Molecular Basis of Downregulation of G-Protein–Coupled Inward Rectifying K+ Current (IK,ACh) in Chronic Human Atrial Fibrillation
Decrease in GIRK4 mRNA Correlates With Reduced IK,ACh and Muscarinic Receptor–Mediated Shortening of Action Potentials
Background Clinical and experimental evidence suggest that the parasympathetic nervous system is involved in the pathogenesis of atrial fibrillation (AF). However, it is unclear whether changes in G-protein-coupled inward rectifying K+ current (IK,ACh) contribute to chronic AF.
Methods and Results In the present study, we used electrophysiological recordings and competitive reverse-transcription polymerase chain reaction to study changes in IK,ACh and the level of the IK,ACh GIRK4 subunit in isolated human atrial myocytes and the atrial tissue of 39 patients with sinus rhythm and 24 patients with chronic AF. The density of IK,ACh was ≈50% smaller in myocytes from patients with AF compared with those in sinus rhythm, and this was accompanied by decreased levels of GIRK4 mRNA. The current density of the inward rectifying K+ current (IK1) was 2-fold larger during AF than in sinus rhythm, in correspondence with an increase in Kir2.1 mRNA. The larger IK1 in AF is consistent with more negative membrane potentials in right atrial trabeculae from AF patients. Moreover, action potential duration was reduced in AF, and the action potential shortening produced by muscarinic receptor stimulation was attenuated, indicating that the changes of IK1 and IK,ACh were functionally relevant.
Conclusions Chronic human AF induces transcriptionally mediated upregulation of IK1 but downregulation of IK,ACh and attenuates the muscarinic receptor–mediated shortening of atrial action potentials. This suggests that atrial myocytes adapt to a chronically high rate by downregulating IK,ACh to counteract the shortening of the atrial effective refractory period due to electrical remodeling.
Received June 22, 2001; revision received September 5, 2001; accepted September 17, 2001.
Clinical1,2 and experimental3–5 evidence suggest that autonomic tone plays an important role in the pathogenesis of atrial fibrillation (AF). Vagal stimulation can initiate AF by increasing the heterogeneity of the effective refractory period (ERP).3,6,7 In dogs, catheter ablation of the cardiac parasympathetic nerves abolishes vagally mediated AF8 and vulnerability of the atria to tachyarrhythmia is reduced in the absence of parasympathetic activity.3 Thus, increased ERP heterogeneity may be important for the initiation and perpetuation of AF. The cellular mechanisms underlying these effects, however, are less well understood.
Stimulation of muscarinic receptors activates the G-protein–coupled inward rectifying K+ current (IK,ACh), which shows altered activity in diseased human atrial tissue.9–12 This current was increased in myocytes from patients with chronic AF,11 and mRNA expression of these channels was reduced.12 At present, it is unclear whether this observation relates to respective changes in human atrial action potentials (APs).
In the present study, we investigated the expression of the inward rectifying K+ current (IK1) and IK,ACh and compared the respective current density in the same tissue from patients with sinus rhythm (SR) and AF. Due to a limited sample size, we investigated only the GIRK4 subunit of IK,ACh. The cardiac IK,ACh channel is composed of GIRK1 (Kir3.1) and GIRK4 (Kir3.4) proteins, with GIRK4 being essential for functional channels.13 Moreover, GIRK4 probably regulates GIRK1 expression.14 Although several different cDNA clones of IK1 have been detected in the human atrium (Kir2.1 through Kir2.4 and TWIK1),15 we selected Kir2.1 because its abundance is suggested to be responsible for >80% of IK1.16 Because the changes in IK,ACh density in human chronic AF seem to be similar to those detected in association with the C825T polymorphism of the gene encoding for the G-protein β3 subunit,17 we genotyped our patients to investigate whether chronic AF and the homozygous 825T allele gene variant are independent contributors to K+ current density. Finally, to examine the functional aspects of the observed current changes in chronic AF, we recorded APs in both single myocytes and trabeculae after pharmacological stimulation of IK,ACh with the muscarinic receptor agonist carbachol.
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 39 patients with SR and 24 patients with chronic AF (AF>6 months; Table 1). Exclusion criteria were paroxysmal AF and use of antiarrhythmic drugs. Significant differences between the 2 groups were found for sex, underlying heart disease, and left atrial diameter. AF patients more often received digitalis, Ca2+ antagonists of nondihydropyridine type, and nitrates (Table 1).
One part of the samples was snap-frozen in liquid nitrogen, and the remainder was used immediately to isolate single myocytes or atrial trabeculae. Some biopsies were too small to permit electrophysiological and mRNA measurements in the same tissue.
Construction of RNA Mimics
RNA mimics were synthesized according to Wang et al16 and verified by reverse-transcription polymerase chain reaction (RT-PCR) and gel electrophoresis. The synthetic RNA mimics had gene-specific primers for the target K+ channel mRNA at both ends and a 460-bp fragment of the human cardiac α-actin in the middle.
Different amounts of RNA mimics were added to 0.5 μg of RNA specimen and denatured at 70°C for 10 minutes. Samples were reverse-transcribed by Superscript RT RNase H− Reverse Transcriptase (Gibco BRL) with random hexamer primers at 25°C for 10 minutes; this was followed by further incubation at 42°C for 50 minutes and a denaturing step at 90°C for 5 minutes. The synthesized cDNA (10 μL) was then used as an amplification template in a 50-μL standard reaction mixture. After an initial denaturing step at 94°C for 5 minutes, PCR mixes were amplified during 30 cycles with denaturing at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 40 seconds (final extension, 72°C for 7 minutes).
Quantification of PCR Products
Ethidium bromide–stained amplicons were separated by gel electrophoresis (2% agarose) and imaged by a CCD camera (Biostep) under ultraviolet light. Size and quantity of gel bands were determined with Phoretix 1D software (version 4.01). Briefly, a DNA mass marker was used to generate a standard curve by linear regression for each experiment. The density of each sample band was then converted to an absolute quantity by calibrating to the standard curve.
The absence of genomic DNA and cDNA contamination in RNA mimics and specimens was shown by lack of signals for RT-negative controls. The equivalence of sample mRNA input in each experiment was confirmed by noncompetitive PCR for α-actin (data not shown). This was based on the assumption that the levels of α-actin are not changed by AF. To prove equal amplification of sample RNA and mimic, known quantities of target and mimic RNA were coamplified in single reaction tubes for 35 cycles and analyzed quantitatively.
Molecular Analysis of the Gβ3 Gene
DNA extraction and genotyping were performed in a blinded manner, as previously described.18
Atrial myocytes were isolated using our previous protocol17 and were suspended in a storage solution consisting of (in mmol/L): KCl 20, KH2PO4 10, glucose 10, K-glutamate 70, β-hydroxybutyrate 10, taurine 10, EGTA 10, and albumin 1 (pH=7.4). Cell yield of well-striated, rod-shaped myocytes was 17.5±1.6% (n=41). Isolated cells were investigated within 6 to 8 hours.
The conventional voltage-clamp technique was used to measure membrane currents.17 pCLAMP 5.5 software (Axon Instruments) was used for data acquisition and analysis. Borosilicate glass microelectrodes had tip resistances of 1 to 2 MΩ when filled with pipette solution, which contained (in mmol/L): K-aspartate 100, NaCl 10, KCl 40, Mg-ATP 5, EGTA 2, GTP-Tris 0.1, and HEPES 10 (pH=7.4). Myocytes were superfused with a solution containing (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10 (pH=7.4 at 22°C to 24°C). Seal resistances were 4 to 8 GΩ. Series resistance and system capacitance were compensated. The presented data were not corrected for the calculated liquid junction potential (−12 mV, software JPCalc).17 The membrane currents were corrected for a linear leak conductance calculated from the reversal potential of IK,ACh.17
Atrial APs were recorded in human right atrial myocytes with patch-electrodes (tip resistance 6 to 8 MΩ) filled with the following solution (in mmol/L): K-aspartate 100, NaCl 8, KCl 40, Mg-ATP 5, EGTA 5, CaCl2 2, GTP-Tris 0.1, and HEPES 10 (pH=7.4); the bath solution contained (in mmol/L): NaCl 150, KCl 5.4, and MgCl2 2 (otherwise as above). AP duration (APD) was also studied in human right atrial trabeculae using the standard microelectrode technique (microelectrode tip resistance, 12 to 15 MΩ; 1 ms stimulus, 25% above threshold intensity). The bath solution contained (in mmol/L): NaCl 125, KCl 5, MgCl2 0.6, CaCl2 1, NaH2PO4 0.4, NaH2CO3 22, and glucose 5.5 and was equilibrated with O2-CO2 (95:5) at 37°C (pH=7.4).
Univariate ANOVAs were applied to associate preoperative variables with channel expression and electrophysiological data (SPSS version 10.0). Differences between continuous data were compared by unpaired Student’s t test. Frequency data were analyzed with χ2 statistics. P<0.05 was considered statistically significant.
Expression of Kir2.1 and GIRK4, density of IK1 and IK,ACh, and AP characteristics were related to selected clinical variables. With univariate ANOVAs, AF was the only predictor of mRNA levels and electrophysiological parameters (data not shown).
All patients of the present study were genotyped for the G-protein β3-subunit C825T polymorphism, and only homozygous and heterozygous C825 allele carriers were included.
Correlation Between mRNA Levels and K+ Current Density
Differences in mRNA levels of Kir2.1 and GIRK4 between the patient groups were evaluated by competitive RT-PCR (Figures 1 and 2⇓). Equal amplification of sample RNA and mimic was verified by coamplification of known RNA quantities of target and mimic in single reaction tubes when subjected to 24 through 35 cycles (Figures 1A and 2⇓A). Representative gels of competitive RT-PCR products are shown in Figures 1B and 2⇓B. Figure 1C shows the abundance of Kir2.1 channel mRNA: the logarithmic ratio of optical intensity (target/mimic) was plotted versus the logarithm of mimic mRNA concentration. The transcripts of Kir2.1 were more and those of GIRK4 less abundant in patients with AF (Figures 1D and 2⇓C).
Absence of noncardiac tissue was confirmed by noncompetitive PCR using gene-specific primers for sucrase-isomaltase (data not shown), an enzyme expressed only in the epithelium of the small intestine.19 To exclude possible contamination of cardiac RNA by neuronal tissue, we measured the expression of the nicotinic acetylcholine receptor β4 subunit by RT-PCR.20 As a positive control, total RNA isolated from rat brain contained mRNA of this subunit, but corresponding message was absent in human atrial samples (Figure 2D).
To correlate channel expression with function, current densities of IK1 and IK,ACh were measured in myocytes from the same biopsies. The myocyte sizes, as measured by membrane capacitance,17 were larger in patients with AF (157±14 pF) than in those with SR (92±12 pF; P<0.05). Mean resting membrane potential (RMP; [K+]o, 20 mmol/L) was more negative in patients with chronic AF than in those with SR (−30.0±1.4 mV, n=23, versus −26.4±2.7 mV, n=12; P<0.05). In SR cells (n=12), the RMP was −25.2±2.3 mV before and −29.8±2.2 mV after carbachol stimulation; in AF cells (n=6), it was −27.5±2.0 mV before and −27.0±1.4 mV after carbachol stimulation (P=NS for both). Accounting for junction potential17 and assuming [K+]i was controlled by the pipette solution (140 mmol/L), the resting membrane potentials were −42 and −39 mV, respectively; the deviation from the potassium equilibrium potential of −49 mV is explained by a voltage drop resulting from the high input resistance of human atrial cells.
Averaged current-voltage relations are shown in Figure 3. At −100 mV, IK1 was significantly larger in patients with AF (−10.2±1.1 pA/pF, n=37 cells from 11 patients) than in those with SR (−4.3±0.7 pA/pF, n=30 cells from 12 patients; P<0.0001), and there was good quantitative agreement between increased IK1 and extent of mRNA upregulation (Figure 3C). In contrast, IK,ACh (defined as carbachol-sensitive current after 2 minutes of application) was smaller in patients with AF (−3.9±1.0 pA/pF, n=37 cells from 11 patients) than in those with SR (−7.3±0.9 pA/pF, n=30 cells from 12 patients; P<0.02), thus corresponding to the respective mRNA levels (Figure 3D). At plateau voltages (−10 mV), IK1 was larger (0.7±0.1 pA/pF for AF versus 0.4±0.1 pA/pF for SR; P<0.05) and IK,ACh was smaller in patients with AF (0.7±0.1 pA/pF for AF versus 1.1±0.1 pA/pF for SR; P<0.03). The effect of carbachol was fully reversible within 2 to 3 minutes of washout. Both currents were completely blocked by Ba2+ (1 mmol/L; Figures 3A and 3B), and in the presence of 1 μmol/L atropine, carbachol failed to activate IK,ACh in SR and AF cells (n=12 cells from 7 patients for SR, n=7 cells from 3 patients for AF; P=NS, data not shown). Atropine had no effect on IK1 in AF cells (−10.5±1.1 pA/pF before and −10.8±1.2 pA/pF after atropine, n=12 cells from 5 patients). In AF cells, the carbachol-stimulated currents had a more negative reversal potential than those in SR cells (−41.0±1.1 mV for AF versus −37.2±1.2 mV for SR; P<0.03). Cumulative concentration-response curves for carbachol (0.01 to 10 μmol/L) were obtained, and individual curve fitting resulted in a mean logEC50 of −6.9±0.1 for SR (n=8 cells from 6 patients) and −6.8±0.1 for AF (n=3 cells from 3 patients; P=NS).
Interestingly, the AF-related decrease in IK,ACh was observed only in large-sized myocytes (>100 pF), whereas the increase of IK1 was independent of cell size (data not shown).
AP Measurements in Human Atrial Myocytes and Trabeculae
Next, we investigated the impact of current changes on atrial APs (Figure 4). In myocytes from AF patients, the late repolarization phase (APD90) was abbreviated and the effect of carbachol was attenuated compared with SR patients (Table 3). We also recorded APs in atrial trabeculae. The RMP in AF was more negative than that in SR (P<0.04; Table 3). These experiments also provided reliable data for the early repolarization phase (APD20), which was longer in AF cells. In contrast, APD90 was shorter in AF, thus confirming the results obtained in single cells (Figure 5 and Table 3). In addition, APD shortening in response to carbachol was significantly smaller in AF than in SR (Figure 5 and Table 3).
The major findings of the present study were as follows. (1) In tissue from the same atrial appendage, mRNAs of Kir2.1 and GIRK4 transcripts quantitatively matched the AF-related increase of IK1 and decrease of IK,ACh. (2) In correspondence with upregulation of IK1 in AF, single myocytes and trabeculae had more negative RMPs and shorter control APs. (3) The abbreviation of the APs produced by muscarinic receptor stimulation was attenuated in both atrial myocytes and trabeculae of AF patients.
Comparison With Previous Studies
We provide direct evidence that the increase of IK111,21 is associated with corresponding changes in Kir2.1 mRNA. In contrast, mRNA concentrations of Kir2.1 and the corresponding currents are not altered in the canine rapid-atrial pacing model.22 Thus, the pathophysiological changes during chronic human AF may be distinct from those associated with rapid pacing in dog atria. Functional consequences of increased IK1 in AF could be shortening of APD90 and hyperpolarization of RMP (see below).
Vagal stimulation shortens APD by cholinergic modulation of repolarization mediated by activation of IK,ACh.15 The resulting increase in ERP heterogeneity may contribute to the perpetuation of AF.7 Therefore, IK,ACh was investigated and found to be reduced in AF, thus confirming previous results (D. Dobrev, MD, et al, unpublished data, 2001). In AF, GIRK4 mRNA was reduced by 32% compared with SR, and this corresponded with the reduction of IK,ACh. While our study was being completed, a similar reduction of GIRK4 mRNA was reported, together with reduced GIRK1 protein, and protein levels of several ion channels, including GIRK1, correlated positively with the ERP and with rate adaptation of this parameter.12
The mean size of the myocytes from AF patients was larger than that in SR.5,23 Moreover, IK,ACh is reduced only in myocytes with >100 pF capacitance (ie, in cells assumed to be hypertrophic; D. Dobrev, MD, et al, unpublished data, 2001). This downregulation of IK,ACh in large cells will limit cholinergic modulation of repolarization. Because carbachol-induced APD shortening was, in fact, attenuated in trabeculae from AF patients, these preparations seem to be dominated by the electrophysiological properties of hypertrophic cells (see below).
APs recorded from atrial tissue of AF patients were shorter. At the molecular level, the AP abbreviation in AF is related to a reduction of the depolarizing inward current and to an increase of hyperpolarizing outward current (L-type Ca2+ current;5 IK111,21). The recently reported downregulation of repolarizing K+ currents such as the transient outward K+ current (Ito) and the ultra-rapid outward K+ current (IKur) in human atrial myocytes21 is in good agreement with the observed prolongation of APD20 in trabeculae from AF patients. APD90 was shorter in AF and responded less well to carbachol, which is consistent with the increased IK1 and decreased IK,ACh measured in myocytes. Because the activation of IK,ACh in myocytes increases ERP heterogeneity and vulnerability to atrial tachyarrhythmia,3,6 the small amplitude of IK,ACh and the attenuated APD90 response to carbachol may be a secondary, compensatory mechanism against the changes that promote the initiation and maintenance of AF. Thus, atrial myocytes probably adapt to a high rate by downregulating IK,ACh to counteract the shortening of ERP.
IK1 and IK,ACh are the main contributors to RMP and may exhibit variable responses in different heart diseases. For example, atrial myocytes from patients with heart failure had reduced IK1 and IK,ACh density,9 whereas in dilated human atria, the density of IK1 was unaltered.24 Here, chronic AF was associated with an increased IK1 density; thus, a more negative RMP would be expected. Indeed, we found that RMP in atrial trabeculae from patients with AF were more negative than those in SR, which may indicate a compensatory response of the atria to the depolarizing effects of the high electrical activity during episodes of AF.
In AF, the carbachol-induced shortening of atrial APD was markedly reduced. However, in human atrium, changes in parasympathetic signaling may also include altered vagal acetylcholine release, altered density of muscarinic receptors, and/or inhibitory G-protein levels. Neurotransmitter release is irrelevant in isolated myocytes and, to the best of our knowledge, acetylcholine release and the density of muscarinic receptors have not been investigated in atria from AF patients, but they are decreased with old age.25,26 The age of our patients was not different between the groups, rendering it unlikely that the attenuated AP response to carbachol was due to age-dependent muscarinic receptor reduction. Because inhibitory G-protein levels are unaffected by AF,23 the observed changes in response to carbachol are probably due to the downregulation of IK,ACh channels.
Valvular heart disease was frequently present in the AF group, but it was absent in the SR group. The subgroup analysis, however, showed that the underlying heart disease did not change atrial ion channel expression or electrophysiological parameters. Digitalis and Ca2+ channel blockers of nondihydropyridine type were more frequently prescribed in AF. Thus, we cannot exclude the possibility that drugs affect ion channel expression or current density.
Parasympathetic stimulation shortens APD and increases ERP heterogeneity and the vulnerability of the atria to tachyarrhythmia in vivo,3 probably contributing to the perpetuation of AF.7 Thus, the reduction of IK,ACh might serve as a compensatory mechanism for the AF-related decrease in APD and ERP. Catheter ablation of cardiac parasympathetic nerves in dogs prevents vagal AF,8 suggesting that, at least in some patients with AF, inhibition of parasympathetic activity may have an antiarrhythmic affect.
The authors thank Manja Schöne and Konstanze Fischer for excellent technical assistance.
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