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The profiles of ion currents during the cardiac action potential can be visualized by the action potential voltage clamp technique. To obtain multiple ion current data from the same cell, the "onion peeling" technique, based on sequential pharmacological dissection of ion currents, has to be applied. Combination of the two methods allows recording of several ion current profiles from the same myocyte under largely physiological conditions. Using this approach, we have studied the densities and integrals of the major cardiac inward (I Ca , I NCX , I Na-late ) and outward (I Kr , I Ks , I K1 ) currents in canine ventricular cells and studied the correlation between them. For this purpose, canine ventricular cardiomyocytes were chosen because their electrophysiological properties are similar to those of human ones. Significant positive correlation was observed between the density and integral of I Ca and I Kr , and positive correlation was found also between the integral of I Ca and I NCX . No further correlations were detected. The Ca 2+ -sensitivity of K + currents was studied by comparing their parameters in the case of normal calcium homeostasis and following blockade of I Ca . Out of the three K + currents studied, only I Ks was Ca 2+ -sensitive. The density and integral of I Ks was significantly greater, while its time-to-peak value was shorter at normal Ca 2+ cycling than following I Ca blockade. No differences were detected for I Kr or I K1 in this regard. Present results indicate that the positive correlation between I Ca and I Kr prominently contribute to the balance between inward and outward fluxes during the action potential plateau in canine myocytes. The results also suggest that the profiles of cardiac ion currents have to be studied under physiological conditions, since their behavior may strongly be influenced by the intracellular Ca 2+ homeostasis and the applied membrane potential protocol., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug's mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

Background and Objective: In spite of the widespread clinical use of articaine and ropivacaine there is little information available on the effects of these drugs on myocardial Ca handling. In the present study, therefore, the concentration-dependent effects of articaine and ropivacaine on the components of intracellular Ca handling were studied and compared in canine ventricular myocardium., Methods: Contractility was measured in ventricular trabeculae, [Ca]i transients were recorded from electrically stimulated isolated ventricular myocytes loaded with the calcium-sensitive dye fura-2, L-type Ca current was recorded under whole cell patch clamp conditions, and the release and reuptake of Ca was monitored in sarcoplasmic reticulum vesicles., Results: Articaine and ropivacaine caused a reversible and concentration-dependent decrease in amplitude of the [Ca]i transient (EC50 = 87.4 +/- 12 and 99.3 +/- 17 micromol l, respectively), which was congruent with the reduction obtained for contractility (EC50 = 73.7 +/- 10 and 72.8 +/- 14 micromol l, respectively). No significant change in diastolic [Ca]i was found. L-type Ca current was significantly reduced by articaine and ropivacaine with EC50 values of 327 +/- 56 and 263 +/- 67 micromol l, respectively. Neither Ca release and Ca uptake nor the ATPase activity of the sarcoplasmic reticulum vesicles was altered by articaine or ropivacaine at concentrations less than 200 micromol l. In summary, articaine and ropivacaine caused no significant changes at the therapeutically relevant concentrations of the micromolar range. No significant differences between the effects of articaine and ropivacaine on contractility, [Ca]i transients, L-type Ca current, and sarcoplasmic reticulum Ca release and uptake were observed., Conclusions: Under conditions of normal application both articaine and ropivacaine are free of cardiodepressant effects; however, a negative inotropic action can be anticipated in cases of accidental intravenous injection or overdose. The observed negative inotropic actions of articaine and ropivacaine are similar in magnitude, and can be mainly attributed to a reduction in net trans-sarcolemmal Ca influx.

SEA0400 is a recently developed inhibitor of the Na+/Ca2+ exchanger (NCX) shown to suppress both forward and reverse mode operation of NCX. Present experiments were designed to study the effect of partial blockade of NCX on Ca handling and contractility in Langendorff-perfused guinea pig hearts loaded with the fluorescent Ca-sensitive dye fura-2. Left ventricular pressure and intracellular calcium concentration ([Ca2+]i) were synchronously recorded before and after cumulative superfusion with 0.3 and 1 muM SEA0400. SEA0400 caused no significant change in the systolic and diastolic values of left ventricular pressure and [Ca2+]i. Accordingly, pulse pressure and amplitude of the [Ca2+]i transient also remained unchanged in the presence of SEA0400. SEA0400 had no influence either on the time required to reach peak values of pressure and [Ca2+)]i or on half relaxation time. On the other hand, both 0.3 and 1 microM SEA0400 significantly increased the decay time constant of [Ca2+]i transients, obtained by fitting its descending limb between 30% and 90% of relaxation, from 127 +/- 7 to 165 +/- 7 and 177 +/- 14 ms, respectively (P < 0.05, n=6). In contrast to the guinea pig hearts, rat hearts responded to SEA0400 treatment with increased [Ca2+]i transients and contractility. These interspecies differences observed in the effect of SEA0400 can be explained by the known differences in calcium handling between the two species.

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.

Aims: This study was designed to evaluate the effects of the Na+/Ca2+ exchange (NCX) inhibitor SEA0400 on Ca2+ handling in isolated canine ventricular myocytes. [ABSTRACT FROM PUBLISHER]

Concentration-dependent (10–1000 μM) effects of terpenoid phenol derivatives were studied on L-type Ca2+ current in isolated canine and human ventricular cardiomyocytes using the whole-cell configuration of patch clamp technique. Carvacrol, thymol and eugenol suppressed peak Ca2+ current at +5 mV, having EC50 values and Hill coefficients of 98±11, 158±7 and 187±15 μM and 1.42±0.05, 2.96±0.43 and 1.6±0.1, respectively, in canine myocytes. Zingerone displayed a weak effect (estimated EC50: 2±0.37 mM, Hill coefficient: 0.73±0.07), while vanillin and guaiacol failed to substantially modify Ca2+ 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 Ca2+ 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 Ca2+ 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. [Copyright &y& Elsevier]

Background: Thymol is widely used as a general antiseptic and antioxidant compound in the medical practice and industry, and also as a stabilizer to several therapeutic agents, including halothane. Thus intoxication with thymol may occur in case of ingestion or improper anesthesia. In the present study, therefore, concentration-dependent effects of thymol (30-600 micro-grams) were studied on calcium and potassium currents in enzymatically isolated rat skeletal muscle fibers using the double vaseline gap voltage clamp technique. Results: Thymol suppressed both Ca and K currents in a concentration-dependent manner, the EC50 values were 193 ± 26 and 93 ± 11 µM, with Hill coefficients of 2.52 ± 0.29 and 1.51 ± 0.18, respectively. Thymol had a biphasic effect on Ca current kinetics: time to peak current and the time constant for inactivation increased at lower (100-200 µM) but decreased below their control values at higher (600 µM) concentrations. Inactivation of K current was also significantly accelerated by thymol (200-300 µM). These effects of thymol developed rapidly and were partially reversible. In spite of the marked effects on the time-dependent properties, thymol caused no change in the current-voltage relationship of Ca and K peak currents. Conclusions: Present results revealed marked suppression of Ca and K currents in skeletal muscle, similar to results obtained previously in cardiac cells. Furthermore, it is possible that part of the suppressive effects of halothane on Ca and K currents, observed experimentally, may be attributed to the concomitant presence of thymol in the superfusate. [ABSTRACT FROM AUTHOR]

Based on earlier pharmacological studies performed using conventional microelectrodes EGIS-7229 (S 21407), the novel antiarrhythmic candidate, was suggested to have a combined mode of action in cardiac tissues isolated from various mammalian species. In order to characterize the electrophysiological effects of the compound, its effects on calcium and potassium currents of isolated canine ventricular cardiomyocytes were studied in the present work using the whole cell configuration of the patch clamp technique. L-type Ca current (ICa) was significantly depressed by EGIS-7229 at concentrations of 3 µM or higher with no concomitant changes in the voltage-dependence of activation and time course of inactivation of ICa. The drug reversibly suppressed the rapid component of the delayed rectifier K current (IKr) in a concentration-dependent manner, having a K0.5 value of 1.1±0.1 µM and a slope factor of close to unity (1.23±0.16), indicating that probably one single binding site of high affinity may be involved in binding of EGIS-7229 to the IKr channel. In contrast, no changes in the slow component of the delayed rectifier K current (IKs) was observed with the compound up to the concentration of 100 µM, even if the current was fully activated by 8-bromo-cAMP. At a concentration of 10 µM or higher, EGIS-7229 caused also a moderate but significant reduction in the inward rectifier K current (IK1) and the transient outward K current (Ito) with no change in the voltage-dependence of activation and steady-state inactivation of Ito. Present results indicate that EGIS-7229 can be considered as a selective IKr blocker at low (1 µM) concentration; however, its combined (class III + IV) mechanism of action is evident at concentrations of 3 µM or higher. Suppression of ICa may explain the lack of development of early afterdepolarizations in the presence of EGIS-7229, predicting a relatively safe clinical application in contrast to pure class III compounds. [ABSTRACT FROM AUTHOR]