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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.)

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 Ca 2+ -activated Cl - current (I Cl(Ca) ) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca 2+ -overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by I Cl(Ca) . Therefore our goal was to examine the role of I Cl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. I Cl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating I Cl(Ca) , was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither I Cl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of I Cl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting I Cl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for I Cl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)