Selective NCX Inhibitor Research Articles

Abstract 12449: Inhibition of the Sodium Potassium ATPase Alpha 3 (ATP1A3) Pump Function Alters Repolarization Time and is Proarrhythmic in Cardiac Myocytes

Introduction: Inhibition of Na + /K + -ATPase (NKA) is arrhythmogenic, but the specific effect of alpha 3 NKA isoform (ATP1A3) inhibition is unknown. Variants in human ATP1A3 can lead to ventricular arrhythmias, yet the mechanism remains unknown. Hypothesis: Loss of NKA function results in shortening of QT interval and increased arrhythmia susceptibility due to intracellular Na+ and Ca2+ overload. Pharmacological inhibition of sodium-calcium exchanger (NCX) can rescue ECG parameter changes and arrhythmic phenotypes by reducing Ca2+ overload in the cell. Methods: Whole-cell patch clamp action potential recordings were conducted on induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from an ostensibly healthy individual to measure action potential duration (APD) and presence of delayed after depolarizations (DADs). Cells were exposed to 10 -7 M ouabain and 10 -5 M ORM-10103 (a selective inhibitor of NCX). ECGs and in vivo intracardiac electrophysiology studies were performed in mice at 12-14 weeks of age after intraperitoneal drug administration of 5 μg/g ouabain and 10 μg/g ORM-10103. Results: APD recordings of iPSC-CMs demonstrated both shortening and lengthening of APD with ouabain exposure. In both models, DADs were found in 6 of 18 cells with ouabain treatment. These findings were rescued with pretreatment using ORM-10103, as 0 of 8 cells developed DADs. On mouse ECG recordings, ouabain led to QRS and QT prolongation. During programmed electrical stimulation, no arrhythmia was noted with vehicle-treated mice. Following ouabain administration, 2 in 12 mice developed induced monomorphic ventricular tachycardia. Pretreatment of ORM-10103 in mice did not rescue QRS and QT prolongation with ouabain administration. Conclusions: Pharmacologic inhibition of ATP1A3 variably altered cardiac repolarization time in a species-specific manner, yet was associated with a substrate for triggered arrhythmias. The arrhythmogenic phenotype was rescued with inhibition of NCX in human cardiac myocytes, suggesting that NCX plays a role in the arrhythmogenic mechanism of ATP1A3. However, these effects were not seen in murine cardiac myocytes. Exploration of specifies-specific differences in NKA isoform expression and cardiac repolarization is needed.

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

SAR340835, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Cardiac Function and Restores Sympathovagal Balance in Heart Failure.

In failing hearts, Na+/Ca2+ exchanger (NCX) overactivity contributes to Ca2+ depletion, leading to contractile dysfunction. Inhibition of NCX is expected to normalize Ca2+ mishandling, to limit afterdepolarization-related arrhythmias, and to improve cardiac function in heart failure (HF). SAR340835/SAR296968 is a selective NCX inhibitor for all NCX isoforms across species, including human, with no effect on the native voltage-dependent calcium and sodium currents in vitro. Additionally, it showed in vitro and in vivo antiarrhythmic properties in several models of early and delayed afterdepolarization-related arrhythmias. Its effect on cardiac function was studied under intravenous infusion at 250,750 or 1500 µg/kg per hour in dogs, which were either normal or submitted to chronic ventricular pacing at 240 bpm (HF dogs). HF dogs were infused with the reference inotrope dobutamine (10 µg/kg per minute, i.v.). In normal dogs, NCX inhibitor increased cardiac contractility (dP/dtmax) and stroke volume (SV) and tended to reduce heart rate (HR). In HF dogs, NCX inhibitor significantly and dose-dependently increased SV from the first dose (+28.5%, +48.8%, and +62% at 250, 750, and 1500 µg/kg per hour, respectively) while significantly increasing dP/dtmax only at 1500 (+33%). Furthermore, NCX inhibitor significantly restored sympathovagal balance and spontaneous baroreflex sensitivity (BRS) from the first dose and reduced HR at the highest dose. In HF dogs, dobutamine significantly increased dP/dtmax and SV (+68.8%) but did not change HR, sympathovagal balance, or BRS. Overall, SAR340835, a selective potent NCX inhibitor, displayed a unique therapeutic profile, combining antiarrhythmic properties, capacity to restore systolic function, sympathovagal balance, and BRS in HF dogs. NCX inhibitors may offer new therapeutic options for acute HF treatment. SIGNIFICANCE STATEMENT: HF is facing growing health and economic burden. Moreover, patients hospitalized for acute heart failure are at high risk of decompensation recurrence, and no current acute decompensated HF therapy definitively improved outcomes. A new potent, Na+/Ca2+ exchanger inhibitor SAR340835 with antiarrhythmic properties improved systolic function of failing hearts without creating hypotension, while reducing heart rate and restoring sympathovagal balance. SAR340835 may offer a unique and attractive pharmacological profile for patients with acute heart failure as compared with current inotrope, such as dobutamine.

Antiarrhythmic effect of ranolazine in combination with selective NCX-inhibition in an experimental model of atrial fibrillation

Abstract Background Previous studies showed an antiarrhythmic effect of ranolazine in different clinical and experimental studies. The aim of this study was to investigate the effects of a combination of ranolazine with different selective NCX-inhibitors in an established experimental model of atrial fibrillation. Methods and results 18 hearts of New Zealand white rabbits were retrogradely perfused. Left and right atrial catheters were used to record monophasic action potentials. Hearts were paced at three different cycle lengths (350ms, 250ms, 150ms). Thereby, cycle-length dependent atrial action potential durations (aAPD90), atrial effective refractory periods (aERP) and atrial post-repolarization refractoriness (aPRR=aERP-aAPD90) were obtained. Vulnerability to AF was tested by a standardized protocol employing several trains of burst pacing. After generating baseline data, the hearts were perfused with a combination of acetylcholine (ACh, 1μM) and isoproterenol (Iso, 1μM) to increase occurrence of AF. Afterwards, the hearts were assigned to two groups and additionally perfused with a combination of 10 μM ranolazine and 1 μM of the selective NCX-inhibitor ORM-10103 (group 1: Rano-ORM) or 10 μM ranolazine and 1 μM of another NCX-inhibitor, SEA0400 (group 2: Rano-SEA). Infusion of ACh/Iso led to a shortening of aAPD90 (group 1/2: −27.2ms/−24.5ms p<0.05), aERP (−29.2ms/−35.6ms p<0.05), aPRR (−12.2ms/−13.7ms p=ns) and the occurrence of AF-Episodes was significantly increased (group 1: baseline 6 episodes to 34 episodes under ACh/Iso p<0.05, group 2: baseline 7 episodes to 26 episodes under ACh/Iso p<0.05). Additional perfusion with ranolazine and ORM-10103 did not alter aAPD90 (−0,47ms) while effective refractory periods (+21.4ms, p<0.05) and aPRR (+43ms, p<0.05) were significantly prolonged and AF episodes were effectively reduced to 15 episodes (p<0.05). In group 2, Rano-SEA led to a slight decrease in aAPD90 (−9.5ms) while aERP (+13.3ms, p<0.05) and aPRR (+19.8ms, p<0.05) were prolonged. The occurrence of AF episodes was reduced to 15 episodes (p=0.1). Conclusion To our knowledge this is the first study investigating the effect of ranolazine combined with different selective NCX-inhibitors in an isolated whole-heart model of AF. Atrial repolarization was not significantly changed with either of the combinations. However, both combinations prolonged aERP and aPRR and thereby suppressed induction of AF. Combining novel cellular targets may therefore lead to new potentially interesting options for antiarrhythmic AF therapy that have to be tested in clinical studies. Funding Acknowledgement Type of funding source: None

Antiarrhythmic Effect of Ranolazine in Combination with Selective NCX-Inhibition in an Experimental Model of Atrial Fibrillation

The aim of this study was to investigate the effects of a combination of ranolazine with different selective inhibitors of the Na+/Ca2+-exchanger (NCX) in an established experimental model of atrial fibrillation (AF). Eighteen hearts of New Zealand white rabbits were retrogradely perfused. Atrial catheters were used to record monophasic action potentials (aPRR). Hearts were paced at three different cycle lengths. Thereby, atrial action potential durations (aAPD90), atrial effective refractory periods (aERP) and atrial post-repolarization refractoriness were obtained. Isoproterenol and acetylcholine were employed to increase the occurrence of AF. Thereafter, the hearts were assigned to two groups (n = 9 each group) and additionally perfused with a combination of 10 µM ranolazine and 1 µM of the selective NCX-inhibitor ORM-10103 (group A: Rano-ORM) or 10 µM ranolazine and 1 µM of another NCX-inhibitor, SEA0400 (group B: Rano-SEA). The infusion of Iso/ACh led to a shortening of aAPD90, aERP, aPRR and the occurrence of AF episodes was significantly increased. Additional perfusion with ranolazine and ORM-10103 (group A) significantly prolonged the refractory periods and aPRR and AF episodes were effectively reduced. In group B, Rano-SEA led to a slight decrease in aAPD90 while aERP and aPRR were prolonged. The occurrence of AF episodes was consecutively reduced. To our knowledge, this is the first study investigating the effect of ranolazine combined with different selective NCX-inhibitors in an isolated whole-heart model of AF. Both combinations prolonged aERP and aPRR and thereby suppressed the induction of AF.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

Background and PurposeThe exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN).Experimental ApproachCurrents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model.Key ResultsORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed.Conclusion and ImplicationsWe confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Long-term effects of Na+ /Ca2+ exchanger inhibition with ORM-11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+ /Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure-volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9± 2 mmHg; NXT (n= 12) vs. NXT + ORM (n= 12); P= 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n= 12) vs. NXT + ORM (n= 12); P< 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure.

Na+/Ca2+ Exchanger 1 in Airway Smooth Muscle of Allergic Inflammation Mouse Model.

Cytosolic free Ca2+ ([Ca2+]cyt) is essential for airway contraction, secretion and remodeling. [Ca2+]cyt homeostasis is controlled by several critical molecules, one of which is the Na+/Ca2+ exchanger 1 (NCX1) in the plasma membrane. Since little is currently known about NCX1 in the airway smooth muscle and its involvement in airway diseases, the present study was designed to investigate the expression and function of NCX1 in normal airway smooth muscle and its relevance to airway inflammation. Western blot analysis, tracheal smooth muscle contraction, and [Ca2+]cyt measurements were performed in mouse tracheal smooth muscle tissues and primary airway smooth muscle cell cultures. Additional studies were performed in a mouse model of allergic airway inflammation. Our data showed that NCX1 proteins were expressed in the human bronchial smooth muscle cells (HBSMCs), murine airway and whole lung. Carbachol raised [Ca2+]cyt in mouse tracheal smooth muscle cells and induced murine tracheal contraction, all of which were significantly attenuated by KB-R7943, a selective NCX inhibitor. Removal of extracellular Na+ increased [Ca2+]cyt in HBSMCs and mouse tracheal SMCs, which was dependent on extracellular Ca2+ and sensitive to KB-R7943. TNF-α treatment of HBSMCs significantly upregulated mRNA and protein expression of NCX1 and enhanced NCX activity. Finally, KB-R7943 abolished the airway hyperresponsiveness to methacholine in an ovalbumin-induced mouse model of allergic airway inflammation. Together, these findings indicate that NCX1 in airway smooth muscle may play an important role in the development of airway hyperresponsiveness, and downregulation or inhibition of NCX1 may serve as a potential therapeutic approach for asthma.

Paradoxical Effects of Sodium-Calcium Exchanger Inhibition on Torsade de Pointes and Early Afterdepolarization in a Heart Failure Rabbit Model.

Calcium homeostasis plays an important role in development of early afterdepolarizations (EADs) and torsade de pointes (TdP). The role of sodium-calcium exchanger (NCX) inhibition in genesis of secondary Ca rise and EAD-TdP is still debated. Dual voltage and intracellular Ca optical mapping were conducted in 6 control and 9 failing rabbit hearts. After baseline electrophysiological and optical mapping studies, E4031 was given to simulate long QT syndrome. ORM-10103 was then administrated to examine the electrophysiological effects on EAD-TdP development. E4031 enhanced secondary Ca rise, EADs development, and TdP inducibility in both control and failing hearts. The results showed that ORM-10103 reduced premature ventricular beats but was unable to suppress the inducibility of TdP or EADs. The electrophysiological effects of ORM-10103 included prolongation of action potential duration (APD) and increased APD heterogeneity in failing hearts. ORM-10103 had a neutral effect on the amplitude of secondary Cai rise in control and heart failure groups. In this model, most EADs generated from long-short APD junction area. In conclusion, highly selective NCX inhibition with ORM-10103 reduced premature ventricular beat burden but was unable to suppress secondary Ca rise, EADs development, or inducibility of TdP. The possible electrophysiological mechanisms include APD prolongation and increased APD heterogeneity.

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Na+/Ca2+ exchanger (NCX) is the main Ca2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca2+ ([Ca2+]i). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1 µM ORM-10962 reduced the Ca2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca2+]i. L-type Ca2+ current (ICa) was not affected by 1 µM ORM-10962 in the absence of SR Ca2+ release, while ICa was suppressed by ORM-10962 during normal Ca2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca2+]i, suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca2+i]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca2+ handling and/or the preserved inducibility of forward NCX by high [Ca2+]i. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

Inotropic effect of NCX inhibition depends on the relative activity of the reverse NCX assessed by a novel inhibitor ORM-10962 on canine ventricular myocytes

Na+/Ca2+ exchanger (NCX) is the main Ca2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca2+ ([Ca2+]i). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca2+]i. L-type Ca2+ current (ICa) was not affected by 1µM ORM-10962 in the absence of SR Ca2+ release, while ICa was suppressed by ORM-10962 during normal Ca2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca2+]i, suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca2+i]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca2+ handling and/or the preserved inducibility of forward NCX by high [Ca2+]i. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

Effect of Docosahexaenoic Acid on Voltage-Independent Ca2+ Entry Pathways in Cultured Vascular Smooth Muscle Cells Stimulated with 5-Hydroxytryptamine.

We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca2+ channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca2+ entry, and Ca2+ entry through Na+/Ca2+ exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca2+ entry, and Ca2+ entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT2A receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca2+]i in cultured VSMCs. Ca2+ influx by adding extracellular Ca2+ (1.3 mM) to the Ca2+-free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca2+ influx when extracellular Ca2+ was added to the Ca2+-free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca2+]i. Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca2+ entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca2+]i.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments.

BackgroundIn this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined.MethodsIon currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 μg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts.ResultsORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 μM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 μM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts.ConclusionsThe present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

Efficacy of selective NCX inhibition by ORM-10103 during simulated ischemia/reperfusion

In this study we evaluated the effects of selective Na+/Ca2+ exchanger (NCX) inhibition by ORM-10103 on the [Ca2+]i transient (CaT), action potential (AP), and cell viability in isolated canine ventricular cardiomyocytes exposed to a simulated ischemia/reperfusion protocol performed either alone (modeling moderate low-flow ischemia) or with simultaneous strophantidine challenge (modeling more severe low-flow ischemia). CaTs were monitored using a Ca2+-sensitive fluorescent dye, APs were recorded by intracellular microelectrodes, and anaerobic shifts in cellular metabolism were verified via monitoring native NADH fluorescence. Simulated ischemia increased the NADH fluorescence, reduced the amplitudes of the AP and CaT and induced membrane depolarization. APs moderately shortened, CaTs prolonged. Diastolic [Ca2+]i ([Ca2+]iD) level increased significantly during ischemia and further elevated following strophantidine application. Reperfusion normalized the NADH level, the amplitude of the AP and duration of the [Ca2+]i transient, but only partially restored action potential triangulation and the amplitude of the CaT. [Ca2+]iD decreased in untreated, but further increased in strophantidine-treated cells. 10µM ORM-10103 significantly reduced the ischemic [Ca2+]i raise in both untreated and strophantidine-treated cells. During reperfusion ORM-10103 decreased [Ca2+]i and eliminated its diastolic elevation in untreated and strophantidine-treated cardiomyocytes. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusion-induced increase in AP and CaT variabilities were substantially reduced, but ischemia-induced shifts in AP morphology were barely influenced. In conclusion, selective NCX inhibition by ORM-10103 is highly effective against ischemia/reperfusion induced pathologic alterations in [Ca2+]i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology.

The Effect of SN-6, a Novel Sodium-Calcium Exchange Inhibitor, on Contractility and Calcium Handling in Isolated Failing Rat Ventricular Myocytes

Specific Na(+) /Ca(2+) exchanger (NCX) inhibition is a potential strategy to correct reduced contractility and depleted sarcoplasmic reticulum (SR) Ca(2+) content in heart failure (HF). SN-6, a benzyloxyphenyl derivative and proposed selective NCX inhibitor, could be used for this purpose. This study aimed to evaluate the effects of SN-6 on contractility and Ca(2+) handling in normal and failing rat cardiomyocytes. HF was induced in rats by coronary artery ligation. Left ventricular myocytes were isolated and superfused with increasing concentrations of SN-6. Sarcomere shortening, induced by field-stimulation, was reduced in amplitude with increasing concentrations of SN-6 compared with control solution. This effect was greater in failing cells. Kinetics of contractility (time to 90% peak and time to 50% relaxation) were significantly faster. Despite this, intracellular Ca(2+) transients demonstrated no change in the peak amplitude at low concentrations of SN-6, suggesting that SN-6 may affect myofilament sensitivity to Ca(2+) . Ten micro molar SN-6 significantly reduced peak Ca(2+) amplitude by 61.57% and 64.73% in normal and failing cells, respectively. Diastolic Ca(2+) was significantly increased at 1μM SN-6. SR Ca(2+) content, assessed by rapid application of caffeine, was reduced in failing cells with 1μM SN-6. Peak ICa , measured by whole-cell patch clamping, was significantly reduced in normal and failing myocytes at 1μM SN-6. Our data suggest that SN-6 is not a selective inhibitor of NCX and impairs contractility and Ca(2+) handling. Its use, together with similar putative NCX blockers, in correcting the contractile abnormalities of heart failure requires further studies.

Protective Effect of Na+/Ca2+ Exchange Blocker KB-R7943 on Myocardial Ischemia–Reperfusion Injury in Hypercholesterolemic Rats

Reverse-mode activation of the Na(+)/Ca(2+) exchanger (NCX) during reperfusion following ischemia contributes to Ca(2+) overload and cardiomyocyte injury. KB-R7943, a selective reverse-mode NCX inhibitor, reduces lethal reperfusion injury under non-ischemic conditions. However, the effectiveness of this compound under ischemic conditions is unclear. In the present study, we studied the effects of KB-R7943 in an animal model of hyperlipidemia. We further assessed whether the K ATP (+) channels are involved in potential protective mechanisms of KB-R7943. Twelve rats were fed normal chow, while 48 animals were fed a high cholesterol diet. The hearts from the control and hypercholesterolemic rats were subjected to 25 min of global ischemia followed by a 120-min reperfusion. Before this, hearts from hypercholesterolemic rats either received no intervention (cholesterol control group) or were pre-treated with 1 μM KB-R7943 and 0.3 μM of K ATP (+) blocker glibenclamide or glibenclamide alone. The infarction sizes (triphenyltetrazolium assay) were 35 ± 5.0 % in the control group, 46 ± 8.7 % in the cholesterol control group (p < 0.05 vs. control group), 28.6 ± 3.3 % in the KB-R7943 group (p < 0.05 vs. cholesterol control group), 44 ± 5 % in the KB-R7943 and glibenclamide group, and 47 ± 8.5 % in the glibenclamide group (p < 0.05 vs. control group). Further, KB-R7943 attenuated the magnitude of cell apoptosis (p < 0.05 vs. cholesterol control group). These beneficial effects were abolished by glibenclamide. In conclusion, diet-induced hypercholesterolemia enhances myocardial injury. Selective reverse-mode NCX inhibitor KB-R7943 reduces the infarction size and apoptosis in hyperlipidemic animals through the activation of K ATP (+) channels.

KB-R7943 Reduces Necrosis and Apoptosis in Hyperlipidemic Animals through the Activation of K+ATP Channels

Reverse-mode activation of the Na+-Ca2+ exchanger (NCX) at the time of reperfusion following ischemia contributes to Ca2+ overload and cardiomyocyte injury, KB-R7943, as a selective reverse-mode NCX inhibitor, reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of KB-R7943 in hyperlipidemic animals and assess if the K+ ATP are involved in the protective mechanisms. Anesthetized rats were randomized into five groups, as follows, and were subjected to 25min global ischemia (GI) followed by 120 min reperfusion (R). Normally fed animals: a Control group (NC) with no additional intervention. Cholesterol fed (6weeks) animals: a Chol group (HC) with no additional interventions, a KB-R7943 group (HKB) treated with KB-R7943, a KB-R7943 and glybenclamide group (HGKB) treated with KB-R7943 and glybenclamide (K+ ATP, blocker, 0.3μM), a glybenclamide group (HGLY) treated with glybenclamide The infarct size was measured by triphenyltetrazolium. The infarct size was 35±5.0% in NC, 46±8.7% in HC and 47±8.5% in HGLY (NC vs. HC, P 0.05). KB-R7943 reduced the infarct size (28.6±3.3% in HKB vs. HC, P<0.05), In addition, KB-R7943 attenuated apoptotic cell (HKB vs. HC, p<0.05), glybenclamide abolished the effect reached by KB-R7943. Thus, diet-induced hypercholesterolemia enhances myocardial injury; KB-R7943 reduces infarct size and apoptosis in hyperlipidemic animals through the activation of K+ ATP channels.

Involvement of Na+/Ca2+ exchanger in migration and contraction of rat cultured tendon fibroblasts

In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca(2+) signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na(+)/Ca(2+) exchangers (NCX). The fluorometric study showed that the intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased by reducing extracellular Na(+) concentration ([Na(+)](o)) in tendon fibroblasts. Selective NCX inhibitors, KB-R7943 and SEA0400, both attenuated [Na(+)](o)-dependent [Ca(2+)](i) elevation and the resting [Ca(2+)](i) in tendon fibroblasts. RT-PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound-healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB-R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast-mediated collagen-matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca(2+) influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.

Potential Therapeutic Effects of Na+/Ca2+ Exchanger Inhibition in Cardiac Diseases

The Na+/Ca2+ exchanger (NCX) has a pivotal role in cardiac Na+ and Ca2+ homeostasis and is an essential pathway for Ca2+ extrusion from cardiomyocytes. Altered NCX function may result in abnormal Ca2+ release from the sarcoplasmic reticulum (SR) and impaired cardiac electrical activity and contractility in several diseases, like arrhythmias, ischemia/reperfusion injury, hypertrophy and heart failure. This review focuses on the role of the exchanger and the major effects of partial NCX blockade in normally functioning and diseased hearts. In healthy cardiac cells consequences of a partial NCX blockade were found to be moderate and species dependent. In rabbit and dog practically no change in the magnitude of the calcium transients and mechanical activity was observed, while elevation of systolic calcium levels and a concomitant positive inotropic action were demonstrated in rat and murine hearts. On the other hand, NCX inhibition represents a novel potential therapeutic strategy in case of a variety of cardiac dysfunctions. Partial NCX blockade was shown to have beneficial effects in animal models of ischemia/reperfusion injury and also antiarrhythmic and positive inotropic actions in the failing heart. Further progress in this field is seriously hampered by the absence of really selective NCX inhibitors. KB-R7943 and SEA0400 are widely used NCX blockers, both drugs, however, have limited selectivity and efficacy, properties required to initiate relevant clinical trials. In summary, there is an increasing demand by both researchers and clinicians for new NCX inhibitors with significantly enhanced selectivity and functionality.

A Na+/Ca2+ exchanger isoform, NCX1, is involved in retinal cell death after N‐methyl‐D‐aspartate injection and ischemia–reperfusion

We investigated the expression of Na(+)/Ca(2+) exchanger (NCX) and the functional role of NCX in retinal damage by using NCX1-heterozygous deficient mice (NCX1(+/-)) and SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline), a selective NCX inhibitor in vivo. We also examined the role of NCX in oxygen-glucose deprivation (OGD) stress with a retinal ganglion cell line (RGC-5) cell culture in vitro. The expression of NCX1 was confirmed and entirely localized in retina by immunoblotting and immunohistochemistry, respectively. NCX1(+/-) mice possessed significant protection against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA). SEA0400 at 3 and 10 mg/kg significantly reduced NMDA- or high intraocular pressure-induced retinal cell damage in mice. Furthermore, SEA0400 reduced the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells and the expression of phosphorylated mitogen-activated protein kinases (ERK1/2, JNK, p38) induced by NMDA injection. In RGC-5, SEA0400 at 0.3 and 1 microM significantly inhibited OGD-induced cell damage. OGD-induced cell damage was aggravated by ouabain (a Na(+),K(+)-ATPase inhibitor) at 100 microM, and this increased damage was significantly reduced by SEA0400 at 1 microM. In conclusion, these results suggest that NCX1 may play a role in retinal cell death induced by NMDA and ischemia-reperfusion.