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Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K + current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (I K1 ) and the rapid delayed rectifier (I Kr ) K + currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. I K1 and I Kr were dissected by 50 µM BaCl 2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased I K1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall I K1 integral. The ISO-induced enhancement of I K1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the I K1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the I Kr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances I K1 in canine left ventricular cells through the CaMKII pathway, while I Kr remains unaffected under the experimental conditions used.

Current density, the membrane current value divided by membrane capacitance (C m ), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that C m and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and C m , and tested if dividing original parameters with C m had any effect. Under conventional voltage clamp conditions, correlations were high for I K1 , moderate for I Kr and I Ca,L , while negligible for I Ks . Correlation between I to1 peak amplitude and C m was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments I K1, I Kr and I Ca,L parameters showed high correlations with C m . For I NCX , I Na,late and I Ks there were low-to-moderate correlations between C m and these current parameters. Dividing the original current parameters with C m reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and C m varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells., (© 2024. The Author(s).)

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.)

Cardiac alternans have crucial importance in the onset of ventricular fibrillation. The early explanation for alternans development was the voltage-driven mechanism, where the action potential (AP) restitution steepness was considered as crucial determining factor. Recent results suggest that restitution slope is an inadequate predictor for alternans development, but several studies still claim the role of membrane potential as underlying mechanism of alternans. These controversial data indicate that the relationship of restitution and alternans development is not completely understood. APs were measured by conventional microelectrode technique from canine right ventricular papillary muscles. Ionic currents combined with fluorescent measurements were recorded by patch-clamp technique. APs combined with fluorescent measurements were monitored by sharp microelectrodes. Rapid pacing evoked restitution-independent AP duration (APD) alternans. When non-alternating AP voltage command was used, Ca 2+ i -transient (CaT) alternans were not observed. When alternating rectangular voltage pulses were applied, CaT alternans were proportional to I CaL amplitude alternans. Selective I CaL inhibition did not influence the fast phase of APD restitution. In this study we found that I CaL has minor contribution in shaping the fast phase of restitution curve suggesting that I CaL -if it plays important role in the alternans mechanism-could be an additional factor that attenuates the reliability of APD restitution slope to predict alternans., (© 2021. The Author(s).)

It has long been known that the electrophysiological effects of many cardioactive drugs strongly depend on the rate dependent frequency. This was recognized first for class I antiarrhythmic agents: their V max suppressive effect was attenuated at long cycle lengths. Later many Ca 2+ channel blockers were also found to follow such kinetics. The explanation was provided by the modulated and the guarded receptor theories. Regarding the duration of cardiac action potentials (APD) an opposite frequency-dependence was observed, i.e. the drug-induced changes in APD were proportional with the cycle length of stimulation, therefore it was referred as "reverse rate-dependency". The beat-to-beat, or short term variability of APD (SV) has been recognized as an important proarrhythmic mechanism, its magnitude can be used as an arrhythmia predictor. SV is modulated by several cardioactive agents, however, these drugs modify also APD itself. In order to clear the drug-specific effects on SV from the concomitant unspecific APD-change related ones, the term of "relative variability" was introduced. Relative variability is increased by ion channel blockers that decrease the negative feedback control of APD (i.e. blockers of I Ca , I Kr and I Ks ) and also by elevation of cytosolic Ca 2+ . Cardiac arrhythmias are also often categorized according to the characteristic heart rate (tachy- and bradyarrhythmias). Tachycardia is proarrhythmic primarily due to the concomitant Ca 2+ overload causing delayed afterdepolarizations. Early afterdepolarizations (EADs) are complications of the bradycardic heart. What is common in the reverse rate-dependent nature of drug action on APD, increased SV and EAD incidence associated with bradycardia., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although late sodium current (I Na-late ) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of I Na-late in canine and guinea pig ventricular cells and compare them to I Na-late recorded in undiseased human hearts. I Na-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human I Na-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine I Na-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo I Na-late profile in guinea pig was due to the slower decay of I Na-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of I Na-late . At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of I Na-late . These results should be taken into account when pharmacological studies with I Na-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with I Na-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of I Na-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca 2+ -dependent activation of TRPM4 channels. Transient outward (I to1 ), rapid delayed rectifier (I Kr ), and inward rectifier (I K1 ) K + currents were suppressed by 10 and 30 μM 9-phenanthrol with the blocking potency for I K1 < I Kr < I to1 and partial reversibility. L-type Ca 2+ current was not affected up to the concentration of 30 μM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 μM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 μM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.

Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca(2+)]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (I Ca, I Ks, and I Kr), while it is increased by I Na and I to. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action.

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.

Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (ICl(Ca)). This study aims to explore which blocker is preferable to study ICl(Ca). Whole-cell voltage-clamp technique was used to record ICa,L, IKs, IKr and IK1, while action potentials were measured using sharp microelectrodes. DIDS- (0.2 mM) and 9-AC-sensitive (0.5 mM) currents were identical in voltage-clamp conditions, regardless of intracellular Ca(2+) buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent, but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization (V max), but DIDS caused marked reduction of V max. Both inhibitors reduced the magnitude of phase-1, but, at slow stimulation rates, this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1 and 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5 mM. The half-effective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5 mM 9-AC. These results suggest that DIDS is equally useful to study ICl(Ca) during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of ICl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (ICa,L, IKr, IKs, IK1); however, ICa,L tracings can be contaminated with ICl(Ca) when measured in voltage-clamp condition.

Tacrolimus is a commonly used immunosuppressive agent which causes cardiovascular complications, e.g., hypertension and hypertrophic cardiomyopathy. In spite of it, there is little information on the cellular cardiac effects of the immunosuppressive agent tacrolimus in larger mammals. In the present study, therefore, the concentration-dependent effects of tacrolimus on action potential morphology and the underlying ion currents were studied in canine ventricular cardiomyocytes. Standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques were applied in myocytes enzymatically dispersed from canine ventricular myocardium. Tacrolimus (3-30 μM) caused a concentration-dependent reduction of maximum velocity of depolarization and repolarization, action potential amplitude, phase-1 repolarization, action potential duration, and plateau potential, while no significant change in the resting membrane potential was observed. Conventional voltage clamp experiments revealed that tacrolimus concentrations ≥3 μM blocked a variety of ion currents, including I(Ca), I(to), I(K1), I(Kr), and I(Ks). Similar results were obtained under action potential voltage clamp conditions. These effects of tacrolimus developed rapidly and were fully reversible upon washout. The blockade of inward currents with the concomitant shortening of action potential duration in canine myocytes is the opposite of those observed previously with tacrolimus in small rodents. It is concluded that although tacrolimus blocks several ion channels at higher concentrations, there is no risk of direct interaction with cardiac ion channels when applying tacrolimus in therapeutic concentrations.

Aim: The aim of this work was to study antagonistic interactions between the effects of various types of Ca(2+) channel blockers and isoproterenol on the amplitude of L-type Ca(2+) current in canine ventricular cells., Methods: Whole-cell version of the patch clamp technique was used to study the effect of isoproterenol on Ca(2+) current in the absence and presence of Ca(2+) channel-blocking agents, including nifedipine, nisoldipine, diltiazem, verapamil, CoCl(2) and MnCl(2) ., Results: Five micromolar Nifedipine, 1 μM nisoldipine, 10 μM diltiazem, 5 μM verapamil, 3 mM CoCl(2) and 5 mM MnCl(2) evoked uniformly a 90-95% blockade of Ca(2+) current in the absence of isoproterenol. Isoproterenol (100 nM) alone increased the amplitude of Ca(2+) current from 6.8 ± 1.3 to 23.7 ± 2.2 pA/pF in a reversible manner. Isoproterenol caused a marked enhancement of Ca(2+) current even in the presence of nifedipine, nisoldipine, diltiazem and verapamil, but not in the presence of CoCl(2) or MnCl(2) ., Conclusion: The results indicate that the action of isoproterenol is different in the presence of organic and inorganic Ca(2+) channel blockers. CoCl(2) and MnCl(2) were able to fully prevent the effect of isoproterenol on Ca(2+) current, while the organic Ca(2+) channel blockers failed to do so. This has to be born in mind when the effects of organic Ca(2+) channel blockers are evaluated either experimentally or clinically under conditions of increased sympathetic tone., (© 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.)

Background and Purpose: While the slow delayed rectifier K(+) current (I(Ks)) is known to be enhanced by the stimulation of β-adrenoceptors in several mammalian species, phosphorylation-dependent regulation of the rapid delayed rectifier K(+) current (I(Kr)) is controversial., Experimental Approach: In the present study, therefore, the effect of isoprenaline (ISO), activators and inhibitors of the protein kinase A (PKA) pathway on I(Kr) and I(Ks) was studied in canine ventricular myocytes using the whole cell patch clamp technique., Key Results: I (Kr) was significantly increased (by 30-50%) following superfusion with ISO, forskolin or intracellular application of PKA activator cAMP analogues (cAMP, 8-Br-cAMP, 6-Bnz-cAMP). Inhibition of PKA by Rp-8-Br-cAMP had no effect on baseline I(Kr). The stimulating effect of ISO on I(Kr) was completely inhibited by selective β₁-adrenoceptor antagonists (metoprolol and CGP-20712A), by the PKA inhibitor Rp-8-Br-cAMP and by the PKA activator cAMP analogues, but not by the EPAC activator 8-pCPT-2'-O-Me-cAMP. In comparison, I(Ks) was increased threefold by the activation of PKA (by ISO or 8-Br-cAMP), and strongly reduced by the PKA inhibitor Rp-8-Br-cAMP. The ISO-induced enhancement of I(Ks) was decreased by Rp-8-Br-cAMP and completely inhibited by 8-Br-cAMP., Conclusions and Implications: The results indicate that the stimulation of β₁-adrenoceptors increases I(Kr), similar to I(Ks), via the activation of PKA in canine ventricular cells., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: In spite of its widespread clinical application, there is little information on the cellular cardiac effects of the antidiabetic drug rosiglitazone in larger experimental animals. In the present study therefore concentration-dependent effects of rosiglitazone on action potential morphology and the underlying ion currents were studied in dog hearts.Experimental Approach: Standard microelectrode techniques, conventional whole cell patch clamp and action potential voltage clamp techniques were applied in enzymatically dispersed ventricular cells from dog hearts.Key Results: At concentrations ≥10 µM rosiglitazone decreased the amplitude of phase-1 repolarization, reduced the maximum velocity of depolarization and caused depression of the plateau potential. These effects developed rapidly and were readily reversible upon washout. Rosiglitazone suppressed several transmembrane ion currents, concentration-dependently, under conventional voltage clamp conditions and altered their kinetic properties. The EC(50) value for this inhibition was 25.2 ± 2.7 µM for the transient outward K(+) current (I(to)), 72.3 ± 9.3 µM for the rapid delayed rectifier K(+) current (I(Kr)) and 82.5 ± 9.4 µM for the L-type Ca(2+) current (I(Ca) ) with Hill coefficients close to unity. The inward rectifier K(+) current (I(K1)) was not affected by rosiglitazone up to concentrations of 100 µM. Suppression of I(to), I(Kr), and I(Ca) was confirmed also under action potential voltage clamp conditions.Conclusions and Implications: Alterations in the densities and kinetic properties of ion currents may carry serious pro-arrhythmic risk in case of overdose with rosiglitazone, especially in patients having multiple cardiovascular risk factors, like elderly diabetic patients. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca2+ current ( ICa), slow delayed rectifier K+ current ( IKs) and fast delayed rectifier K+ current ( IKr) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. EXPERIMENTAL APPROACH Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. KEY RESULTS In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the IKr blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the IKs blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the ICa blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating ICa followed by a rise in IKs, both currents increased with increasing the cycle length. CONCLUSIONS AND IMPLICATIONS The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of IKs- but not IKr- may be responsible for the observed shortening of action potentials. [ABSTRACT FROM AUTHOR]