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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]

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]

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]

Aims Transgenic mice are frequently used to investigate the role of genes involved in cardiac conduction. The QRS duration calculated from the electrocardiogram (ECG) is a commonly used measure for ventricular conduction time. However, the relation between ventricular activation and QRS duration calculated from a mouse surface ECG is not well understood. We aim to relate ventricular activation and repolarization patterns with the mouse ECG. Methods and results Ventricular activation and repolarization patterns generated by high-density optical mapping and a six-lead pseudo-ECG were compared in isolated mouse hearts. In addition, mouse ECGs were simulated in silico. Right-ventricular activation ends later than left-ventricular activation. Final activation coincided with the end of the QRS complex in leads III and aVF, but not in leads I, II, aVR, and aVL. The pattern of early repolarization (at 20% of repolarization, RT20) but not of RT50 or RT80 followed the activation pattern. After sodium channel blockade by ajmaline, total ventricular activation time increased by 10.0 ms, whereas QRS duration increased by only 2.1 ms. In mice carrying a mutation in Scn5a (1798insD), ventricular activation ended after the end of the QRS complex (12.9 ± 0.1 vs. 10.8 ± 0.3). Conclusion In the mouse, ventricular myocardium activation and early repolarization waves are simultaneously present. This hampers unequivocal interpretation of the duration of the QRS complex as a measure of ventricular activation duration, especially when conduction is slowed. Under these conditions mapping of local activation and repolarization patterns is required for correct interpretation of the ECG. [ABSTRACT FROM PUBLISHER]

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]