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Na + /Ca 2+ exchanger (NCX) is the main Ca 2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca 2+ ([Ca 2+ ] 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 Ca 2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca 2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca 2+ ] i . L-type Ca 2+ current (I Ca ) was not affected by 1µM ORM-10962 in the absence of SR Ca 2+ release, while I Ca was suppressed by ORM-10962 during normal Ca 2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca 2+ ] i , suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca 2+ 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 Ca 2+ handling and/or the preserved inducibility of forward NCX by high [Ca 2+ ] 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., (Copyright © 2017. Published by Elsevier B.V.)

Abnormal Ca(2+)homeostasis is often associated with chronic cardiovascular diseases, such as hypertension, heart failure or cardiac arrhythmias, and typically contributes to the basic ethiology of the disease. Pharmacological targeting of cardiac Ca(2+)handling has great therapeutic potential offering invaluable options for the prevention, slowing down the progression or suppression of the harmful outcomes like life threatening cardiac arrhythmias. In this review we outline the existing knowledge on the involvement of malfunction of the ryanodine receptor and the Na(+)-Ca(2+)exchanger in disturbances of Ca(2+)homeostasis and discuss important proof of concept pharmacological studies targeting these mechanisms in context of hypertension, heart failure, atrial fibrillation and ventricular arrhythmias. We emphasize the promising results of preclinical studies underpinning the potential benefits of the therapeutic strategies based on ryanodine receptor or Na(+)-Ca(2+)exchanger inhibition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite its widespread therapeutical use there is little information on the cellular cardiac effects of the antidiabetic drug pioglitazone in larger mammals. In the present study, therefore, the concentration-dependent effects of pioglitazone on ion currents and action potential configuration were studied in isolated canine ventricular myocytes using standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques. Pioglitazone decreased the maximum velocity of depolarization and the amplitude of phase-1 repolarization at concentrations ≥3 μM. Action potentials were shortened by pioglitazone at concentrations ≥10 μM, which effect was accompanied with significant reduction of beat-to-beat variability of action potential duration. Several transmembrane ion currents, including the transient outward K(+) current (Ito), the L-type Ca(2+) current (ICa), the rapid and slow components of the delayed rectifier K(+) current (IKr and IKs, respectively), and the inward rectifier K(+) current (IK1) were inhibited by pioglitazone under conventional voltage clamp conditions. Ito was blocked significantly at concentrations ≥3 μM, ICa, IKr, IKs at concentrations ≥10 μM, while IK1 at concentrations ≥30 μM. Suppression of Ito, ICa, IKr, and IK1 has been confirmed also under action potential voltage clamp conditions. ATP-sensitive K(+) current, when activated by lemakalim, was effectively blocked by pioglitazone. Accordingly, action potentials were prolonged by 10 μM pioglitazone when the drug was applied in the presence of lemakalim. All these effects developed rapidly and were readily reversible upon washout. In conclusion, pioglitazone seems to be a harmless agent at usual therapeutic concentrations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

SEA0400 is a selective inhibitor of the Na(+)/Ca(2+) exchanger having equal potencies to suppress both the forward and reverse mode operation of the Na(+)/Ca(2+) exchanger. Present experiments were designed to study the effect of partial blockade of Na(+)/Ca(2+) exchanger on Ca(2+) handling in isolated rat ventricular myocytes. Intracellular Ca(2+) transient and cell shortening were measured in ventricular myocytes loaded with Fura-2-AM fluorescent dye. Partial blockade of Na(+)/Ca(2+) exchanger was induced by superfusion of the cells with SEA0400 at a concentration of 0.3 microM. Amplitude of the intracellular Ca(2+) transient and cell shortening was significantly increased by SEA0400 in both field stimulated and voltage clamped myocytes, without significant elevation of diastolic Ca(2+) level and the decay time constant of the Ca(2+) transient. In patch clamped myocytes the SEA0400 induced increase in the Ca(2+) transient and cell shortening was accompanied by significant reduction of peak L-type Ca(2+) current. These effects can be explained by the autoregulative nature of cardiac Ca(2+) handling, as the reduced Ca(2+) efflux from the cell results in an increased Ca(2+) load to the sarcoplasmic reticulum leading to increased Ca(2+) release, which in turn may decrease the L-type Ca(2+) current by accelaration of Ca(2+) dependent inactivation of L-type Ca(2+) current. Our results suggest that complex changes in the Ca(2+) cycling can occur after selective pharmacological inhibition of the Na(+)/Ca(2+) exchanger.

Norfluoxetine is the most important active metabolite of the widely used antidepressant fluoxetine but little is known about its pharmacological actions. In this study the anticonvulsant actions of norfluoxetine and fluoxetine were studied and compared to those of phenytoin and clonazepam in pentylenetetrazol-induced mouse epilepsy models. Pretreatment with fluoxetine or norfluoxetine (20mg/kg s.c.), as well as phenytoin (30 mg/kg s.c.) and clonazepam (0.1mg/kg s.c.) significantly increased both the rate and duration of survival, demonstrating a significant protective effect against pentylenetetrazol-induced epilepsy. These effects of norfluoxetine were similar to those of fluoxetine. According to the calculated combined protection scores, both norfluoxetine and fluoxetine were effective from the concentration of 10mg/kg, while the highest protective action was observed with clonazepam. Effects of norfluoxetine and fluoxetine on voltage-gated Ca2+ channels were evaluated by measuring peak Ba2+ current flowing through the Ca2+ channels upon depolarization using whole cell voltage clamp in enzymatically isolated rat cochlear neurons. The current was reduced equally in a concentration-dependent manner by norfluoxetine (EC50=20.4+/-2.7 microM, Hill coefficient=0.86+/-0.1) and fluoxetine (EC50=22.3+/-3.6 microM, Hill coefficient=0.87+/-0.1). It was concluded that the efficacy of the two compounds in neuronal tissues was equal, either in preventing seizure activity or in blocking the neuronal Ca2+ channels.

Concentration-dependent (10-1000 microM) effects of terpenoid phenol derivatives were studied on L-type Ca(2+) current in isolated canine and human ventricular cardiomyocytes using the whole-cell configuration of patch clamp technique. Carvacrol, thymol and eugenol suppressed peak Ca(2+) current at +5 mV, having EC(50) values and Hill coefficients of 98+/-11, 158+/-7 and 187+/-15 microM and 1.42+/-0.05, 2.96+/-0.43 and 1.6+/-0.1, respectively, in canine myocytes. Zingerone displayed a weak effect (estimated EC(50): 2+/-0.37 mM, Hill coefficient: 0.73+/-0.07), while vanillin and guaiacol failed to substantially modify Ca(2+) current up to the concentration of 1 mM. In addition to tonic block, thymol and carvacrol, but not eugenol, evoked marked rate-dependent block at 2 Hz. Carvacrol and eugenol accelerated inactivation of Ca(2+) current and caused leftward shift in the voltage dependence of steady-state inactivation without altering activation kinetics. Carvacrol, but not eugenol, increased the time constant of recovery from inactivation. These effects of carvacrol and eugenol developed rapidly and were largely reversible. In myocytes isolated from undiseased human hearts, the effect of carvacrol was similar to that observed in canine cells. It is concluded that suppression of cardiac Ca(2+) currents by phenol derivatives is influenced by the substituent in the benzene ring, and the blocking effect of these drugs may involve interactions with the inactivation machinery of the channel.

Effects of bimoclomol, the novel heat shock protein (HSP) coinducer, was studied in various mammalian cardiac and rabbit aortic preparations. Bimoclomol decreased the ST-segment elevation induced by coronary occlusion in anesthetized dogs (56% and 80% reduction with 1 and 5 mg/kg, respectively). In isolated working rat hearts, bimoclomol increased coronary flow (CF), decreased the reduction of cardiac output (CO) and left ventricular developed pressure (LVDP) developing after coronary occlusion, and prevented ventricular fibrillation (VF) during reperfusion. In rabbit aortic preparations, precontracted with phenylephrine, bimoclomol induced relaxation (EC(50)=214 microM). Bimoclomol produced partial relaxation against 20 mM KCl, however, bimoclomol failed to relax preparations precontracted with serotonin, PGF(2) or angiotensin II. All these effects were evident within a few minutes after application of bimoclomol. A rapid bimoclomol-induced compartmental translocation of the already preformed HSPs may explain the protective action of the compound.

The effect of citalopram, a selective serotonin reuptake inhibitor (SSRI) antidepressant, was studied on cardiac action potential configuration and compared with that of the tricyclic antidepressant (TCA) clomipramine. Conventional microelectrode techniques were used in right ventricular papillary muscle preparations of the guinea pig. Citalopram caused a concentration-dependent (10-100 microM) shortening of action potential duration (APD), depression of plateau and overshoot potential, and reduction of maximum velocity of depolarization (V(max)). No significant changes in resting membrane potential were observed. Similar results were obtained with clomipramine; however, reduction of V(max) and overshoot was more pronounced with clomipramine, whereas citalopram caused relatively greater shortening of APD. Effects of both drugs were partly reversible. The results indicate that the SSRI antidepressant citalopram, similarly to TCA compounds, alters cardiac action potential configuration in guinea pig ventricular muscle, probably owing to inhibition of cardiac Na(+) and Ca(2+) channels. Differences in cardiac side effects of the two drugs may be related to their different actions on cardiac action potential configuration.

EGIS-7229 is a novel antiarrhythmic candidate having multiple mechanisms of action with class III predominance. In this study, the effects of EGIS-7229 and sotalol on action potential duration (APD) and incidence of early afterdepolarizations (EADs) were studied and compared in rabbit papillary muscle by using conventional microelectrode techniques. In control bathing solution, both drugs increased APD in a concentration-dependent manner; however, the prolongation of APD was greater with sotalol than with EGIS-7229 when the same drug concentrations were compared. EAD developed in 3 of the 11 preparations (27%) bathed with a solution containing 3.6 mmol/l CsCl + 2 mmol/l KCl within the first 120 min of superfusion. The addition of 100 micromol/l sotalol to this superfusate increased the incidence of EAD to 83% (10 from 12), whereas the addition of the same concentration of EGIS-7229 prevented the development of EAD in all of the 9 preparations studied. These differences in incidence of EAD are likely attributable to differences in drug-induced increases of APD-50 in the presence of CsCl. Prolongation of APD-90 showed less correlation with incidence of EAD than changes in APD-50. On the basis of these in vitro results, high concentrations of EGIS-7229 cannot be expected to be torsadogenic in vivo--in contrast with sotalol--presumably owing to the combined class III + IV activity of the compound.

1. Inotropic and electrophysiologic effects of veratrine, vesnarinone, d-sotalol and tetraethylammonium (TEA) were compared. Action-potential duration (APD) and contractility were measured in isolated canine Purkinje fiber and ventricular trabecular muscle preparations by using standard microelectrode techniques. Each drug significantly increased APD and force development in either tissue. 2. Drug-induced increases in force development were normalized to increases in APD. The order of efficacy was found to be vesnarinone>veratrine>TEA in ventricular myocardium, whereas it was veratrine>>vesnarinone=d-sotalol=TEA in Purkinje fibers. 3. The force-APD relation was linear for all drugs in the concentrations used. 4. Simultaneous measurements of APD, force development and intracellular sodium ion activity (a(i)Na) in the presence of either veratrine or lidocaine indicated a linear relation between force development and changes in a(i)Na. 5. The relation between APD and force development was different in ventricular and Purkinje fiber preparations. Differences in the veratrine sensitivity of the force-APD relation observed between Purkinje and ventricular preparations suggest that a(i)Na-dependent changes in Na+/Ca2+ exchange may play a more important role in regulation of force generation in Purkinje fibers than in ventricular myocardium.

1. The cellular electrphysiological effects of EGIS-7229, a novel antiarrhythmic agent, were studied in guinea pig papillary muscles with the use of conventional microelectrode techniques. 2. The drug had a concentration-dependent biphasic effect on action potential duration (APD). APD was significantly lengthened at low concentration (3 mumol/1), whereas it was shortened at concentrations higher than 10 mumol/l. 3. At concentrations higher than 10 mumol/l, the drug decreased the maximum velocity of action potential upstroke (Vmax), the force contraction, and altered the restitution kinetics of APD. 4. The effect of EGIS-7229 on Vmax was frequently dependent; it was most prominent at short pacing cycle lengths (use-dependent block). 5. On the basis of present results, EGIS-7229 appears to carry mixed class I and class III characteristics. Class III properties are present at low concentrations, whereas, at higher concentrations, class I actions may be predominant.

1. The negative inotropic action of 10 microM lidocaine and 100 microM nicorandil was compared as a function of the pacing cycle length, ranging from 300-3000 ms, in isolated canine Purkinje fiber preparations. 2. The applied concentrations of lidocaine and nicorandil produced similar shortening of action potential duration; however, lidocaine compromised contractility stronger than nicorandil at each cycle length. 3. Normalizing the inotropic action of the drugs to their shortening effect on action potential duration, the negative inotropic action of lidocaine can be regarded as a sum of three distinct components: negative inotropy associated to the shortening of action potential duration per se, reduction of contractility likely due to direct inhibition of the fast sodium current and of the "window" sodium current by lidocaine.

1. The effects of methylene blue (MB, 10(-4) M) and ascorbate (ASC, 10(-4) M) on the resting membrane potential of frog skeletal muscle fibers were studied in Cl(-)-free medium at various external K+ concentrations. 2. Muscle fibers formed two distinct populations according to their resting potentials (hyperpolarized to -104 mV or depolarized to -31 mV) after a 90 min period of K+ withdrawal. ASC increased the relative contribution of hyperpolarized fibers, while in the presence of MB, all fibers depolarized during the 90 min of K+ withdrawal. 3. In the presence of 2.5 mM K+, ASC had no significant effect on the resting potential, while MB moved half of the fibers into the depolarized pool. Elevation of [K+]0 to 10 mM caused repolarization of all previously depolarized fibers (to -63 mV) in spite of the continuous presence of MB. 4. Ionic currents were measured during a 60 mV depolarization step using the double sucrose-gap technique. MB significantly increased the peak inward current, while ASC had no effect. The steady-state outward current was not affected by MB or ASC. 5. The results suggest that ionic conductances may be under redox control in frog skeletal muscle.

1. Voltage-clamp experiments were performed to determine the effects of veratrine (1 microgram/ml) on Na and K currents in isolated rabbit ventricular cardiomyocytes. 2. Veratrine did not affect the inward rectifier K current, increased the inactivation time constant of the transient outward current (I(to)) and induced a slowly decaying inward current component (Iv), which was sensitive to tetrodotoxin. 3. Inactivation of fast Na channels by application of short depolarizing prepulses to potentials between -90 and -50 mV prevented the development of Iv.Iv decayed biexponentially with time constants equal to 139 +/- 9.0 ms and 776 +/- 47 ms. The net amplitude of Iv and the time constants for its rapidly and slowly inactivating components were little affected by trains of conditioning prepulses to 0 mV. The contributions, however, of the fast and slow components to the net current were significantly altered by repetitive depolarizations. 4. These components of Iv are likely due to modification of open cardiac Na channels by veratrum alkaloids.

1. Voltage-clamp experiments were performed to determine the effects of veratridine on Na and Ca currents in frog skeletal muscle fibres. 2. Veratridine (1 microM) did not affect the kinetics of the fast Na current but it did induce a slowly inactivating tetrodotoxin-sensitive inward current that was apparent after Na current inactivation. This slow current had a peak amplitude of 6.7 +/- 0.7 microA/cm2 at -20 mV and decayed monoexponentially with a time constant of 606 +/- 77 ms. 3. The slow current had a voltage-dependence for activation that was similar to that of the fast Na current. Single depolarizing prepulses that induced complete inactivation of the fast Na channels, prevented development of the slow current. Trains of brief depolarizations at increasing frequencies increased the amplitude of the slow current. These results suggest that the slow current may be mediated by veratridine modified Na channels that must be in the open position. 4. The low concentration of veratridine (1 microM) did not affect the Ca current, while 100 microM veratridine reversibly suppressed the Ca current and shifted its peak current-voltage relation towards more negative potentials. Thus, veratridine appears not to be a selective fast Na channel modifier as it may also alter Ca channel gating properties in skeletal muscle fibres.

Changes in transmembrane ionic currents induced by OPC-8212 (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinoline) , a recently introduced positive inotropic agent which lengthens cardiac action potential duration, were examined using whole-cell voltage-clamp techniques in single rabbit, guinea pig and human ventricular myocytes. In rabbit, OPC-8212 (12 mumol/l) significantly increased membrane action potential duration measured at 90% of repolarization by an average of 88 ms (from 462 +/- 25 to 550 +/- 35 ms, n = 4; P < 0.05). In rabbit this increase in duration was not associated with significant changes in either the inward rectifier or transient outward K+ currents. The magnitude of the secondary inward current evoked from a holding potential of -50 mV was significantly increased by 97 +/- 8% (n = 6; P < 0.01) while a demonstrable delayed rectifier outward current could not be identified in the rabbit myocytes examined at room temperature. In guinea pig ventricular myocytes, where the delayed rectifier was large, 12 mumol/l OPC-8212 significantly depressed the current by 58 +/- 10% (n = 6; P < 0.01). The effects of OPC-8212 in human ventricular myocytes obtained from the explanted heart of a single patient having an idiopathic cardiomyopathy most closely resembled those observed in isolated rabbit ventricular myocytes. Thus, in rabbit and a few human ventricular myocytes examined at room temperature, OPC-8212 appeared to lengthen cardiac membrane action potential duration primarily by increasing the amplitude of the secondary inward current believed to primarily represent current through L-type Ca2+ channels. In guinea pig preparations, OPC-8212 also decreased the delayed rectifier outward K+ current which also would account for an increase in action potential duration. OPC-8212 could not be demonstrated to affect Na+ current inactivation in a manner similar to that produced by 1 mg/l veratrine, a recognized Na+ channel agonist, which dramatically slowed this process.

1. Using conventional microelectrode techniques a biphasic effect of tetraethylammonium (5 mmol/l) on the configuration of action potentials recorded from isolated canine Purkinje fibres: action potentials were first shortened (early effect) and then lengthened (late effect) by tetraethylammonium. 2. The early effect of tetraethylammonium also included lengthening of phase 1 duration and elevation of the plateau amplitude. These early effects reached steady-state within the first 3 min of superfusion and were readily reversed within 3 min of initiating washout of the drug. 3. The late effect (gradual lengthening of repolarisation during phase 3) failed to reach steady-state within the initial 60 min of superfusion and was not reversible. 4. The early effects of tetraethylammonium were more marked at slow driving rates and were not affected by blockade of alpha- and beta-adrenoceptors using 1 mumol/l phentolamine and 1 mumol/l propranolol. 5. The early effects of tetraethylammonium were mimicked by 4-aminopyridine (0.5 mmol/l), and in the presence of 4-aminopyridine tetraethylammonium failed to induce further changes in action potential morphology. 6. The early effects of tetraethylammonium may be due to inhibition of the transient outward current. 7. The rapid onset and reversibility of these early effects suggest that tetraethylammonium may act from outside the cell membrane.

1. Conventional microelectrode techniques were used to study the effects of antiarrhythmic drugs (quinidine, 2-20 microM; lidocaine, 5-50 microM; verapamil, 2-20 microM), 4-aminopyridine (4-AP, 50-100 microM), and tetrodotoxin (TTX, 1.5-6 nM) on repetitive electrical discharges induced in the muscle membrane in the presence of 1 microM cevadine. 2. Antiarrhythmic drugs and 4-AP produced progressive reduction of the maximum upstroke velocity (V+ max) of the discharges, while the cycle length was prolonged by each drug except 4-AP. 3. The efficacy in depression of V+ max and prolongation of cycle length was not proportional in the case of individual drugs. 4. A simple model of the repetitive activity incorporating drug-effects was presented. The cevadine-modified Na+ channels could be blocked by antiarrhythmic agents, however, the efficacy of these drugs on the normal and cevadine-modified Na+ channels were found to be different.

1. The electrophysiological effects of veratridine, cevadine and aconitine (10(-8)-2 x 10(-4), 2 x 10(-7)-2 x 10(-6) and 2 x 10(-6)-10(-4) mol/l, respectively) were compared on frog muscle membrane using conventional microelectrodes. 2. Veratridine and aconitine were equally effective in depolarizing the resting membrane with the threshold concentration of 5 x 10(-5) mol/l. 3. Volleys of repetitive discharges and slow transient depolarizations were observed when single electrical stimuli were applied in the presence of veratridine (5 x 10(-8)-2 x 10(-5) mol/l), but not aconitine. Volleys with aconitine could be evoked only by repetitive stimulation; however no tendency of repolarization was observed following these volleys. Two orders of magnitude more aconitine than veratridine was required to induce volleys with similar parameters. 4. The effects of cevadine were similar to those of the corresponding concentrations of veratridine. 5. The observed differences between the electrophysiological actions of aconitine and veratrum alkaloids may be explained in part with differences in Na+ channel inactivation produced by these toxins, in addition to differences in their use-dependent behavior.

1. Conventional microelectrode techniques were used to study the effect of quinidine (10 microM), lidocaine (20 microM), and verapamil (3-10 microM) on action potential upstroke (V+ max) in frog skeletal muscle and dog Purkinje fiber. 2. The frequency-dependent nature of V+ max depression induced by these drugs was similar in both preparations, however, quinidine was more potent in skeletal muscle while lidocaine was in Purkinje fibers. 3. In skeletal muscle tetrodotoxin (3 and 15 nM) and low concentrations of antiarrhythmic drugs proportionally reduced the maximum velocity of depolarization and repolarization (V+ max and V- max, respectively), whereas V- max was more depressed than V+ max by high concentrations (50-200 microM) of antiarrhythmics. Decreases in the overshoot potential were proportional to the V+ max block in the case of each drug. 4. These results indicate that therapeutically relevant concentrations of quinidine and lidocaine inhibit skeletal muscle Na+ channels in a use-dependent manner similar to heart, while at higher concentrations the K+ channels may also be blocked. Therapeutic implications of the results are discussed.