Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants.

BACKGROUND: The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which ß-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. METHODS: Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. RESULTS: By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. CONCLUSIONS: Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

Estimating the probability of early afterdepolarizations and predicting arrhythmic risk associated with long QT syndrome type 1 mutations.

Early afterdepolarizations (EADs) are action potential (AP) repolarization abnormalities that can trigger lethal arrhythmias. Simulations using biophysically detailed cardiac myocyte models can reveal how model parameters influence the probability of these cellular arrhythmias; however, such analyses can pose a huge computational burden. We have previously developed a highly simplified approach in which logistic regression models (LRMs) map parameters of complex cell models to the probability of ectopic beats. Here, we extend this approach to predict the probability of EADs (P(EAD)) as a mechanistic metric of arrhythmic risk. We use the LRM to investigate how changes in parameters of the slow-activating delayed rectifier current (IKs) affect P(EAD) for 17 different long QT syndrome type 1 (LQTS1) mutations. In this LQTS1 clinical arrhythmic risk prediction task, we compared P(EAD) for these 17 mutations with two other recently published model-based arrhythmia risk metrics (AP morphology metric across populations of myocyte models and transmural repolarization prolongation based on a one-dimensional [1D] tissue-level model). These model-based risk metrics yield similar prediction performance; however, each fails to stratify clinical risk for a significant number of the 17 studied LQTS1 mutations. Nevertheless, an interpretable ensemble model using multivariate linear regression built by combining all of these model-based risk metrics successfully predicts the clinical risk of 17 mutations. These results illustrate the potential of computational approaches in arrhythmia risk prediction.

Generation of a human induced pluripotent stem cell line (JSPHi002-A) from a patient with long-QT syndrome type 1 caused by KCNQ1 c.773A > T mutation.

We generated an iPSCs line from the peripheral blood mononuclear cells (PBMCs) collected from a patient with long QT syndrome type 1 (LQT1) via a non-integrating system. We identified and verified a missense mutation in the KCNQ1 gene (c.773A > T) by whole-exome sequencing and Sanger sequencing. The established iPSC line was tested for pluripotency, differentiation potential, and karyotype. This cell-based model can help study the molecular mechanism and develop personalized drug therapies for LQT1.

A computational analysis of the effect of sevoflurane in a human ventricular cell model of long QT syndrome: Importance of repolarization reserve in the QT-prolonging effect of sevoflurane.

The slowly and rapidly activating delayed rectifier K+ channels (IKs and IKr, respectively) contribute to the repolarization of ventricular action potential in human heart and thereby determine QT interval on an electrocardiogram. Loss-of-function mutations in genes encoding IKs and IKr cause type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), accompanied by a high risk of malignant ventricular arrhythmias and sudden cardiac death. This study was designed to investigate which cardiac electrophysiological conditions exaggerate QT-prolonging and arrhythmogenic effects of sevoflurane. We used the O'Hara-Rudy dynamic model to reconstruct human ventricular action potential and a pseudo-electrocardiogram, and simulated LQT1 and LQT2 phenotypes by decreasing conductances of IKs and IKr, respectively. Sevoflurane, but not propofol, prolonged ventricular action potential duration and QT interval in wild-type, LQT1 and LQT2 models. The QT-prolonging effect of sevoflurane was more profound in LQT2 than in wild-type and LQT1 models. The potent inhibitory effect of sevoflurane on IKs was primarily responsible for its QT-prolonging effect. In LQT2 model, IKs was considerably enhanced during excessive prolongation of ventricular action potential duration by reduction of IKr and relative contribution of IKs to ventricular repolarization was markedly elevated, which appears to underlie more pronounced QT-prolonging effect of sevoflurane in LQT2 model, compared with wild-type and LQT1 models. This simulation study clearly elucidates the electrophysiological basis underlying the difference in QT-prolonging effect of sevoflurane among wild-type, LQT1 and LQT2 models, and may provide important information for developing anesthetic strategies for patients with long QT syndrome in clinical settings.

A novel mutation KCNQ1p.Thr312del is responsible for long QT syndrome type 1.

Patients with high-risk long QT syndrome (LQTS) mutations may experience life-threatening cardiac events. The present study sought to characterize a novel pathogenic mutation, KCNQ1p.Thr312del, in a Chinese LQT1 family. Clinical and genetic analyses were performed to identify this novel causative gene mutation in this LQTS family. Autosomal dominant inheritance of KCNQ1p.T312del was demonstrated in the three-generation pedigree. All mutation carriers presented with prolonged QT intervals and experienced recurrent syncope during exercise or emotional stress. The functional consequences of the mutant channel were investigated by computer homology modeling as well as whole-cell patch-clamp, western-blot and co-immunoprecipitation techniques using transfected mammalian cells. T312 is in the selectivity filter (SF) of the pore region of the KCNQ1-encoded channel. Homology modeling suggested that secondary structure was altered in the mutant SF compared with the wild-type (WT) SF. There were no significant differences in Kv7.1 expression, membrane trafficking or physical interactions with KCNE1-encoded subunits between the WT and mutant transfected channels. However, the KCNQ1p.T312del channels expressed in transfected cells were non-functional in the absence or presence of auxiliary KCNE1-subunits. Dominant-negative suppression of current density and decelerated activation kinetics were observed in cells expressing KCNQ1WT and KCNQ1p.T312del combined with KCNE1 (KCNQ1WT/p.T312del + KCNE1 channels). Those electrophysiological characteristics underlie the pathogenesis of this novel mutation and also suggest a high risk of cardiac events in patients carrying KCNQ1p.T312del. Although protein kinase A-dependent current increase was preserved, a significant suppression of rate-dependent current facilitation was noted in the KCNQ1WT/p.T312del + KCNE1 channels compared to the WT channels during 1- and 2-Hz stimulation, which was consistent with the patients' phenotype being triggered by exercise. Overall, KCNQ1p.Thr312del induces a loss of function in channel electrophysiology, and it is a high-risk mutation responsible for LQT1.

Human Induced Pluripotent Stem Cell-Derived Engineered Heart Tissue as a Sensitive Test System for QT Prolongation and Arrhythmic Triggers.

BACKGROUND: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of IKs (long QT1) and IKr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM-derived engineered heart tissue (EHT). METHODS: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV. RESULTS: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. IKr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although IKr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K+, and blocking of IKr, IKs, and IK1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, IKs block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline. CONCLUSIONS: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, IKs only contributed to repolarization under adrenergic stimulation.

Complex aberrant splicing in the induced pluripotent stem cell-derived cardiomyocytes from a patient with long QT syndrome carrying KCNQ1-A344Aspl mutation.

BACKGROUND: Long QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1, which encodes the α subunit of the slow delayed rectifier potassium current channel. We previously reported that a synonymous mutation, c.1032G>A, p.A344Aspl, in KCNQ1 is most commonly identified in genotyped patients with LQT1 in Japan and the aberrant splicing was analyzed in the lymphocytes isolated from patients' blood samples. However, the mechanisms underlying the observed processes in human cardiomyocytes remain unclear. OBJECTIVE: The purpose of this study was to establish and analyze patient-specific human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model carrying KCNQ1-A344Aspl. METHODS: We generated hiPSCs from the peripheral blood mononuclear cells obtained from a patient with LQT1 carrying KCNQ1-A344Aspl. Using the differentiated cardiomyocytes, we analyzed splicing variants and performed electrophysiology studies. RESULTS: We identified 7 aberrant RNA variants in A344Aspl hiPSC-CMs, which were more complex compared with those in peripheral lymphocytes. Multielectrode array analysis revealed that 1 µM isoproterenol significantly prolonged the duration of the corrected field potential in A344Aspl hiPSC-CMs as compared with that in control hiPSC-CMs. In addition, 100 nM E-4031, which inhibits the rapid component of the delayed rectifier potassium current, was shown to induce early afterdepolarization-like waveforms in A344Aspl hiPSC-CMs. Action potential durations (APDs) did not significantly differ between the hiPSC-CM groups. After administering 500 nM isoproterenol, APDs of A344Aspl hiPSC-CMs were significantly longer than those of the controls. (R)-N-(4-(4-Methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide and phenylboronic acid, slow delayed rectifier potassium current activators, ameliorated the APDs of hiPSC-CMs. CONCLUSION: We identified complex aberrant messenger RNA variants in the A344Aspl hiPSC-CM model and successfully recapitulated the clinical phenotypes of the patient with concealed LQT1. This model allows the investigation of the underlying mechanisms and development of novel therapies.

Transient Outward K+ Current (Ito) Underlies the Right Ventricular Initiation of Polymorphic Ventricular Tachycardia in a Transgenic Rabbit Model of Long-QT Syndrome Type 1.

BACKGROUND: Sudden death in long-QT syndrome type 1 (LQT1), an inherited disease caused by loss-of-function mutations in KCNQ1, is triggered by early afterdepolarizations (EADs) that initiate polymorphic ventricular tachycardia (pVT). We investigated ionic mechanisms that underlie pVT in LQT1 using a transgenic rabbit model of LQT1. METHODS: Optical mapping, cellular patch clamping, and computer modeling were used to elucidate the mechanisms of EADs in transgenic LQT1 rabbits. RESULTS: The results showed that shorter action potential duration in the right ventricle (RV) was associated with focal activity during pVT initiation. RV cardiomyocytes demonstrated higher incidence of EADs under 50 nmol/L isoproterenol. Voltage-clamp studies revealed that the transient outward potassium current (Ito) magnitude was 28% greater in RV associated with KChiP2 but with no differences in terms of calcium-cycling kinetics and other sarcolemmal currents. Perfusing with the Ito blocker 4-aminopyridine changed the initial focal sites of pVT from the RV to the left ventricle, corroborating the role of Ito in pVT initiation. Computer modeling showed that EADs occur preferentially in the RV because of the larger conductance of the slow-inactivating component of Ito, which repolarizes the membrane potential sufficiently rapidly to allow reactivation of ICa,L before IKr has had sufficient time to activate. CONCLUSIONS: Ito heterogeneity creates both triggers and an arrhythmogenic substrate in LQT1. In the absence of IKs, Ito interactions with ICa,L and IKr promote EADs in the RV while prolonging action potential duration in the left ventricle. This heterogeneity of action potential enhances dispersion of refractoriness and facilitates conduction blocks that initiate pVTs.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi003-A from a patient affected by an autosomal recessive form of Long QT Syndrome type 1.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Using the genome aggregation database, computational pathogenicity prediction tools, and patch clamp heterologous expression studies to demote previously published long QT syndrome type 1 mutations from pathogenic to benign.

BACKGROUND: Mutations in the KCNQ1-encoded Kv7.1 potassium channel cause long QT syndrome (LQTS) type 1 (LQT1). It has been suggested that ∼10%-20% of rare LQTS case-derived variants in the literature may have been published erroneously as LQT1-causative mutations and may be "false positives." OBJECTIVE: The purpose of this study was to determine which previously published KCNQ1 case variants are likely false positives. METHODS: A list of all published, case-derived KCNQ1 missense variants (MVs) was compiled. The occurrence of each MV within the Genome Aggregation Database (gnomAD) was assessed. Eight in silico tools were used to predict each variant's pathogenicity. Case-derived variants that were either (1) too frequently found in gnomAD or (2) absent in gnomAD but predicted to be pathogenic by ≤2 tools were considered potential false positives. Three of these variants were characterized functionally using whole-cell patch clamp technique. RESULTS: Overall, there were 244 KCNQ1 case-derived MVs. Of these, 29 (12%) were seen in ≥10 individuals in gnomAD and are demotable. However, 157 of 244 MVs (64%) were absent in gnomAD. Of these, 7 (4%) were predicted to be pathogenic by ≤2 tools, 3 of which we characterized functionally. There was no significant difference in current density between heterozygous KCNQ1-F127L, -P477L, or -L619M variant-containing channels compared to KCNQ1-WT. CONCLUSION: This study offers preliminary evidence for the demotion of 32 (13%) previously published LQT1 MVs. Of these, 29 were demoted because of their frequent sighting in gnomAD. Additionally, in silico analysis and in vitro functional studies have facilitated the demotion of 3 ultra-rare MVs (F127L, P477L, L619M).

Follow-up of 316 molecularly defined pediatric long-QT syndrome patients: clinical course, treatments, and side effects.

BACKGROUND: Inherited long-QT syndrome (LQTS) is associated with risk of sudden death. We assessed the clinical course and the fulfillment of current treatment strategies in molecularly defined pediatric LQTS type 1 and (LQT1) and type 2 (LQT2) patients. METHODS AND RESULTS: Follow-up data covering a mean of 12 years were collected for 316 genotyped LQT1 and LQT2 patients aged 0 to 18 years. No arrhythmic deaths occurred during the follow-up. Finnish KCNQ1 and KCNH2 founder mutations were associated with fewer cardiac events than other KCNQ1 and KCNH2 mutations (hazard ratio [HR], 0.33; P=0.03 and HR, 0.16; P=0.01, respectively). QTc interval ≥500 ms increased the risk of cardiac events compared with QTc <470 ms (HR, 3.32; P=0.001). Treatment with ß-blocker medication was associated with reduced risk of first cardiac event (HR, 0.23; P=0.001). Noncompliant LQT2 patients were more often symptomatic than compliant LQT2 patients (18% and 0%, respectively; P=0.03). Treatment with implantable cardioverter defibrillator was rare (3%) and resulted in reinterventions in 44% of cases. CONCLUSIONS: Severe cardiac events are uncommon in molecularly defined and appropriately treated pediatric LQTS mutation carriers. ß-Blocker medication reduces the risk of cardiac events and is generally well tolerated in this age group of LQTS patients.

Spatially Discordant Alternans and Arrhythmias in Tachypacing-Induced Cardiac Myopathy in Transgenic LQT1 Rabbits: The Importance of IKs and Ca2+ Cycling.

BACKGROUND: Remodeling of cardiac repolarizing currents, such as the downregulation of slowly activating K+ channels (IKs), could underlie ventricular fibrillation (VF) in heart failure (HF). We evaluated the role of Iks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome. METHODS AND RESULTS: LQT1 and littermate control (LMC) rabbits underwent three weeks of tachypacing to induce cardiac myopathy (TICM). In vivo telemetry demonstrated steepening of the QT/RR slope in LQT1 with TICM (LQT1-TICM; pre: 0.26±0.04, post: 0.52±0.01, P<0.05). In vivo electrophysiology showed that LQT1-TICM had higher incidence of VF than LMC-TICM (6 of 11 vs. 3 of 11, respectively). Optical mapping revealed larger APD dispersion (16±4 vs. 38±6 ms, p<0.05) and steep APD restitution in LQT1-TICM compared to LQT1-sham (0.53±0.12 vs. 1.17±0.13, p<0.05). LQT1-TICM developed spatially discordant alternans (DA), which caused conduction block and higher-frequency VF (15±1 Hz in LQT1-TICM vs. 13±1 Hz in LMC-TICM, p<0.05). Ca2+ DA was highly dynamic and preceded voltage DA in LQT1-TICM. Ryanodine abolished DA in 5 out of 8 LQT1-TICM rabbits, demonstrating the importance of Ca2+ in complex DA formation. Computer simulations suggested that HF remodeling caused Ca2+-driven alternans, which was further potentiated in LQT1-TICM due to the lack of IKs. CONCLUSIONS: Compared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca2+. Our results strongly support the contention that the downregulation of IKs in HF increases Ca2+ dependent alternans and thereby the risk of VF.

Third trimester fetal heart rate predicts phenotype and mutation burden in the type 1 long QT syndrome.

BACKGROUND: Early diagnosis and risk stratification is of clinical importance in the long QT syndrome (LQTS), however, little genotype-specific data are available regarding fetal LQTS. We investigate third trimester fetal heart rate, routinely recorded within public maternal health care, as a possible marker for LQT1 genotype and phenotype. METHODS AND RESULTS: This retrospective study includes 184 fetuses from 2 LQT1 founder populations segregating p.Y111C and p.R518X (74 noncarriers and 110 KCNQ1 mutation carriers, whereof 13 double mutation carriers). Pedigree-based measured genotype analysis revealed significant associations between fetal heart rate, genotype, and phenotype; mean third trimester prelabor fetal heart rates obtained from obstetric records (gestational week 29-41) were lower per added mutation (no mutation, 143±5 beats per minute; single mutation, 134±8 beats per minute; double mutations, 111±6 beats per minute; P<0.0001), and lower in symptomatic versus asymptomatic mutation carriers (122±10 versus 137±9 beats per minute; P<0.0001). Strong correlations between fetal heart rate and neonatal heart rate (r=0.700; P<0.001), and postnatal QTc (r=-0.762; P<0.001) were found. In a multivariable model, fetal genotype explained the majority of variance in fetal heart rate (-10 beats per minute per added mutation; P<1.0×10(-23)). Arrhythmia symptoms and intrauterine ß-blocker exposure each predicted -7 beats per minute, P<0.0001. CONCLUSIONS: In this study including 184 fetuses from 2 LQT1 founder populations, third trimester fetal heart rate discriminated between fetal genotypes and correlated with severity of postnatal cardiac phenotype. This finding strengthens the role of fetal heart rate in the early detection and risk stratification of LQTS, particularly for fetuses with double mutations, at high risk of early life-threatening arrhythmias.

Enhancing the Predictive Power of Mutations in the C-Terminus of the KCNQ1-Encoded Kv7.1 Voltage-Gated Potassium Channel.

Despite the overrepresentation of Kv7.1 mutations among patients with a robust diagnosis of long QT syndrome (LQTS), a background rate of innocuous Kv7.1 missense variants observed in healthy controls creates ambiguity in the interpretation of LQTS genetic test results. A recent study showed that the probability of pathogenicity for rare missense mutations depends in part on the topological location of the variant in Kv7.1's various structure-function domains. Since the Kv7.1's C-terminus accounts for nearly 50 % of the overall protein and nearly 50 % of the overall background rate of rare variants falls within the C-terminus, further enhancement in mutation calling may provide guidance in distinguishing pathogenic long QT syndrome type 1 (LQT1)-causing mutations from rare non-disease-causing variants in the Kv7.1's C-terminus. Therefore, we have used conservation analysis and a large case-control study to generate topology-based estimative predictive values to aid in interpretation, identifying three regions of high conservation within the Kv7.1's C-terminus which have a high probability of LQT1 pathogenicity.

Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of I(Ks) with S3 domain mutations in KCNQ1.

BACKGROUND: The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K(+) channel complex underlie long QT syndrome (LQTS) types 1 and 5. OBJECTIVE: The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1. METHODS: We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected. RESULTS: The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times. CONCLUSION: S3 mutations in KCNQ1 cause diverse kinetic defects in I(Ks), affecting opening and closing properties, and can account for LQT1 phenotypes.

Long-QT mutation p.K557E-Kv7.1: dominant-negative suppression of IKs, but preserved cAMP-dependent up-regulation.

AIMS: Mutations in KCNQ1, encoding for Kv7.1, the α-subunit of the IKs channel, cause long-QT syndrome type 1, potentially predisposing patients to ventricular tachyarrhythmias and sudden cardiac death, in particular, during elevated sympathetic tone. Here, we aim at characterizing the p.Lys557Glu (K557E) Kv7.1 mutation, identified in a Dutch kindred, at baseline and during (mimicked) increased adrenergic tone. METHODS AND RESULTS: K557E carriers had moderate QTc prolongation that augmented significantly during exercise. IKs characteristics were determined after co-expressing Kv7.1-wild-type (WT) and/or K557E with minK and Yotiao in Chinese hamster ovary cells. K557E caused IKs loss of function with slowing of the activation kinetics, acceleration of deactivation kinetics, and a rightward shift of voltage-dependent activation. Together, these contributed to a dominant-negative reduction in IKs density. Confocal microscopy and western blot indicated that trafficking of K557E channels was not impaired. Stimulation of WT IKs by 3'-5'-cyclic adenosine monophosphate (cAMP) generated strong current up-regulation that was preserved for K557E in both hetero- and homozygosis. Accumulation of IKs at fast rates occurred both in WT and in K557E, but was blunted in the latter. In a computational model, K557E showed a loss of action potential shortening during ß-adrenergic stimulation, in accordance with the lack of QT shortening during exercise in patients. CONCLUSION: K557E causes IKs loss of function with reduced fast rate-dependent current accumulation. cAMP-dependent stimulation of mutant IKs is preserved, but incapable of fully compensating for the baseline current reduction, explaining the long QT intervals at baseline and the abnormal QT accommodation during exercise in affected patients.

Electrophysiological phenotype in the LQTS mutations Y111C and R518X in the KCNQ1 gene.

Long QT syndrome is the prototypical disorder of ventricular repolarization (VR), and a genotype-phenotype relation is postulated. Furthermore, although increased VR heterogeneity (dispersion) may be important in the arrhythmogenicity in long QT syndrome, this hypothesis has not been evaluated in humans and cannot be tested by conventional electrocardiography. In contrast, vectorcardiography allows assessment of VR heterogeneity and is more sensitive to VR alterations than electrocardiography. Therefore, vectorcardiography was used to compare the electrophysiological phenotypes of two mutations in the LQT1 gene with different in vitro biophysical properties, and with LQT2 mutation carriers and healthy control subjects. We included 99 LQT1 gene mutation carriers (57 Y111C, 42 R518X) and 19 LQT2 gene mutation carriers. Potassium channel function is in vitro most severely impaired in Y111C. The control group consisted of 121 healthy subjects. QRS, QT, and T-peak to T-end (Tp-e) intervals, measures of the QRS vector and T vector and their relationship, and T-loop morphology parameters were compared at rest. Apart from a longer heart rate-corrected QT interval (QT heart rate corrected according to Bazett) in Y111C mutation carriers, there were no significant differences between the two LQT1 mutations. No signs of increased VR heterogeneity were observed among the LQT1 and LQT2 mutation carriers. QT heart rate corrected according to Bazett and Tp-e were longer, and the Tp-e-to-QT ratio greater in LQT2 than in LQT1 and the control group. In conclusion, there was a marked discrepancy between in vitro potassium channel function and in vivo electrophysiological properties in these two LQT1 mutations. Together with previous observations of the relatively low risk for clinical events in Y111C mutation carriers, our results indicate need for cautiousness in predicting in vivo electrophysiological properties and the propensity for clinical events based on in vitro assessment of ion channel function alone.

Impaired ion channel function related to a common KCNQ1 mutation - implications for risk stratification in long QT syndrome 1.

Long QT syndrome (LQTS) 1 is the most common type of inherited LQTS and is linked to mutations in the KCNQ1 gene. We identified a KCNQ1 missense mutation, KCNQ1 G325R, in an asymptomatic patient presenting with significant QT prolongation (QTc, 448-600ms). Prior clinical reports revealed phenotypic variability ranging from the absence of symptoms to syncope among KCNQ1 G325R mutation carriers. The present study was designed to determine the G325R ion channel phenotype and its association with the clinical LQTS presentation. Electrophysiological testing was performed using the Xenopus oocyte expression system. KCNQ1 G325R channels were non-functional and suppressed wild type (WT) currents by 71.1%. In the presence of the native cardiac regulatory ß-subunit, KCNE1, currents conducted by G325R and WT KCNQ1 were reduced by 52.9%. Co-expression of G325R and WT KCNQ1 with KCNE1 shifted the voltage-dependence of I(Ks) activation by 12.0mV, indicating co-assembly of mutant and WT subunits. The dysfunctional biophysical phenotype validates the pathogenicity of the KCNQ1 G325R mutation and corresponds well with the severe clinical presentation revealed in some reports. However, the index patient and other mutation carriers were asymptomatic, highlighting potential limitations of risk assessment schemes based on ion channel data.