Author + information
- Received June 16, 2013
- Revision received August 5, 2013
- Accepted August 26, 2013
- Published online March 4, 2014.
- Jie Wu, PhD∗,†,‡,
- Nobu Naiki, MD†,
- Wei-Guang Ding, MD, PhD‡,
- Seiko Ohno, MD, PhD†,
- Koichi Kato, MD†,
- Wei-Jin Zang, PhD∗,
- Brian P. Delisle, PhD§,
- Hiroshi Matsuura, MD, PhD‡ and
- Minoru Horie, MD, PhD†∗ ()
- ∗Department of Pharmacology, Medical School of Xi'an Jiaotong University, Xi'an, China
- †Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Japan
- ‡Department of Physiology, Shiga University of Medical Science, Otsu, Japan
- §Department of Physiology, University of Kentucky, Lexington, Kentucky
- ↵∗Reprint requests and correspondence:
Dr. Minoru Horie, Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Seta-Tsukinowa-cho, Otsu, Shiga 520-2192, Japan.
Objectives This study sought to explore molecular mechanisms underlying the adrenergic-induced QT prolongation associated with KCNQ1 mutations.
Background The most frequent type of congenital long QT syndrome is LQT1, which is caused by mutations in the gene (KCNQ1) that encodes the alpha subunit of the slow component of delayed rectifier K+ current (IKs) channel. We identified 11 patients from 4 unrelated families that are heterozygous for KCNQ1-G269S. Most patients remained asymptomatic, and their resting corrected QT intervals ranged from normal to borderline but were prolonged significantly during exercise.
Methods Wild-type (WT) KCNQ1 and/or KCNQ1-G269S (G269S) were expressed in mammalian cells with KCNE1. IKs-like currents were measured in control conditions or after isoproterenol or protein kinase A (PKA) stimulation using the patch-clamp technique. Additionally, experiments that incorporated the phosphomimetic KCNQ1 substitution, S27D, in WT or KCNQ1-G269S were also performed.
Results The coexpression of WT-KCNQ1 with varying amounts of G269S decreased IKs, shifted the current-voltage I-V relation of IKs to more positive potentials, and accelerated the IKs deactivation rates in a concentration-dependent manner. In addition, the coexpression of G269S and WT blunted the activation of IKs in response to isoproterenol or PKA stimulation. Lastly, a phosphomimetic substitution in G269S did not show an increased IKs.
Conclusions G269S modestly affected IKs in control conditions, but it almost completely blunted IKs responsiveness in conditions that simulate or mimic PKA phosphorylation of KCNQ1. This insensitivity to PKA stimulation may explain why patients with G269S mutation showed an excessive prolongation of QT intervals on exercise.
Congenital long QT syndrome (LQTS) is characterized by an abnormal QT interval prolongation on the electrocardiogram (ECG), syncope due to a polymorphic ventricular tachycardia called “torsade de pointes,” and ventricular fibrillation (1,2). At least 13 genes are responsible for different subtypes of the syndrome (LQT1 to LQT13), with LQT1 being the most common and accounting for approximately 40% to 50% of genotyped patients (3,4). LQT1 is caused by mutations in KCNQ1, the alpha subunit of the slow component of delayed rectifier K+ current (IKs), which is a major repolarizing current during the plateau phase of cardiac action potentials (5).
The impaired expression or dysfunction of IKs channels (6,7) can lead to a prolongation in the cardiac action potential and the QT interval on an ECG (7,8). A major role for IKs is to maintain the ventricular action potential duration by offsetting the increase in L-type Ca2+ current (ICa,L) after adrenergic stimulation. Adrenergic stimulation activates protein kinase A (PKA), which directly increases IKs by phosphorylating the KCNQ1 alpha subunit at S27 (8). Not surprisingly, most LQT1 patients experience triggered cardiac events during adrenergic stimulation (i.e., while exercising) (9,10).
More recently, we identified a heterozygous missense KCNQ1 mutation, G269S, in 11 patients from 4 unrelated families. Similar to previous clinical reports (10–12), most of our patients have normal to borderline corrected QT (QTc) intervals at rest, but their QTc intervals are significantly prolonged after exercise. We characterize the functional consequences of the IKs channel reconstituted with G269S in mammalian cells and provide important insight into molecular mechanisms underlying the adrenergic-induced LQTS. Specifically, we found G269S modestly affected IKs but severely blunted the increase in IKs with isoproterenol, pharmacological activators of PKA, and with the PKA phosphomimetic mutation KCNQ1-S27D. These findings may explain why patients with G269S mutation showed an excessive prolongation of QT intervals on exercise and suggest a potential benefit of beta-blocker therapy.
Clinical investigation and genetic testing
The clinical diagnosis of LQTS was referred to the criteria of Schwartz et al. (2). The protocol for genetic analysis was approved by the institutional ethics committee and performed under its guidelines. Written informed consent was obtained from every subject before the analysis. Genomic deoxyribonucleic acid (DNA) used for genetic evaluation was isolated from venous blood lymphocytes. In addition to KCNQ1, genetic screening for mutations in other LQTS-related genes including SCN5A, KCNH2, KCNE1, KCNE2, and KCNJ2 was conducted by denaturing high-performance liquid chromatography (WAVE system, Transgenomic Inc., Omaha, Nebraska). For abnormal screening patterns, sequencing was performed with an automated sequencer (ABI PRISM 3100x, Applied Biosystems, Foster City, California).
Heterologous expression of cDNA in CHO and HEK293 cells
Full-length complementary deoxyribonucleic acid (cDNA) encoding human wild-type (WT) KCNQ1 (GenBank AF000571, Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France) was subcloned into a pIRES2-EGFP expression vector. KCNQ1-G269S, KCNQ1-S27D, and KCNQ1-(S27D-G269S) mutants were constructed using a Quick Change II XL site-directed mutagenesis kit (Stratagene, La Jolla, California), and they were also subcloned into the pIRES2-EGFP expression vector. Full-length cDNA encoding human KCNE1 (GenBank M26685) subcloned into the pCDNA3.1 expression vector was obtained by polymerase chain reaction from human heart cDNA library (Clontech, Mountain View, California). Full-length cDNA encoding human A-kinase-anchoring protein 9 (Yotiao or AKAP9) was subcloned into pCDNA3.1 expression vector (Department of Bio-Informational Pharmacology, Tokyo Medical and Dental University, Japan). KCNQ1-WT and/or its mutants, KCNE1 and Yotiao cDNA were transiently transfected into Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells using Lipofectamine (Invitrogen Life Technologies Inc., Carlsbad, California) according to the manufacturer's instructions.
Solutions and chemicals
The pipette solution contained (in mmol/l) 70 potassium aspartate, 40 KCl, 10 KH2PO4, 1 MgSO4, 3 Na2 adenosine triphosphate (Sigma, St. Louis, Missouri), 0.1 Li2 Guanosine-5'-triphosphate (Roche Diagnostics GmbH, Mannheim, Germany), 5 ethylene glycol tetraacetic acid, and 5 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; and the pH was adjusted to 7.2 with KOH. The extracellular solution contained (in mmol/l) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5.0 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; the pH was adjusted to 7.4 with NaOH. Isoproterenol (Sigma) was dissolved in distilled water (containing 1 mmol/l ascorbic acid) to yield 10 mmol/l stock solution and kept in the dark at 4°C. Forskolin (FK, Sigma) and 3-isobutyl-l-methyl-xanthine (IBMX, Sigma) were respectively dissolved in dimethyl sulfoxide (Sigma) to yield stock solutions of 5 mmol/l and 15 mmol/l, respectively.
Electrophysiological recordings and data analysis
Forty-eight hours after transfection, cells attached to a glass coverslip were transferred to a 0.5-ml bath chamber perfused with extracellular solution and maintained at 25°C. Patch-clamp experiments were conducted on green fluorescent protein-positive cells. Whole-cell membrane currents were recorded with an EPC-8 patch-clamp amplifier (HEKA, Lambrecht, Germany). IKs were evoked by depolarizing voltage-clamp steps given from a holding potential of –80 mV to various test potentials. IKs amplitude was determined by measuring the amplitude of tail current elicited on repolarization to –50 mV following 2-s depolarization to 30 mV every 10 s, and currents were normalized to the cell membrane capacitance to obtain current densities (pA/pF). Voltage-dependence of IKs activation was evaluated by fitting the I-V relation of the tail currents to a Boltzmann equation: IK,tail = 1/[1+exp([Vh – Vm]/k)], where IK,tail is the tail current amplitude density, Vh is the voltage at half-maximal activation, Vm is the test potential, and k is the slope factor. The deactivation kinetics of IKs after depolarization was determined by a single exponential fit of tail current trace.
Forty-eight hours after transfection, CHO cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature, rinsed once in PBS, and permeabilized with 0.2% triton X-100 in PBS. Cells were blocked with 5% bovine serum albumin in PBS for 30 min and then incubated overnight at 4°C with anti-Kv7.1 (KCNQ1, Santa Cruz Biotechnology, Inc., Santa Cruz, California) antibody against its C-terminus at 1:2,000 dilution. Following incubation, cells were labeled with AlexaFluor 488-conjugated goat antirabbit immunoglobulin G (Molecular Probes, Eugene, Oregon) at 1:500 dilution. Immunofluorescence stained cells were captured using a confocal laser scanning microscope (LSM META 510, Carl Zeiss, Berlin, Germany).
All data are expressed as mean ± SE (QTc as mean ± SD), with the number of experiments in parentheses. Statistical comparisons were analyzed using unpaired Student t test and 1-way or 2-way analysis of variance with Newman-Keuls post hoc test. A p value of <0.05 was considered statistically significant.
Genotype analyses of 551 consecutive LQTS probands identified 4 unrelated carriers with the KCNQ1-G269S mutation. Figure 1A shows 4 family pedigrees for the different probands (arrows). Figure 1B shows the location of the mutation (transmembrane domain S5) in the KCNQ1 alpha subunit, and the lower panel in Figure 1C shows representative results of a genotype-positive patient's sequencing data (805G>A).
Figure 1D depicts 2 sets of 12-lead ECG recorded from a 7-year-old boy in Family #2 (Fig. 1A, bold arrow). His resting QTc interval was 437 ms, but it was prolonged to 494 ms after exercise. The boy was asymptomatic, but his maternal grandfather died suddenly at the age of 28 years (the detailed clinical information is not available). In Family #4, the 22-year-old female proband experienced sudden syncope, which recovered soon and was suspected to be due to ventricular tachyarrhythmia, while dancing, but her symptoms have disappeared after receiving beta-adrenergic blocker metoprolol tartrate for 42 months.
We performed an exercise tolerance test in 8 of the 11 mutation carriers (Fig. 1A). The mean resting QTc intervals were 441.6 ± 44.5 ms but significantly (p < 0.05) prolonged to 485.5 ± 14.4 ms after exercise. Therefore, KCNQ1-G269S might minimally affect IKs at rest but impair the up-regulation of IKs by PKA, because the exercise stress (PKA activation) does not prolong the QTc interval in control subjects (13).
KCNQ1-G269S mutation causes a mild loss-of-function in IKs in control conditions
Figures 2A to 2D show 4 sets of representative current traces recorded from CHO cells expressing KCNE1 with KCNQ1-WT, KCNQ1-WT + KCNQ1-G269S, and KCNQ1-G269S, respectively. Cells transfected with varying amounts of G269S cDNA reduced both the steady state and tail IKs amplitudes in a concentration-dependent manner.
Figure 2E shows the current-voltage (I-V) relations for tail IKs elicited after the voltage-step to –50 mV from various test potentials, and Figure 2F summarizes tail IKs densities under 4 different conditions. Compared with WT, G269S alone or WT + G269S significantly decreased the mean IKs density for voltages between –20 mV and 50 mV and the mutation displayed a moderate dominant-negative suppression on WT-KCNQ1 function.
Figure 3A shows the normalized I-V relations for tail IKs in Figure 2E. The individual I-V relations were described by the Boltzmann equation to calculate the voltage for half-maximal activation (Vh) and slope factor (k) values (Table 1). Coexpressing WT with different amounts of G269S increased the Vh and k in a concentration-dependent manner.
Deactivation rates for IKs were measured by depolarizing cells to 30 mV for 2 s, followed by a tail-pulse from –60 mV to –30 mV in 10-mV increments. Figure 3B shows the time constant for deactivation plotted as a function of tail-pulse potential. Compared with WT, G269S significantly accelerated the deactivation rates between –60 mV and –30 mV. In addition, coexpressing WT with increasing amounts of G269S showed a concentration-dependent acceleration of IKs deactivation kinetics.
Effects of G269S mutant subunits on the expression of KCNQ1 tetramers
Figure 4 shows microscopic phase contrast (Fig. 4A) and confocal images (Fig. 4B) of 3 CHO cells expressing KCNE1 with KCNQ1-WT, WT + G269S mutation, and G269S alone. KCNQ1-WT proteins were amply transported to the cell membrane. In cells expressing G269S alone, G269S proteins were mostly distributed in the cytosol. In cells expressing WT + G269S, WT + G269S proteins were expressed both on the cell membrane and in the cytosol, suggesting that the cell membrane expression of channel proteins is increased by the coexpression of WT subunits.
IKs reconstituted with G269S mutant reduces its responses to PKA stimulation
To explain how G269S may be causing adrenergic-induced prolongation of QTc intervals, we tested whether G269S might impair the PKA-mediated regulation of IKs in HEK293 cells expressing KCNQ1-WT and/or G269S with KCNE1 and Yotiao protein (8) (we failed to record the response of IKs to beta-adrenergic agonist isoproterenol or PKA stimulation in CHO cells).
In cells expressing WT, both isoproterenol (100 nmol/l) (Fig. 5A) and a PKA-stimulating cocktail (5 μmol/l FK + 15 μmol/l IBMX) (Fig. 5B) increased tail IKs density by ∼100%. In contrast, cells expressing G269S or WT + G269S dramatically reduced the response of the peak tail IKs to isoproterenol or the PKA stimulation. Figure 5C shows the bar graphs that summarize the percent increase in tail IKs densities. These data suggest that G269S blunted the response of IKs to beta-adrenergic agonist or PKA stimulation.
G269S prevents the increase in IKs caused by the phosphomimetic S27D mutation
To elucidate further the mechanism underlying the attenuation of this PKA-mediated phosphorylation by G269S mutation, we engineered the phosphomimetic KCNQ1-S27D mutation with or without G269S mutation (14) and examined IKs recapitulated by coexpressing KCNE1 and Yotiao (Fig. 6).
Compared with WT, S27D increased IKs amplitudes considerably (Figs. 6A and 6B), which is similar to what was reported previously (14,15). Figure 6C shows that, consistent with S27D mimicking a PKA phosphorylated IKs channel, the application of 100 nmol/l of isoproterenol only slightly affected the peak tail IKs (14.1 ± 5.5%, n = 7). In contrast, compared with G269S, S27D-G269S did not increase peak tail IKs (Figs. 6D and 6E). The data are summarized in Figure 6F. Therefore, G269S prevents PKA up-regulation of IKs even if S27 is phosphorylated.
The present study revealed that G269S causes adrenergic-induced LQT, exerts a moderate dominant-negative suppression on the IKs channel, and prevents up-regulation in response to PKA stimulation. The exercise-dependent unmasking of QTc prolongation observed in our G269S patients is likely due to an adrenergic up-regulation of ICa,L without concomitant up-regulation of IKs.
Approximately 20% to 25% of genotype-confirmed LQTS patients have a normal range of QTc (16,17) and the percentage of these silent mutation carriers is significantly higher in LQT1 (36%) than in the other 2 major types of LQTS (LQT2, 19%, and LQT3, 10%). Lethal arrhythmias can occur in these apparently healthy silent mutation carriers without any premonitory sign (4,18). Several cases of physical-exertion–triggered cardiac events (including death) have been reported in silent G269S carriers (10–12).
Mutations in KCNQ1 are responsible for defects in IKs, which is the main outward component in maintaining cardiac repolarization reserve and highly sensitive to catecholamines (5,19). Functional analysis showed that G269S mutation reduced IKs density independent of changes in gating, shifted the I-V relation to a more depolarizing direction, and accelerated the deactivation time course. Taken together, G269S mutation exerted “loss-of-function” effects on the IKs channel.
Immunocytochemical studies (Fig. 4) indicated that G269S decreased cell surface expression of channel proteins. However, the trafficking-deficiency was partially rescued by coexpression of the WT subunits, resulting in the increased expression of channel proteins on the cell membrane. In contrast to the trafficking-deficient LQT1 mutations KCNQ1-ΔS276 and T587M (7,20), G269S appeared to form heteromultimers with WT and traffic to the cell membrane. We suspect that the partial correction of the G269S trafficking-deficient phenotype by coexpression with WT might partially explain why the clinical phenotype of KCNQ1-G269S mutation carriers is mild despite the observation that the channels are functionally defective.
In human ventricular myocytes, the rapid component of delayed rectifier K+ current, IKr, and ICa,L normally play the dominant role in regulating the ventricular action potential at rest (21,22). Therefore, KCNQ1 mutations (e.g., G269S) that cause a mild-to-moderate functional defect in IKs might ordinarily have little effect on the ventricular action potential. In contrast, IKs plays a major role in regulating the ventricular action potential after adrenergic stimulation, which activates IKs to prevent excessive ventricular action potential duration or QT prolongation by ICa,L (8,23). The role that IKs plays during adrenergic stimulation may explain why 62% of cardiac events in LQT1 patients occur during exercise (8,9). The blunted response of G269S channels to both beta-adrenergic agonist and PKA stimulation (Fig. 5) may result in excessive ventricular action potential duration and QTc prolongation specifically during exercise.
Consistent with previous reports (14,15), our experiments also demonstrated that the S27D mutant mimicked the PKA-mediated phosphorylation of IKs channels and increased IKs (Fig. 6). In contrast, S27D did not mimic PKA phosphorylation of IKs with G269S mutation. Therefore, G269S appears to prevent the functional effect that phosphorylation at S27 has on IKs.
Several groups have recently reported that different LQT1 mutations reduce the PKA sensitivity of IKs. Heijman et al. (15) reported that the A341V, which predisposes patients to a severe form of LQT1 (24), is also less sensitive to PKA stimulation. The decreased sensitivity to PKA is because A341V blunts phosphorylation at S27. Consistent with this, compared with cells expressing A341V, cells expressing S27D-A341V mimicked PKA stimulation and caused an up-regulation in IKs. We show a novel mechanism for PKA insensitivity. Unlike S27D-A341V, S27D-G269S did not mimic PKA stimulation of IKs, suggesting that G269S dissociates the link between phosphorylation at S27 and up-regulation of IKs.
Additionally, Barsheshet et al. (25) showed that patients with LQT1 mutations in the cytoplasmic loops between the S2 and S3 or S4 and S5 have a high risk for life-threatening events and derive a benefit from treatment with beta-adrenergic blockers. These mutations generate IKs, which is also resistant to PKA activation. Our findings first demonstrated that a “latent” LQT1 mutation could also be insensitive to PKA stimulation, which may explain the unmasking of the latent LQT1 phenotype that occurs during exercise. Consistent with the previous findings (25), the cardiac events of our symptomatic G269S patient have disappeared since beginning beta-blocker therapy for 42 months. This suggests that beta-adrenergic therapy would also be effective in patients with latent LQT1 mutations such as G269S. We speculate that beta-blocker therapy is effective because it would inhibit adrenergic-induced increases in ICa,L, which would minimize QTc prolongation with exercise.
In the present study, we screened the mutations that are responsible for LQT1, 2, 3, 5, 6, and 7. Therefore, the comorbidity of other types of LQTS was not completely excluded, although their frequency was quite low. In addition, only one patient was symptomatic of cardiac event and received beta-adrenergic blocker. Further studies are required to confirm that beta-blocker therapy may protect individuals who carry KCNQ1 mutations insensitive to PKA regulation.
We found that G269S caused moderate functional dysfunction in control conditions but almost completely disrupted PKA mediated up-regulation in IKs. This is likely why the G269S patients have borderline resting QTc intervals but significantly longer QTc while exercising.
The authors thank Dr. Daniel C. Bartos (Department of Physiology, University of Kentucky, Kentucky) for the KCNQ1-S27D mutation; Dr. J. Barhanin (Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France) for the gift of KCNQ1-WT; and Dr. J Kurokawa (Department of Bio-Informational Pharmacology, Tokyo Medical and Dental University, Japan) for the full-length cDNA encoding human A-kinase-anchoring protein 9 subcloned into pCDNA3.1 expression vector.
This work was supported by research grants from the Ministry of Education, Culture, Science, and Technology of Japan (to Dr. Horie); Health Science Research Grants from the Ministry of Health, Labor and Welfare of Japan for Clinical Research on Measures for Intractable Diseases (to Dr. Horie); Translational Research Funds from Japan Circulation Society (to Dr. Horie); and National Natural Science Foundation of China (#81273501 to Drs. Wu and Ding). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Wu and Naiki contributed equally to this paper.
- Abbreviations and Acronyms
- complementary deoxyribonucleic acid
- Chinese hamster ovary
- deoxyribonucleic acid
- human embryonic kidney 293
- L-type Ca2+ current
- the slow component of delayed rectifier K+ current
- long QT syndrome
- corrected QT
- phosphate-buffered saline
- protein kinase A
- wild type
- Received June 16, 2013.
- Revision received August 5, 2013.
- Accepted August 26, 2013.
- American College of Cardiology Foundation
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