The G Protein–Gated Potassium Current IK,ACh Is Constitutively Active
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《循环学杂志》
the Department of Pharmacology and Toxicology (D.D., A.F., N.V., E.W., T.C., U.R.) and Cardiovascular Center (M.K.), Medical Faculty, Dresden University of Technology, Dresden, Germany
Department of Pharmacology and Pharmacotherapy (N.J.), Medical University of Szeged, Szeged, Hungary.
Abstract
Background— The molecular mechanism of increased background inward rectifier current (IK1) in atrial fibrillation (AF) is not fully understood. We tested whether constitutively active acetylcholine (ACh)-activated IK,ACh contributes to enhanced basal conductance in chronic AF (cAF).
Methods and Results— Whole-cell and single-channel currents were measured with standard voltage-clamp techniques in atrial myocytes from patients with sinus rhythm (SR) and cAF. The selective IK,ACh blocker tertiapin was used for inhibition of IK,ACh. Whole-cell basal current was larger in cAF than in SR, whereas carbachol (CCh)-activated IK,ACh was lower in cAF than in SR. Tertiapin (0.1 to 100 nmol/L) reduced IK,ACh in a concentration-dependent manner with greater potency in cAF than in SR (–logIC50: 9.1 versus 8.2; P<0.05). Basal current contained a tertiapin-sensitive component that was larger in cAF than in SR (tertiapin [10 nmol/L]-sensitive current at –100 mV: cAF, –6.7±1.2 pA/pF, n=16/5 [myocytes/patients] versus SR, –1.7±0.5 pA/pF, n=24/8), suggesting contribution of constitutively active IK,ACh to basal current. In single-channel recordings, constitutively active IK,ACh was prominent in cAF but not in SR (channel open probability: cAF, 5.4±0.7%, n=19/9 versus SR, 0.1±0.05%, n=16/9; P<0.05). Moreover, IK1 channel open probability was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05) without changes in other channel characteristics.
Conclusions— Our results demonstrate that larger basal inward rectifier K+ current in cAF consists of increased IK1 activity and constitutively active IK,ACh. Blockade of IK,ACh may represent a new therapeutic target in AF.
Key Words: arrhythmia fibrillation ion channels remodeling signal transduction
Introduction
Atrial fibrillation (AF) is the most frequent cardiac arrhythmia in the clinical setting. It is associated with shorter action potential duration (APD) and effective refractory period and a loss of rate-dependent APD adaptation that involve concomitant alterations in ion current activity.1,2 Ion channel remodeling in patients with chronic AF (cAF) includes decreased density of L-type Ca2+ currents (ICa,L) and increased amplitude of the background inward rectifier K+ current IK1.3–8 The changes in atrial electrical properties (electrical remodeling) promote induction of AF and the tendency of the arrhythmia to be sustained.1 The autonomic nervous system may also contribute to initiation and persistence of AF. Vagal stimulation reduces atrial APD and effective refractory period and increases dispersion of atrial repolarization.9 This creates an arrhythmogenic substrate for reentry of the excitation wavefront, which promotes the duration of the AF episodes.1 Although it is generally accepted that vagal nerve activation contributes to initiation of AF, its precise role in cAF and the underlying molecular mechanisms are currently unknown.
Clinical Perspective p 3706
Vagally released acetylcholine (ACh) stimulates muscarinic receptors (M-receptors) and activates the atrial ACh-regulated potassium current IK,ACh.10 The resulting shortening of APD in response to M-receptor stimulation is mediated by IK,ACh because in knockout mice lacking this channel, M-receptor stimulation did not induce AF.11 Therefore, increased activity of IK,ACh was expected to contribute to induction or perpetuation of AF. Studies in atrial biopsies from patients with cAF revealed, however, that both expression of the Kir3.1 and Kir3.4 channel subunits and activation of whole-cell IK,ACh in response to M-receptor stimulation were lower in AF than in sinus rhythm (SR).6,7,12,13 This was interpreted as an adaptation of atrial myocytes to the high beating rate by reducing IK,ACh to counteract the shortening of APD.2
Reduced M-receptor–mediated activation of IK,ACh in AF compared with SR is associated with higher IK1 amplitude.3,4,6,7,14,15 Although larger amplitude of IK1 during AF is paralleled by increased mRNA and protein levels of the Kir2.1 channel subunit,6,15 the molecular basis of increased IK1 remains incompletely understood. One possibility is that IK,ACh or the ATP-regulated inward rectifier IK,ATP, which cannot be distinguished electrophysiologically at the whole-cell level, contributes to background IK1 by developing spontaneous activity. Therefore, throughout the remainder of this report, inward rectifier current in the absence of agonists is called basal current rather than IK1.
Theoretically, each component of the M-receptor–mediated signal transduction, including IK,ACh, may develop spontaneous activity in the absence of abnormal vagal tone. For instance, dog atrial myocytes possess a constitutively active IK,ACh-like current that develops higher amplitude after 1 week of atrial tachypacing.16 Hence, constitutively active IK,ACh channels may also exist in atria of AF patients.
The present study tested the hypothesis that increased IK1 in patients with cAF consists of enhanced IK1 and constitutively active IK,ACh current components.
Methods
Human Samples
The study was approved by the local ethics committee of the university (No. EK790799), and each patient gave written informed consent.
Right atrial appendages were obtained from 46 patients with SR and 33 patients with cAF (cAF >6 months; Table 1).
Electrophysiological Recordings
Atrial myocytes were isolated with the use of our previous protocol17 and were suspended in storage solution (in mmol/L: KCl 20, KH2PO4 10, glucose 10, K-glutamate 70, -hydroxybutyrate 10, taurine 10, EGTA 10, albumin 1, pH 7.4). Membrane currents were measured with standard whole-cell voltage-clamp techniques. ISO-2 software (MFK) was used for whole-cell and single-channel data acquisition and analysis.
For whole-cell measurements, borosilicate glass microelectrodes had tip resistances of 1 to 2 M when filled with pipette solution (in mmol/L: K-aspartate 100, NaCl 10, KCl 40, Mg-ATP 5, EGTA 2, GTP-Tris 0.1, HEPES 10, pH 7.4). Myocytes were superfused with a solution containing the following (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH 7.4 at 22°C to 24°C. Seal resistances were 4 to 8 G. Series resistance and cell capacitance were compensated. Basal current was measured by applying a depolarizing ramp pulse from –100 to +40 mV (holding potential –80 mV; see inset in Figure 1A). IK,ACh was stimulated with the nonselective M-receptor agonist carbachol (CCh) (2 μmol/L) and was identified with the selective bee venom toxin tertiapin.18 The nature of basal current and IK,ACh as inward rectifier currents was proven in each myocyte by applying Ba2+ (1 mmol/L) at the end of the experiments. The myocytes were superfused with tyrode solution via a peristaltic pump throughout the experiments. Drugs were applied via an additional rapid solution exchange system (ALA Scientific Instruments). During drug-free periods, the rapid solution exchange system supplied tyrode solution only. Data were not corrected for the calculated liquid junction potential (–12 mV; software JPCalc, version 2.2). To control for variability in myocyte size, the currents are expressed as densities (pA/pF).
Single-channel currents were recorded in cell-attached configuration (Axopatch 200B, Axon Instruments). The bath solution contained the following (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH 7.4 with NaOH. Borosilicate glass microelectrodes were coated with Sylgard (Dow Corning) and had tip resistances of 3 to 8 M when filled with the pipette solution (in mmol/L: KCl 140, HEPES 10, pH 7.2 with KOH). IK,ACh was activated by including 10 μmol/L CCh into the pipette solution; identity of IK,ACh in the absence of CCh application was tested by applying 10 nmol/L tertiapin to the bath solution. The single-channel characteristics of IK1 and IK,ACh were analyzed at –120 mV. The total recording time was 20 seconds for each voltage or drug application. The open probability of the channels was calculated from the total number of openings during 20 seconds of recording time. The average open probability per 1-second tracing was used for statistical evaluation. The single-channel slope conductances of IK1 and IK,ACh were calculated from the individual current-voltage relationships by linear regression analysis.
Molecular Analysis of the G3 Gene
Direct activation of IK,ACh is mediated by -subunits of G proteins. The G protein 3 subunit gene (GNB3) contains a C825T polymorphism, whereby homozygous 825T-allele carriers exhibit larger basal inward rectifier K+ current densities possibly because of enhanced signal transduction.17 To exclude confounding by homozygous 825T-allele carriers, all patients were genotyped for the C825T polymorphism. DNA extraction and genotyping were performed in a blinded manner as described.7 In this report the patient sample includes homozygous and heterozygous C825-allele carriers only.
Statistical Analysis
Throughout this report, n refers to the number of myocytes/patients, unless otherwise stated. For quantitative comparisons, average values were calculated from multiple data obtained from 1 patient. Because the number of cells investigated varied widely in each patient (unbalanced observations), we calculated means from average values for each patient.
One-way ANOVAs were applied to determine the sources of K+ current variation (SPSS version 12.0). Independent variables were cAF, selected clinical variables, and medication (Table 1). Differences between group means for continuous data were compared by unpaired Student t test or when patients with SR or cAF were stratified by underlying disease or medication by 1-way ANOVA and post hoc multiple comparisons tests (Bonferroni t test). The concentration-dependent effects of tertiapin on basal and CCh-activated current were evaluated with a mixed linear model (with fixed factors for rhythm status and concentration of tertiapin and random factor for subjects) followed by post hoc Tukey-Kramer t test (SAS version 9.1). To calculate the concentration of tertiapin blocking the CCh-activated IK,ACh by 50% (IC50) in SR and cAF, the concentration-response curve of the mean values was fitted to a 4-parameter logistic function (Prism version 4.0). Frequency data were analyzed with 2 statistics. P<0.05 was considered statistically significant.
Results
Amplitudes of basal current and IK,ACh were related to selected clinical variables. Significant differences between the 2 groups were found for hyperlipidemia and left atrial diameter. Patients with cAF more often received digitalis and diuretics, whereas lipid-lowering drugs were more frequently used in those with SR (Table 1). The patients used for the tertiapin studies showed clinical characteristics comparable to those of the other groups. With 1-way ANOVAs, AF was the only predictor of basal current and IK,ACh current density (data not shown).
Basal Current and IK,ACh in Atrial Myocytes of SR and cAF Patients
In voltage-clamped human atrial myocytes, cell capacitances averaged 80.3±4.7 pF (n=136) for SR and 89.6±4.1 pF (n=76) for cAF myocytes (P<0.05). Basal current in the absence of agonist (Figure 1A) was confirmed to be larger in cAF than in SR (at –100 mV: –22.9±2.1 pA/pF, n=76/25 [myocytes/patients] versus –12.7±1.0 pA/pF, n=136/38; P<0.05).
Application of CCh resulted in rapid initial increase (Peak) of IK,ACh amplitude, which was followed by a decrease to a quasi–steady state level (Qss) despite the continuous presence of CCh (desensitization).10 There was no difference in desensitization of IK,ACh between myocytes from SR and cAF patients (Figure 1B), the Peak/Qss ratio of IK,ACh being 0.58±0.06 (n=76/25) for AF versus 0.54±0.03 (n=136/38) for SR (P=NS). As expected, the CCh-activated Peak- and Qss-IK,ACh currents were smaller in cAF than in SR groups (Figure 1B, 1C).
Effects of Tertiapin on Basal Current and IK,ACh
The selective IK,ACh blocker tertiapin18 was used to discriminate between IK1 and IK,ACh contribution to enhanced basal current in AF. Tertiapin blocks IK,ACh with IC50 values between 8 and 30 nmol/L without any effect on IK1 up to 1 μmol/L.16,19,20
For this set of experiments we applied CCh (2 μmol/L) twice with 4 minutes of washing in between (S1, S2). S1 served as internal control, whereas S2 was measured in the presence of tertiapin or the nonselective M-receptor blocker atropine. Even under control conditions, IK,ACh was lower during S2 than during S1, suggesting incomplete recovery from desensitization (Figure 2A, 2B). However, the degrees of IK,ACh desensitization were similar in SR and cAF myocytes (Figure 2C).
Application of tertiapin (0.1 to 100 nmol/L) during S2 reduced the S2/S1 ratio in a concentration-dependent manner, with greater potency in cAF than in SR (–logIC50: 9.1 for AF versus 8.2 for SR; P<0.05; Figure 3A to 3C). In addition, tertiapin concentration-dependently impaired basal current in cAF, yielding a tertiapin-sensitive component of –6.7±1.2 pA/pF at 10 nmol/L (n=16/5; Figure 3D, 3E). After subtraction of the tertiapin-sensitive current component, basal current of cAF patients remained increased (Figure 3D), suggesting enhanced IK1 in these patients.
Tertiapin also suppressed a component of basal current in SR; however, this amounted to only –1.7±0.5 pA/pF at 10 nmol/L (n=24/8). Application of drug-free tyrode solution alone caused a small decrease of basal current by –1.5±0.6 pA/pF (n=5/4) in SR and –1.6±1.2 pA/pF (n=4/3) in cAF. Our results suggest constitutively active IK,ACh channels in cAF only (Figure 3A, 3C).
The cAF patients involved in the experiments with tertiapin did not have a history of vagally mediated AF. Compared with SR, they had larger left atria and a higher prevalence of diabetes, and they more frequently took diuretics (Table 1). However, basal currents were similar in cAF patients with and without valvular heart disease (28.7±4.2 pA/pF, n=21/5 versus –26.2±4.4 pA/pF, n=17/4) or diabetes (27.1±3.4 pA/pF, n=28/7 versus –29.2±7.5 pA/pF, n=10/2). The same holds true for treatment with diuretics (27.9±3.5 pA/pF, n=24/6 versus –26.9±6.2 pA/pF, n=14/3). The tertiapin (10 nmol/L)-sensitive current component was 6.4±1.5 pA/pF (n=12/3) and 6.3±1.6 pA/pF (n=12/4) in the absence of diuretics and digitalis, respectively, suggesting a lack of drug effects on constitutively active IK,ACh channels in cAF.
Next we excluded the contribution of spontaneously active M-receptors to enhanced basal current in cAF. Atropine (1 μmol/L) abolished the CCh-activated IK,ACh in both groups without effect on basal currents. Thus, tertiapin-sensitive constitutive activity of IK,ACh is independent of M-receptors (Figure 4).
Single-Channel Activity of IK1 and IK,ACh in SR and Chronic AF Patients
For a more direct proof of constitutive activity of IK,ACh channels in cAF, we measured single-channel activity in the cell-attached mode. Constitutive activity of IK,ACh was detected in cAF but was minute in SR (channel open probability: cAF, 5.4±0.8%, n=19/9 versus SR, 0.1±0.01%, n=16/9; P<0.05; Figure 5A to 5C). Moreover, open probability of IK1 was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05; Figure 5D) without changes in other channel characteristics (Table 2). Exclusion of cAF patients taking diuretics or digitalis had no impact on constitutive activity of IK,ACh. Open probability of IK,ACh was 6.1±0.7% (n=8/4) and 5.2±0.5% (n=9/4) in the absence of diuretics and digitalis, respectively.
Inclusion of 10 μmol/L CCh in the pipette solution caused single-channel openings in periodic bursts in all SR and cAF myocytes studied. Amplitudes and open times of constitutively active IK,ACh channels were not different from those of channels activated with CCh (Table 2).
Exposure of myocytes from SR patients with tertiapin (10 nmol/L) reduced open probability of CCh-activated IK,ACh (Figure 6A, 6C), which is in agreement with previous results obtained in rabbit atrial myocytes.20 Consistent with the reduction of whole-cell basal current shown in Figure 3C, open probability of constitutively active IK,ACh during cAF was lower in the presence of 10 nmol/L tertiapin without concomitant effects on IK1 (Figure 6B, 6D).
Discussion
In the present study we demonstrate that in addition to enhanced subunit expression, increased open probability of IK1 channels contributes to higher basal inward rectifier K+ current in patients with cAF. We provide evidence for involvement of agonist-independent constitutive activity of IK,ACh channels from several observations: (1) the selective IK,ACh inhibitor tertiapin reduced basal current in cAF but not in SR; (2) single-channel measurements demonstrated spontaneous openings of IK,ACh channels in myocytes from cAF patients only; and (3) the open probability of constitutively active single IK,ACh channels during cAF was lower in the presence of tertiapin. Evidence for agonist-independent constitutive activity of IK,ACh was provided by the lack of block of these channels by the M-receptor antagonist atropine. Our results suggest that in cAF the higher basal inward rectifier K+ current results from increased expression and open probability of IK1 and constitutively active IK,ACh channels, which may contribute to the perpetuation of the arrhythmia.
Comparison With Previous Studies
At the whole-cell level, several studies reported higher amplitude of basal inward rectifier K+ current in cAF than in SR patients.3,4,6,7,14,15 Pacing-induced tachycardia in dogs also results in higher IK1 amplitude,16,21 suggesting that the increase in IK1 is a consequence of the arrhythmia. Because expression of the corresponding channel subunits at mRNA and protein levels followed the increase in current amplitude,6,15 it was concluded that modified channel subunit transcription and/or translation is the only mechanism of increased IK1 during human cAF. Our results at the single-channel level, however, showed that open probability of IK1 is slightly but significantly higher in cAF than in SR, suggesting that the molecular basis of increased IK1 is more complex and involves not only increased channel subunit expression but also additional modifications of channel regulation. The molecular mechanisms of enhanced open channel probability of IK1 are currently unknown but may involve impaired phosphorylation-dependent regulation. The Kir2.x channel subunits of IK1 possess phosphorylation sites for different kinases, and higher channel subunit phosphorylation results in lower IK1 amplitude.22,23 Because cAF is associated with higher activity of the counterbalancing type 1 and type 2A serine/threonine protein phosphatases,8 it cannot be excluded that the expected stronger channel dephosphorylation contributes to higher IK1 activity in AF. Further studies are needed to identify the molecular mechanisms of modified IK1 regulation in cAF patients.
Consistent with previous studies,4,6,7 we found that the M-receptor–mediated activation of IK,ACh is smaller in cAF than in SR patients. Moreover, reduced channel subunit expression at the mRNA and protein level corresponds well to the lower activation of IK,ACh in cAF.6,12,13,15 Nevertheless, we clearly demonstrated that IK,ACh becomes constitutively active when cAF develops. The selective IK,ACh blocker tertiapin concentration-dependently inhibited whole-cell CCh-activated IK,ACh, with higher potency in AF than in SR. Tertiapin blocks native IK,ACh channels with IC50 values between 8 and 30 nmol/L16,19,20 without significant effects on other ion channels.19,20 The effective concentration range of tertiapin found in the present study (Figure 3) is in good agreement with the reported potency. In human atria, however, tertiapin unmasked a robust component of constitutively active IK,ACh in cAF and a substantially smaller one in SR. Recent findings in dog atria and pulmonary veins detected a tertiapin-sensitive current component contributing to basal inward rectifier current, and, consistent with our results, pacing-induced tachycardia was associated with higher whole-cell current density of this current component.16 Thus, the development of constitutively active IK,ACh currents in human cAF is probably a consequence of atrial remodeling contributing to the perpetuation rather than the initiation of the arrhythmia.
The molecular basis of constitutively active IK,ACh is currently unknown. At the single-channel level, basal IK,ACh activity in cAF resulted from a higher number of channel openings and subsequent increase in channel open probability without concomitant changes in basic current properties. The basic properties of IK1 and IK,ACh were in good agreement with previous results.24,25 Thus, the reduced channel subunit expression in cAF6,12,13,15 is probably part of the modified IK,ACh regulation and can be interpreted as an adaptation on the higher frequency of IK,ACh openings in these patients.
The regulation of IK,ACh is complex, suggesting several putative mechanisms for constitutive activity of IK,ACh. Because the latter was not affected by blocking M-receptors with atropine, basal activity of IK,ACh is an agonist-independent process. Increased agonist-independent dissociation of G- and G-subunits of inhibitory G proteins appears unlikely because in dog atrial myocytes neither pertussis toxin treatment nor absence of GTP affected the tertiapin-sensitive component of basal current.16
Evidence for constitutive activity of atrial IK,ACh channels was provided by earlier studies26,27 that showed that agonist-independent activation of IK,ACh requires ATP. Interestingly, consistent with the results from atria of dogs with tachypacing,16 pertussis toxin treatment failed to prevent the ATP activation of IK,ACh.26 Because activation of IK,ACh requires ATP,26–28 modified phosphorylation-dependent channel regulation may contribute to the development of constitutively active IK,ACh in cAF. Consistent with this idea, recent publications have shown that in atrial myocardium Kir3.1 forms a macromolecular complex, allowing for local regulation of IK,ACh function.28,29 The atrial GIRK1 macromolecular complex is composed of the catalytic subunits of PKA, PKC, and CaMKII and the protein phosphatases PP1 and PP2A.28,29 Thus, the qualitative and quantitative composition of the GIRK1 macromolecular complex may change during AF, resulting in abnormal regulation of IK,ACh. Further work is needed to verify these hypotheses.
Study Limitations
In the present investigation we studied regulation of IK1 and IK,ACh only in isolated myocytes in vitro. The situation in multicellular preparations and in vivo is more complex. Thus, our data should be extrapolated with caution to the in situ atria. It is well known that the atria are heterogeneous tissues. Therefore, our data obtained in right atrial appendages may not necessarily reflect alterations in the rest of the atria. Digitalis, diuretics, and lipid-lowering drugs were more frequently prescribed in cAF. Thus, we cannot exclude that drugs affect density of IK1 and IK,ACh. Finally, our current recordings were performed at room temperature only. Their regulation may differ at physiological temperature.
Potential Clinical Implications
Our results show that the molecular basis for the higher amplitude of basal inward rectifier current in AF involves increased Kir2.x channel subunit expression6,15 and channel open probability (present study). Two-dimensional computer simulations of human atrial tissue showed that IK1 affects spiral wave dynamics.30 An increase of IK1 during cAF resulted in reduced APD and hyperpolarization of the resting membrane potential, which is consistent with the shorter APD and the more negative resting membrane potential of multicellular trabeculae from patients with cAF.6 The changes in APD and resting membrane potential were associated with higher availability of the Na+ current (INa) and enhanced excitability, leading to faster rotation frequencies and stabilization of functional reentry. Most important, reduction of IK1 in the model was associated with APD prolongation and depolarization, resulting in decreased rotation frequency and spiral wave termination.30 Consistent with these findings, mice with overexpression of the IK1 channel subunit Kir2.131 and humans with a gain-of-function mutation of this channel develop cAF.32 Thus, blockade of IK1 may be a viable antiarrhythmic option.
In analogy to IK1, constitutively active IK,ACh may contribute to stabilization of functional reentry because in ACh-induced AF in sheep, the higher activation frequencies in left atria are associated with larger density of IK,ACh.33 In vivo, vagal nerve stimulation abbreviates APD and increases APD heterogeneity and atrial vulnerability to tachyarrhythmia,34 which perpetuate AF.9 In knockout mice lacking the Kir3.4 channel subunit of IK,ACh, M-receptor stimulation did not induce AF,11 suggesting that the effects of vagal nerve activation are mediated by IK,ACh. Thus, the agonist-independent constitutive activation of IK,ACh during cAF is expected to increase atrial vulnerability to tachyarrhythmia and to sustain AF. Because some antiarrhythmic drugs such as amiodarone, flecainide, quinidine, and verapamil, which effectively terminate AF,35 are also inhibitors of IK,ACh,36–38 it cannot be excluded that their effectiveness in AF results in part from blockade of IK,ACh and that at least in some patients with AF the inhibition of constitutively active IK,ACh may have an antiarrhythmic affect.
Finally, there is evidence of circulating M2 autoantibodies in AF patients, which may exert a tonic activation of M-receptor signal transduction.39 Furthermore, the ACh concentrations in the synaptic clefts could be elevated because of reduced activity of its degrading enzyme acetylcholine esterase.40 Because atropine had no effect on basal current in cAF, the development of constitutively active IK,ACh channels occurs in a receptor-independent manner. Thus, it is likely that the elevated synaptic ACh concentrations and the presence of M2 autoantibodies and constitutively active IK,ACh channels reinforce each other in promoting AF. Further studies are needed to test these hypotheses.
Conclusions
We demonstrated that the molecular basis of higher basal inward rectifier K+ current in patients with cAF involves increased density of IK1 and development of constitutively active IK,ACh. Our results provide a mechanistic insight into the regulation of IK1 and IK,ACh in cAF that may help to design new therapeutic options for AF.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DO 769/1 to Dr Dobrev). The authors thank Trautlinde Thurm, Ulrike Heinrich, and Manja Schne for excellent technical assistance and Eberhard Kuhlisch for statistical advice.
Disclosures
None.
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Department of Pharmacology and Pharmacotherapy (N.J.), Medical University of Szeged, Szeged, Hungary.
Abstract
Background— The molecular mechanism of increased background inward rectifier current (IK1) in atrial fibrillation (AF) is not fully understood. We tested whether constitutively active acetylcholine (ACh)-activated IK,ACh contributes to enhanced basal conductance in chronic AF (cAF).
Methods and Results— Whole-cell and single-channel currents were measured with standard voltage-clamp techniques in atrial myocytes from patients with sinus rhythm (SR) and cAF. The selective IK,ACh blocker tertiapin was used for inhibition of IK,ACh. Whole-cell basal current was larger in cAF than in SR, whereas carbachol (CCh)-activated IK,ACh was lower in cAF than in SR. Tertiapin (0.1 to 100 nmol/L) reduced IK,ACh in a concentration-dependent manner with greater potency in cAF than in SR (–logIC50: 9.1 versus 8.2; P<0.05). Basal current contained a tertiapin-sensitive component that was larger in cAF than in SR (tertiapin [10 nmol/L]-sensitive current at –100 mV: cAF, –6.7±1.2 pA/pF, n=16/5 [myocytes/patients] versus SR, –1.7±0.5 pA/pF, n=24/8), suggesting contribution of constitutively active IK,ACh to basal current. In single-channel recordings, constitutively active IK,ACh was prominent in cAF but not in SR (channel open probability: cAF, 5.4±0.7%, n=19/9 versus SR, 0.1±0.05%, n=16/9; P<0.05). Moreover, IK1 channel open probability was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05) without changes in other channel characteristics.
Conclusions— Our results demonstrate that larger basal inward rectifier K+ current in cAF consists of increased IK1 activity and constitutively active IK,ACh. Blockade of IK,ACh may represent a new therapeutic target in AF.
Key Words: arrhythmia fibrillation ion channels remodeling signal transduction
Introduction
Atrial fibrillation (AF) is the most frequent cardiac arrhythmia in the clinical setting. It is associated with shorter action potential duration (APD) and effective refractory period and a loss of rate-dependent APD adaptation that involve concomitant alterations in ion current activity.1,2 Ion channel remodeling in patients with chronic AF (cAF) includes decreased density of L-type Ca2+ currents (ICa,L) and increased amplitude of the background inward rectifier K+ current IK1.3–8 The changes in atrial electrical properties (electrical remodeling) promote induction of AF and the tendency of the arrhythmia to be sustained.1 The autonomic nervous system may also contribute to initiation and persistence of AF. Vagal stimulation reduces atrial APD and effective refractory period and increases dispersion of atrial repolarization.9 This creates an arrhythmogenic substrate for reentry of the excitation wavefront, which promotes the duration of the AF episodes.1 Although it is generally accepted that vagal nerve activation contributes to initiation of AF, its precise role in cAF and the underlying molecular mechanisms are currently unknown.
Clinical Perspective p 3706
Vagally released acetylcholine (ACh) stimulates muscarinic receptors (M-receptors) and activates the atrial ACh-regulated potassium current IK,ACh.10 The resulting shortening of APD in response to M-receptor stimulation is mediated by IK,ACh because in knockout mice lacking this channel, M-receptor stimulation did not induce AF.11 Therefore, increased activity of IK,ACh was expected to contribute to induction or perpetuation of AF. Studies in atrial biopsies from patients with cAF revealed, however, that both expression of the Kir3.1 and Kir3.4 channel subunits and activation of whole-cell IK,ACh in response to M-receptor stimulation were lower in AF than in sinus rhythm (SR).6,7,12,13 This was interpreted as an adaptation of atrial myocytes to the high beating rate by reducing IK,ACh to counteract the shortening of APD.2
Reduced M-receptor–mediated activation of IK,ACh in AF compared with SR is associated with higher IK1 amplitude.3,4,6,7,14,15 Although larger amplitude of IK1 during AF is paralleled by increased mRNA and protein levels of the Kir2.1 channel subunit,6,15 the molecular basis of increased IK1 remains incompletely understood. One possibility is that IK,ACh or the ATP-regulated inward rectifier IK,ATP, which cannot be distinguished electrophysiologically at the whole-cell level, contributes to background IK1 by developing spontaneous activity. Therefore, throughout the remainder of this report, inward rectifier current in the absence of agonists is called basal current rather than IK1.
Theoretically, each component of the M-receptor–mediated signal transduction, including IK,ACh, may develop spontaneous activity in the absence of abnormal vagal tone. For instance, dog atrial myocytes possess a constitutively active IK,ACh-like current that develops higher amplitude after 1 week of atrial tachypacing.16 Hence, constitutively active IK,ACh channels may also exist in atria of AF patients.
The present study tested the hypothesis that increased IK1 in patients with cAF consists of enhanced IK1 and constitutively active IK,ACh current components.
Methods
Human Samples
The study was approved by the local ethics committee of the university (No. EK790799), and each patient gave written informed consent.
Right atrial appendages were obtained from 46 patients with SR and 33 patients with cAF (cAF >6 months; Table 1).
Electrophysiological Recordings
Atrial myocytes were isolated with the use of our previous protocol17 and were suspended in storage solution (in mmol/L: KCl 20, KH2PO4 10, glucose 10, K-glutamate 70, -hydroxybutyrate 10, taurine 10, EGTA 10, albumin 1, pH 7.4). Membrane currents were measured with standard whole-cell voltage-clamp techniques. ISO-2 software (MFK) was used for whole-cell and single-channel data acquisition and analysis.
For whole-cell measurements, borosilicate glass microelectrodes had tip resistances of 1 to 2 M when filled with pipette solution (in mmol/L: K-aspartate 100, NaCl 10, KCl 40, Mg-ATP 5, EGTA 2, GTP-Tris 0.1, HEPES 10, pH 7.4). Myocytes were superfused with a solution containing the following (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH 7.4 at 22°C to 24°C. Seal resistances were 4 to 8 G. Series resistance and cell capacitance were compensated. Basal current was measured by applying a depolarizing ramp pulse from –100 to +40 mV (holding potential –80 mV; see inset in Figure 1A). IK,ACh was stimulated with the nonselective M-receptor agonist carbachol (CCh) (2 μmol/L) and was identified with the selective bee venom toxin tertiapin.18 The nature of basal current and IK,ACh as inward rectifier currents was proven in each myocyte by applying Ba2+ (1 mmol/L) at the end of the experiments. The myocytes were superfused with tyrode solution via a peristaltic pump throughout the experiments. Drugs were applied via an additional rapid solution exchange system (ALA Scientific Instruments). During drug-free periods, the rapid solution exchange system supplied tyrode solution only. Data were not corrected for the calculated liquid junction potential (–12 mV; software JPCalc, version 2.2). To control for variability in myocyte size, the currents are expressed as densities (pA/pF).
Single-channel currents were recorded in cell-attached configuration (Axopatch 200B, Axon Instruments). The bath solution contained the following (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH 7.4 with NaOH. Borosilicate glass microelectrodes were coated with Sylgard (Dow Corning) and had tip resistances of 3 to 8 M when filled with the pipette solution (in mmol/L: KCl 140, HEPES 10, pH 7.2 with KOH). IK,ACh was activated by including 10 μmol/L CCh into the pipette solution; identity of IK,ACh in the absence of CCh application was tested by applying 10 nmol/L tertiapin to the bath solution. The single-channel characteristics of IK1 and IK,ACh were analyzed at –120 mV. The total recording time was 20 seconds for each voltage or drug application. The open probability of the channels was calculated from the total number of openings during 20 seconds of recording time. The average open probability per 1-second tracing was used for statistical evaluation. The single-channel slope conductances of IK1 and IK,ACh were calculated from the individual current-voltage relationships by linear regression analysis.
Molecular Analysis of the G3 Gene
Direct activation of IK,ACh is mediated by -subunits of G proteins. The G protein 3 subunit gene (GNB3) contains a C825T polymorphism, whereby homozygous 825T-allele carriers exhibit larger basal inward rectifier K+ current densities possibly because of enhanced signal transduction.17 To exclude confounding by homozygous 825T-allele carriers, all patients were genotyped for the C825T polymorphism. DNA extraction and genotyping were performed in a blinded manner as described.7 In this report the patient sample includes homozygous and heterozygous C825-allele carriers only.
Statistical Analysis
Throughout this report, n refers to the number of myocytes/patients, unless otherwise stated. For quantitative comparisons, average values were calculated from multiple data obtained from 1 patient. Because the number of cells investigated varied widely in each patient (unbalanced observations), we calculated means from average values for each patient.
One-way ANOVAs were applied to determine the sources of K+ current variation (SPSS version 12.0). Independent variables were cAF, selected clinical variables, and medication (Table 1). Differences between group means for continuous data were compared by unpaired Student t test or when patients with SR or cAF were stratified by underlying disease or medication by 1-way ANOVA and post hoc multiple comparisons tests (Bonferroni t test). The concentration-dependent effects of tertiapin on basal and CCh-activated current were evaluated with a mixed linear model (with fixed factors for rhythm status and concentration of tertiapin and random factor for subjects) followed by post hoc Tukey-Kramer t test (SAS version 9.1). To calculate the concentration of tertiapin blocking the CCh-activated IK,ACh by 50% (IC50) in SR and cAF, the concentration-response curve of the mean values was fitted to a 4-parameter logistic function (Prism version 4.0). Frequency data were analyzed with 2 statistics. P<0.05 was considered statistically significant.
Results
Amplitudes of basal current and IK,ACh were related to selected clinical variables. Significant differences between the 2 groups were found for hyperlipidemia and left atrial diameter. Patients with cAF more often received digitalis and diuretics, whereas lipid-lowering drugs were more frequently used in those with SR (Table 1). The patients used for the tertiapin studies showed clinical characteristics comparable to those of the other groups. With 1-way ANOVAs, AF was the only predictor of basal current and IK,ACh current density (data not shown).
Basal Current and IK,ACh in Atrial Myocytes of SR and cAF Patients
In voltage-clamped human atrial myocytes, cell capacitances averaged 80.3±4.7 pF (n=136) for SR and 89.6±4.1 pF (n=76) for cAF myocytes (P<0.05). Basal current in the absence of agonist (Figure 1A) was confirmed to be larger in cAF than in SR (at –100 mV: –22.9±2.1 pA/pF, n=76/25 [myocytes/patients] versus –12.7±1.0 pA/pF, n=136/38; P<0.05).
Application of CCh resulted in rapid initial increase (Peak) of IK,ACh amplitude, which was followed by a decrease to a quasi–steady state level (Qss) despite the continuous presence of CCh (desensitization).10 There was no difference in desensitization of IK,ACh between myocytes from SR and cAF patients (Figure 1B), the Peak/Qss ratio of IK,ACh being 0.58±0.06 (n=76/25) for AF versus 0.54±0.03 (n=136/38) for SR (P=NS). As expected, the CCh-activated Peak- and Qss-IK,ACh currents were smaller in cAF than in SR groups (Figure 1B, 1C).
Effects of Tertiapin on Basal Current and IK,ACh
The selective IK,ACh blocker tertiapin18 was used to discriminate between IK1 and IK,ACh contribution to enhanced basal current in AF. Tertiapin blocks IK,ACh with IC50 values between 8 and 30 nmol/L without any effect on IK1 up to 1 μmol/L.16,19,20
For this set of experiments we applied CCh (2 μmol/L) twice with 4 minutes of washing in between (S1, S2). S1 served as internal control, whereas S2 was measured in the presence of tertiapin or the nonselective M-receptor blocker atropine. Even under control conditions, IK,ACh was lower during S2 than during S1, suggesting incomplete recovery from desensitization (Figure 2A, 2B). However, the degrees of IK,ACh desensitization were similar in SR and cAF myocytes (Figure 2C).
Application of tertiapin (0.1 to 100 nmol/L) during S2 reduced the S2/S1 ratio in a concentration-dependent manner, with greater potency in cAF than in SR (–logIC50: 9.1 for AF versus 8.2 for SR; P<0.05; Figure 3A to 3C). In addition, tertiapin concentration-dependently impaired basal current in cAF, yielding a tertiapin-sensitive component of –6.7±1.2 pA/pF at 10 nmol/L (n=16/5; Figure 3D, 3E). After subtraction of the tertiapin-sensitive current component, basal current of cAF patients remained increased (Figure 3D), suggesting enhanced IK1 in these patients.
Tertiapin also suppressed a component of basal current in SR; however, this amounted to only –1.7±0.5 pA/pF at 10 nmol/L (n=24/8). Application of drug-free tyrode solution alone caused a small decrease of basal current by –1.5±0.6 pA/pF (n=5/4) in SR and –1.6±1.2 pA/pF (n=4/3) in cAF. Our results suggest constitutively active IK,ACh channels in cAF only (Figure 3A, 3C).
The cAF patients involved in the experiments with tertiapin did not have a history of vagally mediated AF. Compared with SR, they had larger left atria and a higher prevalence of diabetes, and they more frequently took diuretics (Table 1). However, basal currents were similar in cAF patients with and without valvular heart disease (28.7±4.2 pA/pF, n=21/5 versus –26.2±4.4 pA/pF, n=17/4) or diabetes (27.1±3.4 pA/pF, n=28/7 versus –29.2±7.5 pA/pF, n=10/2). The same holds true for treatment with diuretics (27.9±3.5 pA/pF, n=24/6 versus –26.9±6.2 pA/pF, n=14/3). The tertiapin (10 nmol/L)-sensitive current component was 6.4±1.5 pA/pF (n=12/3) and 6.3±1.6 pA/pF (n=12/4) in the absence of diuretics and digitalis, respectively, suggesting a lack of drug effects on constitutively active IK,ACh channels in cAF.
Next we excluded the contribution of spontaneously active M-receptors to enhanced basal current in cAF. Atropine (1 μmol/L) abolished the CCh-activated IK,ACh in both groups without effect on basal currents. Thus, tertiapin-sensitive constitutive activity of IK,ACh is independent of M-receptors (Figure 4).
Single-Channel Activity of IK1 and IK,ACh in SR and Chronic AF Patients
For a more direct proof of constitutive activity of IK,ACh channels in cAF, we measured single-channel activity in the cell-attached mode. Constitutive activity of IK,ACh was detected in cAF but was minute in SR (channel open probability: cAF, 5.4±0.8%, n=19/9 versus SR, 0.1±0.01%, n=16/9; P<0.05; Figure 5A to 5C). Moreover, open probability of IK1 was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05; Figure 5D) without changes in other channel characteristics (Table 2). Exclusion of cAF patients taking diuretics or digitalis had no impact on constitutive activity of IK,ACh. Open probability of IK,ACh was 6.1±0.7% (n=8/4) and 5.2±0.5% (n=9/4) in the absence of diuretics and digitalis, respectively.
Inclusion of 10 μmol/L CCh in the pipette solution caused single-channel openings in periodic bursts in all SR and cAF myocytes studied. Amplitudes and open times of constitutively active IK,ACh channels were not different from those of channels activated with CCh (Table 2).
Exposure of myocytes from SR patients with tertiapin (10 nmol/L) reduced open probability of CCh-activated IK,ACh (Figure 6A, 6C), which is in agreement with previous results obtained in rabbit atrial myocytes.20 Consistent with the reduction of whole-cell basal current shown in Figure 3C, open probability of constitutively active IK,ACh during cAF was lower in the presence of 10 nmol/L tertiapin without concomitant effects on IK1 (Figure 6B, 6D).
Discussion
In the present study we demonstrate that in addition to enhanced subunit expression, increased open probability of IK1 channels contributes to higher basal inward rectifier K+ current in patients with cAF. We provide evidence for involvement of agonist-independent constitutive activity of IK,ACh channels from several observations: (1) the selective IK,ACh inhibitor tertiapin reduced basal current in cAF but not in SR; (2) single-channel measurements demonstrated spontaneous openings of IK,ACh channels in myocytes from cAF patients only; and (3) the open probability of constitutively active single IK,ACh channels during cAF was lower in the presence of tertiapin. Evidence for agonist-independent constitutive activity of IK,ACh was provided by the lack of block of these channels by the M-receptor antagonist atropine. Our results suggest that in cAF the higher basal inward rectifier K+ current results from increased expression and open probability of IK1 and constitutively active IK,ACh channels, which may contribute to the perpetuation of the arrhythmia.
Comparison With Previous Studies
At the whole-cell level, several studies reported higher amplitude of basal inward rectifier K+ current in cAF than in SR patients.3,4,6,7,14,15 Pacing-induced tachycardia in dogs also results in higher IK1 amplitude,16,21 suggesting that the increase in IK1 is a consequence of the arrhythmia. Because expression of the corresponding channel subunits at mRNA and protein levels followed the increase in current amplitude,6,15 it was concluded that modified channel subunit transcription and/or translation is the only mechanism of increased IK1 during human cAF. Our results at the single-channel level, however, showed that open probability of IK1 is slightly but significantly higher in cAF than in SR, suggesting that the molecular basis of increased IK1 is more complex and involves not only increased channel subunit expression but also additional modifications of channel regulation. The molecular mechanisms of enhanced open channel probability of IK1 are currently unknown but may involve impaired phosphorylation-dependent regulation. The Kir2.x channel subunits of IK1 possess phosphorylation sites for different kinases, and higher channel subunit phosphorylation results in lower IK1 amplitude.22,23 Because cAF is associated with higher activity of the counterbalancing type 1 and type 2A serine/threonine protein phosphatases,8 it cannot be excluded that the expected stronger channel dephosphorylation contributes to higher IK1 activity in AF. Further studies are needed to identify the molecular mechanisms of modified IK1 regulation in cAF patients.
Consistent with previous studies,4,6,7 we found that the M-receptor–mediated activation of IK,ACh is smaller in cAF than in SR patients. Moreover, reduced channel subunit expression at the mRNA and protein level corresponds well to the lower activation of IK,ACh in cAF.6,12,13,15 Nevertheless, we clearly demonstrated that IK,ACh becomes constitutively active when cAF develops. The selective IK,ACh blocker tertiapin concentration-dependently inhibited whole-cell CCh-activated IK,ACh, with higher potency in AF than in SR. Tertiapin blocks native IK,ACh channels with IC50 values between 8 and 30 nmol/L16,19,20 without significant effects on other ion channels.19,20 The effective concentration range of tertiapin found in the present study (Figure 3) is in good agreement with the reported potency. In human atria, however, tertiapin unmasked a robust component of constitutively active IK,ACh in cAF and a substantially smaller one in SR. Recent findings in dog atria and pulmonary veins detected a tertiapin-sensitive current component contributing to basal inward rectifier current, and, consistent with our results, pacing-induced tachycardia was associated with higher whole-cell current density of this current component.16 Thus, the development of constitutively active IK,ACh currents in human cAF is probably a consequence of atrial remodeling contributing to the perpetuation rather than the initiation of the arrhythmia.
The molecular basis of constitutively active IK,ACh is currently unknown. At the single-channel level, basal IK,ACh activity in cAF resulted from a higher number of channel openings and subsequent increase in channel open probability without concomitant changes in basic current properties. The basic properties of IK1 and IK,ACh were in good agreement with previous results.24,25 Thus, the reduced channel subunit expression in cAF6,12,13,15 is probably part of the modified IK,ACh regulation and can be interpreted as an adaptation on the higher frequency of IK,ACh openings in these patients.
The regulation of IK,ACh is complex, suggesting several putative mechanisms for constitutive activity of IK,ACh. Because the latter was not affected by blocking M-receptors with atropine, basal activity of IK,ACh is an agonist-independent process. Increased agonist-independent dissociation of G- and G-subunits of inhibitory G proteins appears unlikely because in dog atrial myocytes neither pertussis toxin treatment nor absence of GTP affected the tertiapin-sensitive component of basal current.16
Evidence for constitutive activity of atrial IK,ACh channels was provided by earlier studies26,27 that showed that agonist-independent activation of IK,ACh requires ATP. Interestingly, consistent with the results from atria of dogs with tachypacing,16 pertussis toxin treatment failed to prevent the ATP activation of IK,ACh.26 Because activation of IK,ACh requires ATP,26–28 modified phosphorylation-dependent channel regulation may contribute to the development of constitutively active IK,ACh in cAF. Consistent with this idea, recent publications have shown that in atrial myocardium Kir3.1 forms a macromolecular complex, allowing for local regulation of IK,ACh function.28,29 The atrial GIRK1 macromolecular complex is composed of the catalytic subunits of PKA, PKC, and CaMKII and the protein phosphatases PP1 and PP2A.28,29 Thus, the qualitative and quantitative composition of the GIRK1 macromolecular complex may change during AF, resulting in abnormal regulation of IK,ACh. Further work is needed to verify these hypotheses.
Study Limitations
In the present investigation we studied regulation of IK1 and IK,ACh only in isolated myocytes in vitro. The situation in multicellular preparations and in vivo is more complex. Thus, our data should be extrapolated with caution to the in situ atria. It is well known that the atria are heterogeneous tissues. Therefore, our data obtained in right atrial appendages may not necessarily reflect alterations in the rest of the atria. Digitalis, diuretics, and lipid-lowering drugs were more frequently prescribed in cAF. Thus, we cannot exclude that drugs affect density of IK1 and IK,ACh. Finally, our current recordings were performed at room temperature only. Their regulation may differ at physiological temperature.
Potential Clinical Implications
Our results show that the molecular basis for the higher amplitude of basal inward rectifier current in AF involves increased Kir2.x channel subunit expression6,15 and channel open probability (present study). Two-dimensional computer simulations of human atrial tissue showed that IK1 affects spiral wave dynamics.30 An increase of IK1 during cAF resulted in reduced APD and hyperpolarization of the resting membrane potential, which is consistent with the shorter APD and the more negative resting membrane potential of multicellular trabeculae from patients with cAF.6 The changes in APD and resting membrane potential were associated with higher availability of the Na+ current (INa) and enhanced excitability, leading to faster rotation frequencies and stabilization of functional reentry. Most important, reduction of IK1 in the model was associated with APD prolongation and depolarization, resulting in decreased rotation frequency and spiral wave termination.30 Consistent with these findings, mice with overexpression of the IK1 channel subunit Kir2.131 and humans with a gain-of-function mutation of this channel develop cAF.32 Thus, blockade of IK1 may be a viable antiarrhythmic option.
In analogy to IK1, constitutively active IK,ACh may contribute to stabilization of functional reentry because in ACh-induced AF in sheep, the higher activation frequencies in left atria are associated with larger density of IK,ACh.33 In vivo, vagal nerve stimulation abbreviates APD and increases APD heterogeneity and atrial vulnerability to tachyarrhythmia,34 which perpetuate AF.9 In knockout mice lacking the Kir3.4 channel subunit of IK,ACh, M-receptor stimulation did not induce AF,11 suggesting that the effects of vagal nerve activation are mediated by IK,ACh. Thus, the agonist-independent constitutive activation of IK,ACh during cAF is expected to increase atrial vulnerability to tachyarrhythmia and to sustain AF. Because some antiarrhythmic drugs such as amiodarone, flecainide, quinidine, and verapamil, which effectively terminate AF,35 are also inhibitors of IK,ACh,36–38 it cannot be excluded that their effectiveness in AF results in part from blockade of IK,ACh and that at least in some patients with AF the inhibition of constitutively active IK,ACh may have an antiarrhythmic affect.
Finally, there is evidence of circulating M2 autoantibodies in AF patients, which may exert a tonic activation of M-receptor signal transduction.39 Furthermore, the ACh concentrations in the synaptic clefts could be elevated because of reduced activity of its degrading enzyme acetylcholine esterase.40 Because atropine had no effect on basal current in cAF, the development of constitutively active IK,ACh channels occurs in a receptor-independent manner. Thus, it is likely that the elevated synaptic ACh concentrations and the presence of M2 autoantibodies and constitutively active IK,ACh channels reinforce each other in promoting AF. Further studies are needed to test these hypotheses.
Conclusions
We demonstrated that the molecular basis of higher basal inward rectifier K+ current in patients with cAF involves increased density of IK1 and development of constitutively active IK,ACh. Our results provide a mechanistic insight into the regulation of IK1 and IK,ACh in cAF that may help to design new therapeutic options for AF.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DO 769/1 to Dr Dobrev). The authors thank Trautlinde Thurm, Ulrike Heinrich, and Manja Schne for excellent technical assistance and Eberhard Kuhlisch for statistical advice.
Disclosures
None.
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