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 ] 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.

Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to β-AR stimulation.

Highlights• Ca 2+ -entry via I Ca,L is essential for the activation of I Cl(Ca) • I Cl(Ca) can be activated even in the absence of CICR• TMEM16A and Bestrophin-3 are expressed on human left ventricular muscle• TMEM16A and Bestrophin-3 co-localize with each other and with Ca v 1.2 channels (I Cl(Ca) ) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of I Cl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of I Cl(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 Ca v 1.2 was also studied. The profile of I Cl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltageclamp 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 (1.1 µM) decreased I Cl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of I Cl(Ca) . Ca 2+ -entry through L-type Ca 2+ channels was essential for activation of I Cl(Ca) . TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Ca v 1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of I Cl(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 I Cl(Ca) requires high Ca 2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

The role of Ca2+-activated Cl− current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(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. ICl(Ca) was blocked by 0.5 mmol/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 ICl(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 ICl(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 ICl(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 ICl(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 ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

a b s t r a c tIn this study we evaluated the effects of selective Na þ /Ca 2 þ exchanger (NCX) inhibition by ORM-10103 on the [Ca 2 þ ] 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 Ca 2 þ -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. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusioninduced 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 [Ca 2 þ ] i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology.