Introduction

Sympathetic activation of the heart is an important mechanism for fight-or-flight response, encompassing changes in cellular electrophysiology, calcium handling, and contractility. The action potential (AP) configuration is modified based on the changes of several inward and outward ion currents. One of these outward currents, the slow delayed rectifier K+ current (IKs), is a crucial component of the repolarization reserve1,2 and is also robustly increased by sympathetic stimulation in the canine heart3,4. However, the precise regulatory mechanisms of IKs are not fully elucidated yet. It is widely accepted that many effects of catecholamines on cardiac K+ channels are mediated via the protein kinase A (PKA) pathway, while others are mediated through distinct, partially independent mechanisms such as the Ca2+-sensitive calcium/calmodulin-dependent protein kinase II (CaMKII) pathway5,6,7. Notably, sympathetic activation leads to an increase in intracellular calcium concentration ([Ca2+]i), at least partly due to a PKA-, and Rad-mediated increase in L-type Ca2+ current (ICa,L). The reported findings on the ß-adrenergic regulation of cardiac IKs are highly dependent on the specific experimental conditions and species studied, further complicated by the extensive crosstalk between the downstream signaling pathways of ß-adrenergic receptor activation8.

This study was designed to delineate the contributions of the PKA and CaMKII pathways in the acute ß-adrenergic receptor mediated modulation of IKs in canine ventricular cardiomyocytes under physiological conditions. These conditions included (1) unbuffered [Ca2+]i for the physiological intracellular calcium homeostasis, (2) the use of action potential voltage clamp (APVC) technique to visualize the IKs current under an actual ventricular AP, and (3) activating ß-adrenergic receptors with low (not saturating) concentration of isoproterenol (10 nM). Canine myocytes were chosen for their electrophysiological properties, which closely resemble those of human cardiomyocytes9,10,11,12.

Results

We studied the effects of ß-adrenergic receptor activation on the parameters of IKs by using 10 nM isoproterenol (ISO). In this relatively low concentration of ISO, as shown in Fig. 1A, the peak density of IKs was larger (0.32 ± 0.06 A/F vs 1.36 ± 0.34 A/F; see also Fig. 2C) and the time-to-peak value was shorter (153.5 ± 4.7 ms vs 128.9 ± 6.5 ms, Fig. 1A inset) compared to control conditions. Opposite differences were observed when calcium entry, and therefore [Ca2+]i was reduced by the application of 1 µM nisoldipine (NISO). As demonstrated in Fig. 1B, peak IKs density was lower (0.32 ± 0.06 A/F vs 0.19 ± 0.02 A/F; see also Fig. 3C) and the time to peak IKs density took longer time (153.5 ± 4.7 ms vs 170.9 ± 4.1 ms, Fig. 1B inset). The true current–voltage relationship of IKs under a ventricular action potential is shown in black in Fig. 1C, D, under control conditions, and in the presence of ISO (purple trace in Fig. 1C) or NISO (blue trace in Fig. 1D). These curves also indicate that IKs activates earlier (i.e. at more positive membrane potentials on the graph of Fig. 1C) under ß-adrenergic receptor activation and later (i.e. at more negative voltages in Fig. 1D) when [Ca2+]i was decreased by nisoldipine. In addition, the mid-plateau current density (current density measured at the halving point between the AP peak and 90% repolarization level) was also larger in the ISO-treated cells (0.23 ± 0.03 A/F vs 1.04 ± 0.26 A/F; Fig. 2B) and smaller in cells treated with NISO (0.23 ± 0.03 A/F vs 0.06 ± 0.01 A/F; Fig. 3B), similarly to the current integral, which was more than fourfold larger in ISO (28.2 ± 5.3 mC/F vs 135.7 ± 32.1 mC/F; Fig. 2D) and almost 50% smaller in NISO (28.2 ± 5.3 mC/F vs 15.4 ± 1.3 mC/F; Fig. 3D) compared to control conditions. All these differences evoked by ISO or NISO in the parameters of IKs were statistically significant (p < 0.05).

Fig. 1
figure 1

Effect of 10 nM isoproterenol (ISO) and 1 µM nisoldipine (NISO) on IKs in canine ventricular cardiomyocytes. (A, B) Command action potentials (above), representative averaged IKs current traces (below), and IKs time to peak values (inserts below) obtained under APVC conditions in untreated control (black), in the presence of 10 nM ISO (violet), and 1 µM NISO (blue). (C, D) Current–voltage relationships obtained from the experiment shown on panel A and B (untreated control: black, ISO: violet, NISO: blue). Red curves were generated by scaling the control traces up or down to the amplitude of those obtained with ISO or NISO, respectively. On the relative scale, 100% is the maximal current density obtained either in CTRL (red) and in ISO (violet) (C), or in CTRL (red) and NISO (blue) (D).

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Fig. 2
figure 2

Effect of pretreatment with 1 µM nisoldipine (NISO), 1 µM KN-93, 3 µM H-89 or KN-93 + H-89 on the isoproterenol (ISO) induced changes in IKs. (A) Command action potential (above) and representative averaged IKs current traces (below) obtained under APVC conditions in untreated controls, in the presence of 10 nM ISO, and in the further 4 groups when ISO was applied after pretreatment with 1 µM NISO, 1 µM KN-93, 3 µM H-89, or KN-93 + H-89. (B) Mid-plateau current densities, (C) peak current densities, (D) current integrals under the conditions on panel (A). Symbols and bars represent mean ± SEM, small dots represent individual data, numbers in parentheses indicate the number of cells studied. Asterisks indicate significant differences (p < 0.05) between groups determined using Dunnett’s post-hoc test comparing data to the ISO group.

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Fig. 3
figure 3

Effect of 1 µM nisoldipine (NISO), 1 µM KN-93, and 3 µM H-89, on the parameters of IKs under baseline conditions. (A) Command action potential (above) and representative averaged IKs current traces (below) obtained under APVC conditions in untreated controls, in the presence of 1 µM NISO (blue), 1 µM KN-93, and 3 µM H-89. (B) Mid-plateau current densities, (C) peak current densities, and (D) current integrals under the conditions on panel (A). Symbols and bars are mean ± SEM, small dots represent individual data, numbers in parenthesis indicate the number of cells studied. Asterisks indicate significant differences (p < 0.05) between groups determined using Dunnett’s post-hoc test comparing data to the control group.

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To investigate the signal transduction pathways mediating the effect of ß-adrenergic receptor stimulation by ISO, cells were pretreated either with the CaMKII inhibitor 1 µM KN-93, or with the PKA inhibitor 3 µM H-89, or with both, before adding 10 nM ISO to the bath. The effect of ISO on the parameters of IKs was significantly smaller in cases when CaMKII was inhibited, either by 1 µM KN-93 alone or in combination with 1 µM KN-93 and 3 µM H-89 together, as represented in Fig. 2A. The peak current densities (Fig. 2C) were 1.36 ± 0.34 A/F in ISO, whereas 0.66 ± 0.16 A/F when KN-93 was applied before ISO, and 0.62 ± 0.12 A/F when both KN-93 and H-89 was applied before ISO. The mid-plateau densities (Fig. 2B) were 1.04 ± 0.26 A/F, 0.41 ± 0.11 A/F and 0.29 ± 0.07 A/F, whereas the current integrals (Fig. 2D) were 135.7 ± 32.1 mC/F, 65.2 ± 11.9 mC/F and 56.7 ± 12.8 mC/F, respectively.

While PKA blockade alone by 3 µM H-89 seemingly reduced the effect of ISO on IKs throughout the applied command AP pulse (as it appears on the representative traces of Fig. 2A), this effect did not reach statistical significance in any of the examined parameters of IKs peak density (0.80 ± 0.12 A/F), IKs mid-plateau density (0.56 ± 0.11 A/F), or the current integral (81.5 ± 15.5 mC/F).

Pretreating the cells with 1 µM NISO failed to modify the effects of ISO on IKs (Fig. 2) indicating that pathways independent of Ca2+-entry dominate the ß-adrenergic receptor activated signal transduction in enhancing IKs.

We also tested the effects of 1 µM KN-93, 3 µM H-89 and 1 µM NISO on IKs without ß-adrenergic receptor activation. The mid-plateau IKs density was significantly lower either in the presence of KN-93 (0.10 ± 0.03 A/F) or H-89 (0.11 ± 0.03 A/F) than in the untreated control cells (Fig. 3A, B). Magnitudes of IKs peak current densities (Fig. 3C) and IKs current integrals Fig. 3D) were not significantly different in the presence of either KN-93 or H-89 compared to baseline conditions. In the presence of NISO, however, all these parameters were significantly smaller than in the untreated cell group.

While KN-93 is a potent and widely used inhibitor of CaMKII, it affects many other Ca/CaM-associated targets independently of CaMKII13. Therefore, we conducted some experiments with autocamtide-2 related inhibitory peptide (AIP), a non-CaM-associated CaMKII inhibitor (Supplemetary Fig. 1) with and without β-adrenergic receptor activation. Data obtained with AIP reinforced our results obtained with KN-93 (see the Supplementary Results).

Discussion

Our study was designed to delineate the contributions of the PKA and CaMKII pathways in the ß-adrenergic receptor mediated modulation of IKs in canine ventricular cardiomyocytes under physiological conditions. Based on our results, four conclusions can be made. (1) In canine ventricular cardiomyocytes, CaMKII is predominantly involved in mediating the effect of ß-adrenergic receptor activation on IKs, while the contribution of PKA may be limited. (2) The potential pathways mediating the effect of ß-adrenergic receptor activation on IKs are independent of [Ca2+]i elevation, as 1 µM nisoldipine did not prevent the effects of isoproterenol on the parameters of IKs. (3) Under baseline conditions (without ß-adrenergic receptor activation), both PKA and CaMKII are somewhat involved in regulating IKs. (4) The baseline regulation of IKs is highly sensitive to [Ca2+]i, as all parameters of IKs were significantly smaller in the presence of 1 µM nisoldipine. The possible arrangement that meets all these four conditions is discussed in Fig. 4, summarizing the potential underlying signal transduction mechanisms involved in the regulation of IKs.

Fig. 4
figure 4

Signal transduction pathways possibly involved in the regulation of IKs in canine ventricular cells. IKs: slow delayed rectifier K+ current, Kv7.1: voltage-gated potassium channel subfamily Q member 1 (KCNQ1), ß-AR: beta-adrenergic receptor, Gs: stimulatory G protein, AC: adenylate cyclase, cAMP: cyclic adenosine monophosphate, EPAC: exchange protein activated by cAMP, Rap: Ras-proximate GTP binding protein, PCLε: phospholipase C epsilon, PKC: protein kinase C, Akt: serin/threonine kinase isolated from the AKR mouse spontaneous thymoma cells (also known as protein kinase B), NOS: nitrogen monoxide synthase, NO; nitrogen monoxide, PKA: protein kinase A, ICa,L: L-type Ca2+ current, Cav1.2: voltage-gated calcium channel subunit alpha1C (CACNA1C), Rad: Ras-related glycolysis inhibitor and calcium channel regulator protein, RyR: ryanodine receptor, [Ca2+]i: intracellular Ca2+ concentration, CaM: calmodulin, PP1: protein phosphatase 1. “P” in yellow circle symbolizes protein phosphorylation.

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Under baseline conditions—theoretically—more than one mechanism depicted in Fig. 4 may be involved in regulating IKs. The [Ca2+]i sensitivity of IKs observed in our experiments, which is similar to results reported in guinea pig14, may be the consequence of contribution of the PKA → ICa,L/RyR activation → [Ca2+]i increase → Ca2+/CaM complex → CaMKII activation pathway. In addition to this, CaM increases IKs directly by binding to the Kv7.1 (KCNQ1) channel5,15,16. This CaM-dependent increase in IKs relies on PKA activation, but it is independent of CaMKII17, and it may be responsible for the [Ca2+]i sensitivity of IKs under baseline conditions, explaining why IKs was smaller in the presence of nisoldipine. Here, it must also be noted that [Ca2+]i in the submembrane compartment (close to the ion channels located in the sarcolemma) reaches much higher levels and undergoes more rapid changes than the bulk cytosolic [Ca2+]i. According to this, Saucerman and Bers18 suggested that local submembrane CaM and CaMKII are activated at each heartbeat, whereas cytosolic CaM and CaMKII are quite insensitive to cytosolic [Ca2+]i. In contrast, Bartos et al.19 concluded that micromolar [Ca2+]i in the submembrane space is not required to maximize [Ca2+]i-dependent IKs activation during normal Ca2+ transients in rabbit ventricular cells. Their assumption is based on that IKs recorded during physiological Ca2+ transients was similar to IKs measured with [Ca2+]i clamped at 500–600 nM. Our results also indicate that under baseline conditions in paced myocytes, IKs is partially activated in a Ca2+-dependent fashion.

Additionally, PKA activation may reduce protein phosphatase 1 (PP1) activity leading to an overall increased phosphorylation level of CaMKII20,21. The contribution of this pathway to the regulation of baseline IKs may be limited due to its [Ca2+]i insensitivity, even though PKA activation by various ways was able to significantly increase canine IKs in the presence of nifedipine4.

In contrast to baseline conditions, the ISO-induced ß-adrenergic receptor activation mediated changes in IKs (1) were not sensitive to [Ca2+]i; (2) were minimally affected by PKA inhibition; but (3) were markedly suppressed by CaMKII blockade. This suggests two potential mechanisms responsible for mediating the effect of ISO, both based on the cAMP-induced activation of the guanine nucleotide exchange protein activated by cAMP (EPAC). One of these is the EPAC → Akt → NOS → NO → CaMKII pathway22,23, while the other one is the EPAC → Rap → PLCε → PKC → CaMKII pathway24,25. In both pathways, CaMKII activation is independent of PKA activation and the magnitude of [Ca2+]i. Supporting this explanation, β-adrenergic stimulation activated CaMKII in a PKA-independent manner in cultured murine cardiac cells26.

Another possibility for the seemingly minimal effect of PKA inhibition on the ISO-induced IKs enhancement is that 3 μM H-89 might reduce but not completely eliminate the ß-adrenergic receptor mediated PKA activation. The initial report on H-8927 determined its dissociation constant for PKA to be 0.048 nM in a cell-free in vitro assay, and H-89 is usually used at concentrations of 1–10 μM for PKA blockade17,28,29. Using a fluorescence resonance energy transfer-based PKA activity assay, Bobin et al.30 found that 10 μM H-89 was necessary to effectively reduce PKA activity induced by 1 nM ISO in adult rat ventricular myocytes, whereas 1 µM H-89 had only minor, insignificant effects.

We avoided using higher H-89 concentrations to prevent potential non-specific CaMKII blockade, which could occur due to H-89 interacting with the ATP binding site of CaMKII. Indeed, H-89 has been shown to inhibit CaMKII with a dissociation constant of approximately 30 μM in cell-free in vitro assays27. However, in pheochromocytoma cell line lysates, 30 μM H-89 did not significantly reduce CaMKII activity27. It is important to note that the effect of H-89 heavily depends on substrate and ATP concentrations31, as well as the presence of potential activators28. Additionally, pheochromocytoma cells primarily express the neuronal CaMKIIα isoform, while CaMKIIδ is the major cytosolic isoform found in cardiac cells, which may exhibit different affinity for H-89.

It was demonstrated that to abolish the ISO-induced positive inotropic effect in the intact rat heart, H-89 was required in 30 μM concentration17, which is rather high. This suggests that in addition to PKA, high concentrations of H-89 might interfere with other kinases, such as CaMKII, involved in transducing β-adrenergic receptor activation to target proteins. This interference could affect processes like the phosphorylation of the ryanodine receptor and phospholamban, as seen in the experiments by Ferrero et al.17, or activation of IKs in the present study.

It might seem to be surprising that none of the ISO-mediated effects could be prevented by pretreating the cells with 1 µM NISO. One might suppose that ISO could activate ICa,L to such a great extent that it overcomes the ICa,L blocking effect of NISO. This possibility can be excluded by previous results showing that even a higher ISO concentration (100 nM) was not able to increase ICa,L substantially in the presence of 1 µM NISO32 and no mechanical activity of the cardiomyocytes was observed under these experimental conditions, suggesting very low [Ca2+]i levels.

In conclusion, many different signaling pathways are involved in regulating IKs. Under baseline conditions the regulation is strongly [Ca2+]i-dependent, with PKA and CaM-CaMKII involved, whereas during ß-adrenergic stimulation it is [Ca2+]i-independent and supposes a pivotal role of EPAC-mediated activation of CaMKII.

Study limitations

Our study was designed to describe the contributions of the PKA and CaMKII pathways in the acute (short-term) ß-adrenergic receptor mediated modulation of IKs in canine ventricular cardiomyocytes under physiological conditions. The limitations of the study originate from its design: (1) the highly complex nature of adrenergic signaling in the heart, (2) possible species-specific differences (3) time course of ß-adrenergic receptor activation, and (4) changes under pathological conditions.

β-adrenergic signaling is complex, involving not only PKA and CaMKII, but also many other pathways. β-adrenergic agonists can activate both β1- and β2-adrenergic receptors. While β1-receptors regulate cardiac function solely through the “classical” cAMP-PKA-CaMKII pathway, β2-receptors can be coupled to both the “classical” and the “non-classical” pathway33. This latter involves switching the β2-receptor coupling from Gs to Gi and consequently activating two main signaling pathways: the phosphatidylinositol 3-kinase—Akt, and the mitogen-activated protein kinase cascade33,34. Exploring these pathways in their entire complexity was well beyond the aim and focus of the current study. It should also be mentioned that several other pathways beyond β-adrenergic signaling (e.g. ATP, phosphatidylinositol 4,5-bisphosphate, protein kinase G) can also regulate IKs.

While canine models are commonly used in cardiac research, and channel proteins responsible for generating IKs (both the pore-forming subunit and the regulatory subunit) are similarly distributed in canine and human myocardium12, there can be species-specific differences in channel proteins and in signaling pathways between human and canine hearts. Therefore, results from canine myocytes may not fully translate to human physiology. Ideally, similar studies in human-derived cells (or in humanized models) shall be conducted to confirm whether the observed mechanisms of the present study also exist in human cardiomyocytes.

Chronic (long-term) effects of sustained β-adrenergic receptor activation (such as in chronic stress, heart failure or under other chronic cardiovascular conditions) may differ. Chronic β-adrenergic stimulation can lead to desensitization of receptors and substantial changes in intracellular signaling pathways. Further studies are needed to explore the effects of chronic β-adrenergic activation, and to investigate different cardiac disease conditions (e.g. heart failure), as the adaptations observed under these conditions could reveal additional mechanisms in IKs regulation or potential changes in the role of PKA and CaMKII. Here are a few examples of how long-term β-receptor activation may affect IKs. In cultured adult guinea pig cardiomyocytes, Zhang et al.35 found that sustained β-adrenergic activation by isoproterenol for 48 h reduced IKs, contrary to acute isoproterenol exposure. This can be explained either by the desensitization of the adrenergic receptors, or by the downregulation of IKs channel protein expression. Shrugg et al.36 found that in cultured HEK-293B cells 12–24 h of sustained β-adrenergic receptor stimulation also reduced IKs, which was prevented by CaMKII inhibition. Similarly, CaMKIIδ overexpression reduced IKs. This effect did not occur in S484A KCNQ1 mutant channels, which cannot be phosphorylated by CaMKII. In a canine model of cardiac hypertrophy, Stengl et al.37 found a reduction of IKs, together with a blunted β-adrenergic response in left ventricular cells 30 days after the induction of hypertrophy by ablation of the AV node.

Methods

The present study is reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Animals

Adult mongrel dogs of either sex, bred for research purposes were purchased from a local breeder. The animals were 12–18 months old, weighing 8–14 kg at the time of sacrifice. Experimental dogs were anesthetized with intramuscular injections of 10 mg/kg ketamine hydrochloride (Calypsol, Richter Gedeon, Hungary) + 1 mg/kg xylazine hydrochloride (Sedaxylan, Eurovet Animal Health BV, The Netherlands) according to the protocol approved by the Institutional Animal Care Committee of the University of Debrecen (license No: 2/2020/DEMÁB, 9/2015/DEMÁB). All animal procedures conformed to the guidelines from Directive 2010/63/EU of the European Parliament.

Isolation of cardiomyocytes

Single canine myocytes were obtained by enzymatic dispersion using the segment perfusion technique, as described previously38. A wedge-shaped section of the ventricular wall, supplied by the LAD coronary artery, was cannulated and perfused with a nominally Ca2+-free Joklik solution (Minimum Essential Medium Eagle, Joklik Modification). After a 5 min period required for washing out the blood, this solution was supplemented with collagenase (Type II, 1 mg/ml, final activity of 224 U/ml, Worthington Biochemical Co., Lakewood, NJ, USA) and 0.2% bovine serum albumin (Fraction V., Sigma) containing 50 µM Ca2+; this perfusion lasted for 30 min typically. Finally, the tissue was minced to small chunks, the cells were released by gentle agitation and the normal extracellular Ca2+ concentration was restored. Before use the cells were stored in refrigerator at 15 °C in Minimum Essential Medium Eagle. This procedure yielded dominantly midmyocardial myocytes showing clear cross-striations.

Electrophysiology

Cells were perfused in a lucid chamber under an inverted microscope with a modified Tyrode solution at a rate of 1–2 ml/min. This solution contained (in mM): NaCl 121, KCl 4, CaCl2 1.3, MgCl2 1, HEPES 10, NaHCO3 25, and glucose 10; at pH = 7.35, with an osmolarity of 300 ± 3 mOsm. The bath temperature was set to 37 ºC using a temperature controller (Cell MicroControls, Norfolk, VA, USA). Electrical signals were amplified and recorded (MultiClamp 700B or 700A, Molecular Devices, Sunnyvale, CA, USA) under the control of a pClamp 10 software (Molecular Devices) following analogue–digital conversion (Digidata 1440A or 1332, Molecular Devices). Electrodes having tip resistances of 2–3 MΩ when filled with pipette solution were fabricated from borosilicate glass. The pipette solution contained (in mM) K-aspartate 120, KCl 30, KOH 10, MgATP 3, HEPES 10, Na2-phosphocreatine 3, EGTA 0.01, and cAMP 0.002; at pH = 7.3, osmolality = 285 ± 2 mOsm. The series resistance ranged typically between 4 and 8 MΩ. The experiment was discarded in all cases when the series resistance above 12 MΩ or increased by more than 50% during the experiment. For further details see references12,39.

Experiments were performed using the APVC technique as previously described40,41. Cells were paced at a constant cycle length of 0.7 s, corresponding to the normal heart rate of dogs. To diminish the consequences of cell-to-cell variations in AP duration, “canonic” APs were applied as command signals, instead of using the own AP of the cell. The canonic AP was chosen to represent a midmyocardial canine AP with average parameters. This approach allowed the comparison of the individual current traces. IKs was defined as a 0.5 µM HMR-1556 sensitive current. In order to reduce the noise and trace-to-trace fluctuations, 20 consecutive current traces were recorded and averaged before and after the 5 min application of HMR-1556. IKs was obtained by subtracting the average post-drug current trace from the average pre-drug one. The trace subtraction and averaging were carried out by a different person than the experiment, therefore investigators were blinded to the applied chemicals during the analysis of raw data.

IKs was characterized by maximal current density (peak current), its density measured at the half-duration of the command AP (mid-plateau current), and the total charge carried by the current (integral). All parameters were normalized to cell capacitance, determined using hyperpolarizing pulses from + 10 to − 10 mV lasting for 15 ms.

Chemicals

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), except for HMR-1556, KN-93 and H-89 which were purchased from Tocris Bioscience (Bristol, UK). HMR-1556 was used in the bath solution, whereas both kinase inhibitors (1 µM KN-93 and 3 µM H-89) were applied in the pipette solution.

ISO stock solution of 10 µM was freshly created right before each experiment, and this was diluted to 10 nM final concentration in the bath solution. This relatively low concentration of isoproterenol causes a significant but not saturating increase in IKs4. Similarly, Prabhakar et al.42 found that in mouse hearts, 10 nM ISO caused a significant, but not saturating increase in both + dP/dt and − dP/dt. Recently, Saha et al.43 found that in HEK-293B cells, the EC50 value of ISO on cAMP production increase was 7.7 nM.

Statistics

The sample size calculation was done with GPower 3.1 and was based on previous studies on the effect of ISO on IKs4. The used parameters resulted in a calculated sample size of 7 for each group (one-way ANOVA; 6 groups; effect size = 0.64; α = 0.05; desired power = 0.8). Actual sample sizes for the different groups varied between 7 and 11. For the NISO + ISO group, one cell was excluded from data analysis after the subtraction (therefore n = 6 in this group), because it showed a partially inward IKs current trace, which is impossibl

Results are expressed as mean ± SEM values, n denotes the number of cells studied. Experimental groups were statistically compared with one-way ANOVA, followed by Dunnett’s post-hoc test. In the experiments discussed in Figs. 1 and 3, the untreated control group was used as a reference, while in the “ISO-activated” comparison (Fig. 2), each group was compared to the 10 nM ISO treated group in the Dunnett’s post-hoc test. Differences were considered statistically significant when p < 0.05.