against ischemia-induced reentrant arrhythmias because of altered ion channel function.Methods and Results: ECGs were measured in low-density lipoprotein receptor knockout (LDLr(-/-)), apolipoprotein A1 knockout (ApoA1(-/-)), and wild-type (WT) mice. Action potentials, calcium handling, and ion currents were recorded in ventricular myocytes. Gene expression was determined by quantitative polymerase chain reaction and Western blot. In isolated perfused hearts, regional ischemia was induced and arrhythmia inducibility was tested. Serum low-density lipoprotein (LDL) cholesterol was higher in LDLr(-/-) mice than in WT mice (2.6 versus 0.4 mmol/L), and high-density lipoprotein cholesterol was significantly lower in ApoA1(-/-) mice than in WT mice (0.3 versus 1.8 mmol/L). LDLr(-/-) and ApoA1(-/-) myocytes contained more cholesterol than WT (34.4±2.8 and 36.5±2.4 versus 25.5±0.4 μmol/g protein). The major potassium currents were not different in LDLr(-/-) and ApoA1(-/-) compared with WT mice. The L-type calcium current (I(Ca)), however, was larger in LDLr(-/-) and ApoA1(-/-) than in WT (12.1±0.7 and 12.8±0.8 versus 9.4±1.1 pA/pF). Calcium transient amplitude and fractional sarcoplasmic reticulum calcium release were larger and action potential and QTc duration longer in LDLr(-/-) and ApoA1(-/-) than in WT mice (action potential duration at 90% of repolarization: 102±4 and 106±3 versus 84±3.1 ms; QTc: 50.9±1.3 and 52.8±0.8 versus 43.5±1.2 ms). During ischemia, ventricular tachycardia/ventricular fibrillation inducibility was larger in WT than in LDLr(-/-) and ApoA1(-/-) hearts. Expression of sodium channel and Ca-handling genes were not significantly different between groups.Conclusions: Dyscholesterolemia is associated with action potential prolongation because of increased I(Ca) and reduces occurrence of reentrant arrhythmias during ischemia. [ABSTRACT FROM AUTHOR]

31P MRS provides a unique non-invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small-sized, fast-beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three-dimensional image-selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4 T. Cardiac 31P MR spectra were acquired in vivo in healthy mice ( n = 9) and in transverse aortic constricted (TAC) mice ( n = 8) using respiratory-gated, cardiac-triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′-triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one-dimensional (1D) ISIS resulted in two-fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory-gated, cardiac-triggered 3D ISIS allows for non-invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The article discusses the results of a study on the role of a diet rich in unsaturated fatty acids in the prevention of heart failure (HF). The study used rabbits as a model of pressure and volume overload. The results showed that rabbits fed with high oleic sunflower oil or fish oil had larger myocardial fatty acid oxidation capacity than rabbits fed with no supplement. Unsaturated fatty acids contained in sunflower oil or fish oil can prevent HF and hypertrophy and HF-induced arrhythmias.

Abstract: Objective: Dietary supplementation with fish oil-derived n-3 fatty acids reduces mortality in patients with myocardial infarction, but may have adverse effects in angina patients. The underlying electrophysiologic mechanisms are poorly understood. We studied the arrhythmias and the electrophysiologic changes during regional ischemia in hearts from pigs fed a diet rich in fish oil. Methods: Pigs received diets rich in fish oil, in sunflower oil, or a control diet for 8 weeks. Hearts were isolated and perfused. Ischemia was created by occluding the left anterior descending artery. Diastolic stimulation threshold, refractory period, conduction velocity, activation recovery intervals and the maximum downstroke velocity of 176 electrograms were measured in the ischemic zone. Spontaneous arrhythmias during 75 min of regional ischemia were counted. Results: More episodes of spontaneous ischemia-induced sustained ventricular tachycardia and ventricular fibrillation occurred in the fish oil and sunflower oil group than in the control group. More inexcitable myocardium was present in the ischemic zone in the group fed fish oil or sunflower oil than in the control group after 20 min of ischemia. After 40 min of ischemia, more block occurred in the control group than in the other groups. The downstroke velocity of the electrograms in the ischemic border zone was lower in the fish oil group and sunflower oil group than in the control after 20 min. Conclusions: A diet rich in fish oil results in proarrhythmia compared to a control diet during regional ischemia in pigs. Myocardial excitability is reduced in the fish oil and sunflower oil group during the early phase of arrhythmogenesis. In the late phase of arrhythmogenesis, excitability is more reduced in the control group than in the fish oil and sunflower oil group. [Copyright &y& Elsevier]

Abstract: Background: Omega-3 polyunsaturated fatty acids (ω3-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered ω3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to ω3-PUFA-incorporation into the sarcolemma on membrane currents are unknown. Methods: Pigs received a diet either rich in ω3-PUFAs or in ω9-fatty acids for 8weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies. Results: ω3-VMs contained higher amounts of ω3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In ω3 VMs, L-type Ca2+ current (I Ca,L) and Na+–Ca2+ exchange current (I NCX) were reduced by approximately 20% and 60%, respectively, and inward rectifier K+ current (I K1) and slow delayed rectifier K+ current (I Ks) were increased by approximately 50% and 70%, respectively, compared to control. Densities of rapid delayed rectifier K+ current, Ca2+-activated Cl− current, and Na+ current (I Na) were unchanged, although voltage-dependence of I Na inactivation was more negative in ω3 VMs. Conclusions: A fish oil diet increases ω3-PUFA content in the ventricular sarcolemma, decreases I Ca,L and I NCX, and increases I K1 and I Ks, resulting in AP shortening. Incorporation of ω3-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating ω3-PUFAs. [Copyright &y& Elsevier]

Abstract: Objective: In patients with heart disease, the transition from compensatory hypertrophy to heart failure (HF) is associated with altered calcium handling. Up-regulated Na+/H+-exchanger (NHE-1) activity underlies increased [Na+]i and disturbance of cellular calcium handling in HF. We hypothesize that chronic inhibition of NHE-1 activity prevents the hypertrophic response, cellular remodeling, and development of HF. Methods: Rabbits received a control or cariporide (inhibitor of NHE-1) diet for 3 months, starting after induction of combined volume and pressure overload. Age-matched animals served as control. Development of HF was examined echocardiographically and electrocardiographically after 3 months. [Na+]i, [Ca2+]i, pHi, and action potentials were measured in left ventricular midmural myocytes with SBFI, indo-1, SNARF, and di-4-anepps. Sarcoplasmic reticulum calcium content was calculated from the response of [Ca2+]i to rapid cooling. Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. Results: Chronic treatment of rabbits with the specific Na+/H+-exchanger activity inhibitor cariporide greatly attenuated development of hypertrophy and entirely abolished development of HF; the heart/body weight ratio increased only little, no change in lung weight occurred, left ventricular dimensions and fractional shortening changed mildly, ascites was not present, QT duration did not increase, and sudden death did not occur. Chronic cariporide treatment also prevented cellular electrical and ionic remodeling. Myocyte dimensions were unaltered, action potentials were not prolonged, cytoplasmic sodium and NHE-1 activity did not increase, cytoplasmic and SR calcium handling remained undisturbed, and no increase of the incidence of calcium after-transient dependent delayed after depolarizations (DADs) occurred. Conclusion: We conclude that enhanced activity of NHE-1 underlies cardiac cellular electrical and ionic remodeling in experimental heart failure, and that chronic dietary treatment with cariporide attenuates hypertrophy, development of HF, and cellular remodeling. [Copyright &y& Elsevier]

Objective: Myocardial ischemia and ventricular arrhythmias often complicate congestive heart failure. Ischemia-induced dispersion in repolarization is an important arrhythmogenic factor that might be caused by intrinsic cellular differences in response to simulated ischemia (SI) or by changed coupling of myocytes. We hypothesized that intrinsic heterogeneity in action potential duration (APD) or the occurrence of rigor is larger in failing than in normal rabbit myocytes during SI. Methods: Heart failure (HF) was induced with volume and pressure overload. Left ventricular myocytes from apex, free wall and base were enzymatically isolated and exposed to SI with NaCN. Results: There were no baseline differences in APD before SI. During SI no differences in time to inexcitability occurred but the range in APD increased more in HF than in normal cells. Rigor occurred after 16.8±3.5 and 23.0±7.5 min (P<0.05) in normal and HF myocytes, with no differences between apical, free wall or base cells. Variance in time to rigor was larger in HF than in normal cells (55.7 versus 12.4 min2). Blockade of anaerobic reserve decreased variance in time to rigor, also when normalized to mean, in HF and normal myocytes. In coupled normal and HF cell pairs, no delay in action potential propagation or differences in APD occurred during SI, and time to rigor was synchronized (P<0.05 vs. single cells). Conclusions: Intercellular differences in APD and in time of rigor arise in normal and HF myocytes subjected to SI, and are inhibited by blockade of anaerobic glycolysis. Dispersion in APD and tolerance to SI is increased in HF cells. APD and time to rigor are completely synchronized in coupled cell pairs. [Copyright &y& Elsevier]

Objective: After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart failure (HF). Methods: We measured the stimulation rate dependence (0.2–3 Hz) of diastolic calcium, calcium transient amplitude and SR calcium content in left ventricular myocytes isolated from hearts of rabbits with pressure and volume overload-induced HF and age-matched control animals. Cytosolic calcium was measured with indo-1. In some experiments, delayed after-depolarizations (DADs) were monitored with the voltage sensitive dye di-4-Annepps. SR calcium content was estimated from the response to rapid cooling (RC). After-transients were elicited in the presence of norepinephrine (100 nmol/l) after cessation of burst pacing. Results: With increasing stimulation rate (0.2–3.0 Hz): (1) steady state diastolic [Ca]i increased from 102 to 174 nmol/l in HF and from 44 to 103 nmol/l in control, (2) calcium transient amplitudes decreased from 310 to 254 nmol/l in HF and increased from 186 to 429 nmol/l in control, (3) SR calcium content decreased from 1.25 to 1.09 mmol/l in HF and increased from 1.51 to 2.48 mmol/l in control, (4) in HF and control, the end diastolic SR membrane calcium gradient decreased by about 30%; at any stimulation rate, the magnitude of gradient in HF was one-third of control, (5) systolic depletion of SR was 85% in HF and 60% in control. In HF, noradrenaline (100 nmol/l) increased SR calcium content and SR membrane gradient by 40% versus about 7% in control. Calcium after-transients were observed in 14 out of 18 HF rabbits, and none in eight control animals and were associated with DADs. Calcium after-transients were associated with a 35% decrease in SR calcium content. The frequency of occurrence of calcium after-transients was related to diastolic calcium. Conclusions: in HF, diastolic calcium is increased and both SR calcium content and SR membrane calcium gradient are decreased in a stimulation rate-dependent manner. In HF, β-adrenergic stimulation can partly restore the SR calcium content and SR membrane gradient at higher stimulation rates in a meta-stable condition; upon transition to low stimulation rates, the SR membrane can no longer maintain this high unbalanced SR calcium load at increased diastolic calcium, the magnitude of which is causally related to the occurrence of calcium after-transients. [Copyright &y& Elsevier]

Abstract: By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO2) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO2 within the in vivo heart?, 2) is mitoPO2 heterogeneously distributed?, and 3) how does mitoPO2 of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO2 was 35±5 mm Hg. The mitoPO2 was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO2 between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0–10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO2. For Langendorff-perfused rat hearts, mean mitoPO2 was 29±5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO2 between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO2 is considerably heterogeneous, and 3) that mitoPO2 of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart. [Copyright &y& Elsevier]

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. [Display omitted] • TNF-α increases intracellular sodium and activates NHE-1 in human endothelial cells. • Increasing intracellular sodium drives ROS formation in human endothelial cells. • Empagliflozin lowers ROS through NHE-1 inhibition and cellular sodium lowering. [ABSTRACT FROM AUTHOR]