Genetic variant annotation scores in congenital long QT syndrome.

BACKGROUND: Congenital Long QT Syndrome (LQTS) is a hereditary arrhythmic disorder. We aimed to assess the performance of current genetic variant annotation scores among LQTS patients and their predictive impact. METHODS: We evaluated 2025 patients with unique mutations for LQT1-LQT3. A patient-specific score was calculated for each of four established genetic variant annotation algorithms: CADD, SIFT, REVEL, and PolyPhen-2. The scores were tested for the identification of LQTS and their predictive performance for cardiac events (CE) and life-threatening events (LTE) and then compared with the predictive performance of LQTS categorization based on mutation location/function. Score performance was tested using Harrell's C-index. RESULTS: A total of 917 subjects were classified as LQT1, 838 as LQT2, and 270 as LQT3. The identification of a pathogenic variant occurred in 99% with CADD, 92% with SIFT, 100% with REVEL, and 86% with PolyPhen-2. However, none of the genetic scores correlated with the risk of CE (Harrell's C-index: CADD = 0.50, SIFT = 0.51, REVEL = 0.50, and PolyPhen-2 = 0.52) or LTE (Harrell's C-index: CADD = 0.50, SIFT = 0.53, REVEL = 0.54, and PolyPhen-2 = 0.52). In contrast, high-risk mutation categorization based on location/function was a powerful independent predictor of CE (HR = 1.88; p < .001) and LTE (HR = 1.89, p < .001). CONCLUSION: In congenital LQTS patients, well-established algorithms (CADD, SIFT, REVEL, and PolyPhen-2) were able to identify the majority of the causal variants as pathogenic. However, the scores did not predict clinical outcomes. These results indicate that mutation location/functional assays are essential for accurate interpretation of the risk associated with LQTS mutations.

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression.

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.

The auxiliary subunit KCNE1 regulates KCNQ1 channel response to sustained calcium-dependent PKC activation.

The slow cardiac delayed rectifier current (IKs) is formed by KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Decrease of IKs currents either due to inherited mutations or pathological remodeling is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in heart disease and diabetes. Recently, we found that sustained stimulation of the calcium-dependent PKCßII isoform leads to decrease in KCNQ1 subunit membrane localization and KCNQ1/KCNE1 channel activity, although the role of KCNE1 in this regulation was not explored. Here, we show that the auxiliary KCNE1 subunit expression is necessary for channel internalization. A mutation in a KCNE1 phosphorylation site (KCNE1(S102A)) abolished channel internalization in both heterologous expression systems and cardiomyocytes. Altogether, our results suggest that KCNE1(S102) phosphorylation by PKCßII leads to KCNQ1/KCNE1 channel internalization in response to sustained PKC stimulus, while leaving KCNQ1 homomeric channels in the membrane. This preferential internalization is expected to have strong impact on cardiac repolarization. Our results suggest that KCNE1(S102) is an important anti-arrhythmic drug target to prevent IKs pathological remodeling leading to cardiac arrhythmias.

PKCßII specifically regulates KCNQ1/KCNE1 channel membrane localization.

The slow voltage-gated potassium channel (IKs) is composed of the KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Activation of protein kinase C (PKC) has been linked to cardiac arrhythmias. Although PKC has been shown to be a regulator of a number of cardiac channels, including IKs, little is known about regulation of the channel by specific isoforms of PKC. Here we studied the role of different PKC isoforms on IKs channel membrane localization and function. Our studies focused on PKC isoforms that translocate to the plasma membrane in response to Gq-coupled receptor (GqPCR) stimulation: PKCα, PKCßI, PKCßII and PKCε. Prolonged stimulation of GqPCRs has been shown to decrease IKs membrane expression, but the specific role of each PKC isoform is unclear. Here we show that stimulation of calcium-dependent isoforms of PKC (cPKC) but not PKCε mimic receptor activation. In addition, we show that general PKCß (LY-333531) and PKCßII inhibitors but not PKCα or PKCßI inhibitors blocked the effect of cPKC on the KCNQ1/KCNE1 channel. PKCß inhibitors also blocked GqPCR-mediated decrease in channel membrane expression in cardiomyocytes. Direct activation of PKCßII using constitutively active PKCßII construct mimicked agonist-induced decrease in membrane expression and channel function, while dominant negative PKCßII showed no effect. This suggests that the KCNQ1/KCNE1 channel was not regulated by basal levels of PKCßII activity. Our results indicate that PKCßII is a specific regulator of IKs membrane localization. PKCßII expression and activation are strongly increased in many disease states, including heart disease and diabetes. Thus, our results suggest that PKCßII inhibition may protect against acquired QT prolongation associated with heart disease.

Statin-specific inhibition of Rab-GTPase regulates cPKC-mediated IKs internalization.

Statins are prescribed for prevention and treatment of coronary artery disease. Statins have different cholesterol lowering abilities, with rosuvastatin and atorvastatin being the most effective, while statins like simvastatin and fluvastatin having lower effectiveness. Statins, in addition to their cholesterol lowering effects, can prevent isoprenylation of Rab-GTPase proteins, a protein family important for the regulation of membrane-bound protein trafficking. Here we show that endosomal localization of Rab-GTPases (Rab5, Rab7 and Rab11) was inhibited in a statin-specific manner, with stronger effects by fluvastatin, followed by simvastatin and atorvastatin, and with a limited effect by rosuvastatin. Fluvastatin inhibition of Rab5 has been shown to mediate cPKC-dependent trafficking regulation of the cardiac delayed rectifier KCNQ1/KCNE1 channels. We observed statin-specific inhibition of channel regulation consistent with statin-specific Rab-GTPase inhibition both in heterologous systems and cardiomyocytes. Our results uncover a non-cholesterol-reducing statin-specific effect of statins. Because Rab-GTPases are important regulators of membrane trafficking they may underlie statin specific pleiotropic effects. Therefore, statin-specificity may allow better treatment tailoring.

Fluvastatin inhibits Rab5-mediated IKs internalization caused by chronic Ca2+-dependent PKC activation.

Statins, in addition to their cholesterol lowering effects, can prevent isoprenylation of Rab GTPase proteins, a key protein family for the regulation of protein trafficking. Rab-GTPases have been shown to be involved in the control of membrane expression level of ion channels, including one of the major cardiac repolarizing channels, IKs. Decreased IKs function has been observed in a number of disease states and associated with increased propensity for arrhythmias, but the mechanism underlying IKs decrease remains elusive. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in variety of human diseases and have been suggested to acutely regulate IKs function. We hypothesize that chronic cPKC stimulation leads to Rab-mediated decrease in IKs membrane expression, and that can be prevented by statins. In this study we show that chronic cPKC stimulation caused a dramatic Rab5 GTPase-dependent decrease in plasma membrane localization of the IKs pore forming subunit KCNQ1, reducing IKs function. Our data indicates fluvastatin inhibition of Rab5 restores channel localization and function after cPKC-mediated channel internalization. Our results indicate a novel statin anti-arrhythmic effect that would be expected to inhibit pathological electrical remodeling in a number of disease states associated with high cPKC activation. Because Rab-GTPases are important regulators of membrane trafficking they may underlie other statin pleiotropic effects.

Loss-of-Function KCNE2 Variants: True Monogenic Culprits of Long-QT Syndrome or Proarrhythmic Variants Requiring Secondary Provocation?

BACKGROUND: Insight into type 6 long-QT syndrome (LQT6), stemming from mutations in the KCNE2-encoded voltage-gated channel ß-subunit, is limited. We sought to further characterize its clinical phenotype. METHODS AND RESULTS: Individuals with reported pathogenic KCNE2 mutations identified during arrhythmia evaluation were collected from inherited arrhythmia clinics and the Rochester long-QT syndrome (LQTS) registry. Previously reported LQT6 cases were identified through a search of the MEDLINE database. Clinical features were assessed, while reported KCNE2 mutations were evaluated for genotype-phenotype segregation and classified according to the contemporary American College of Medical Genetics guidelines. Twenty-seven probands possessed reported pathogenic KCNE2 mutations, while a MEDLINE search identified 17 additional LQT6 cases providing clinical and genetic data. Sixteen probands had normal resting QTc values and only developed QT prolongation and malignant arrhythmias after exposure to QT-prolonging stressors, 10 had other LQTS pathogenic mutations, and 10 did not have an LQTS phenotype. Although the remaining 8 subjects had an LQTS phenotype, evidence suggested that the KCNE2 variant was not the underlying culprit. The collective frequency of KCNE2 variants implicated in LQT6 in the Exome Aggregation Consortium database was 1.4%, in comparison with a 0.0005% estimated clinical prevalence for LQT6. CONCLUSIONS: On the basis of clinical phenotype, the high allelic frequencies of LQT6 mutations in the Exome Aggregation Consortium database, and absence of previous documentation of genotype-phenotype segregation, our findings suggest that many KCNE2 variants, and perhaps all, have been erroneously designated as LQTS-causative mutations. Instead, KCNE2 variants may confer proarrhythmic susceptibility when provoked by additional environmental/acquired or genetic factors, or both.

Stop-codon and C-terminal nonsense mutations are associated with a lower risk of cardiac events in patients with long QT syndrome type 1.

BACKGROUND: In long QT syndrome type 1 (LQT1), the location and type of mutations have been shown to affect the clinical outcome. Although haploinsufficiency, including stop-codon and frameshift mutations, has been associated with a lower risk of cardiac events in patients with LQT1, nonsense mutations have been presumed functionally equivalent. OBJECTIVE: The purpose of this study was to evaluate clinical differences between patients with nonsense mutations. METHODS: The study sample comprised 1090 patients with genetically confirmed mutations. Patients were categorized into 5 groups, depending on mutation type and location: missense not located in the high-risk cytoplasmic loop (c-loop) (n = 698), which is used as reference; missense c-loop (n = 192); stop-codon (n = 67); frameshift (n = 39); and others (n = 94). The primary outcome was a composite end point of syncope, aborted cardiac arrest, and long QT syndrome-related death (cardiac events). Outcomes were evaluated using the multivariate Cox proportional hazards regression analysis. Standard patch clamp techniques were used. RESULTS: Compared to patients with missense non-c-loop mutations, the risk of cardiac events was reduced significantly in patients with stop-codon mutations (hazard ratio [HR] 0.57; 95% confidence interval [CI] 0.34-0.96; P = .035), but not in patients with frameshift mutations (HR 1.01; 95% CI 0.58-1.77; P = .97). Our data suggest that currents of the most common stop-codon mutant channel (Q530X) were larger than those of haploinsufficient channels (wild type: 42 ± 6 pA/pF, n = 20; Q530X+wild type: 79 ± 14 pA/pF, n = 20; P < .05) and voltage dependence of activation was altered. CONCLUSION: Stop-codon mutations are associated with a lower risk of cardiac events in patients with LQT1, while frameshift mutations are associated with the same risk as the majority of the missense mutations. Our data indicate functional differences between these previously considered equivalent mutation subtypes.

Development of data acquisition components for simultaneous recording of 3D epicardial and surface ECG signals in the langendorff perfusion apparatus.

Sudden Cardiac Death (SCD) claims 7 million lives per year. The importance of myocardial electrogram (EGM) repolarization alternans and surface electrocardiogram (ECG) T-wave alternans is gaining traction for understanding the underlying SCD mechanisms. However, the relationship between the 3D spatial distribution of myocardial EGMs and surface ECG with respect to SCD has yet to be investigated. To make this happen, a modified data acquisition system has been developed and fabricated in conjunction with the Langendorff perfusion system to enable simultaneous recording and analysis of the 3D spatial distribution of myocardial EGMs and the surface ECG. Two devices have been fabricated: a basket catheter, which obtains 3D EGM data; and an ECG chamber, capable of keeping the constraints of the Langendorff system. Noise analysis confirmed, for all devices, a signal to noise ratio (SNR) of median (µ) >= 51.7dB and standard deviation (σ) <;= 2.1dB. A Langendorff rat heart experiment further confirmed successful recording of 3D EGM and surface ECG data with an acceptable SNR. The developed system can be used to study the relationship between 3D EGM and surface ECG data, which can be utilized to understand the mechanisms of SCD.

Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, ßI, ßII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCßI and PKCßII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, ß and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.

Impaired IKs channel activation by Ca(2+)-dependent PKC shows correlation with emotion/arousal-triggered events in LQT1.

BACKGROUND: The most common inherited cardiac arrhythmia, LQT1, is due to IKs potassium channel mutations and is linked to high risk of adrenergic-triggered cardiac events. We recently showed that although exercise-triggered events are very well treated by ß-blockers for these patients, acute arousal-triggered event rate were not significantly reduced after beta-blocker treatment, suggesting that the mechanisms underlying arousal-triggered arrhythmias may be different from those during exercise. IKs is strongly regulated by ß-adrenergic receptor (ß-AR) signaling, but little is known about the role of α1-AR-mediated regulation. METHODS AND RESULTS: Here we show, using a combination of cellular electrophysiology and computational modeling, that IKs phosphorylation and α1-AR regulation via activation of calcium-dependent PKC isoforms (cPKC) may be a key mechanism to control channel voltage-dependent activation and consequently action potential duration (APD) in response to adrenergic-stimulus. We show that simulated mutation-specific combined adrenergic effects (ß+α) on APD were strongly correlated to acute stress-triggered cardiac event rate for patients while ß-AR effects alone were not. CONCLUSION: We were able to show that calcium-dependent PKC signaling is key to normal QT shortening during acute arousal and when impaired, correlates with increased rate of sudden arousal-triggered cardiac events. Our study suggests that the acute α1-AR-cPKC regulation of IKs is important for QT shortening in "fight-or-flight" response and is linked to decreased risk of sudden emotion/arousal-triggered cardiac events in LQT1 patients.

Prognostic implications of mutation-specific QTc standard deviation in congenital long QT syndrome.

BACKGROUND: Individual corrected QT interval (QTc) may vary widely among carriers of the same long QT syndrome (LQTS) mutation. Currently, neither the mechanism nor the implications of this variable penetrance are well understood. OBJECTIVES: To hypothesize that the assessment of QTc variance in patients with congenital LQTS who carry the same mutation provides incremental prognostic information on the patient-specific QTc. METHODS: The study population comprised 1206 patients with LQTS with 95 different mutations and ≥ 5 individuals who carry the same mutation. Multivariate Cox proportional hazards regression analysis was used to assess the effect of mutation-specific standard deviation of QTc (QTcSD) on the risk of cardiac events (comprising syncope, aborted cardiac arrest, and sudden cardiac death) from birth through age 40 years in the total population and by genotype. RESULTS: Assessment of mutation-specific QTcSD showed large differences among carriers of the same mutations (median QTcSD 45 ms). Multivariate analysis showed that each 20 ms increment in QTcSD was associated with a significant 33% (P = .002) increase in the risk of cardiac events after adjustment for the patient-specific QTc duration and the family effect on QTc. The risk associated with QTcSD was pronounced among patients with long QT syndrome type 1 (hazard ratio 1.55 per 20 ms increment; P<.001), whereas among patients with long QT syndrome type 2, the risk associated with QTcSD was not statistically significant (hazard ratio 0.99; P = .95; P value for QTcSD-by-genotype interaction = .002). CONCLUSIONS: Our findings suggest that mutations with a wider variation in QTc duration are associated with increased risk of cardiac events. These findings appear to be genotype-specific, with a pronounced effect among patients with the long QT syndrome type 1 genotype.

Risk of life-threatening cardiac events among patients with long QT syndrome and multiple mutations.

BACKGROUND: Patients with long QT syndrome (LQTS) who harbor multiple mutations (i.e. ≥ 2 mutations in ≥ 1 LQTS-susceptibility gene) may experience increased risk for life-threatening cardiac events. OBJECTIVES: The present study was designed to compare the clinical course of LQTS patients with multiple mutations to those with a single mutation. METHODS: The risk for life-threatening cardiac events (comprising aborted cardiac arrest, implantable defibrillator shock, or sudden cardiac death) from birth through age 40 years, by the presence of multiple vs. single mutations, was assessed among 403 patients from the LQTS Registry. RESULTS: Patients with multiple mutations (n=57) exhibited a longer QTc at enrollment compared with those with a single mutation (mean ± SD: 506 ± 72 vs. 480 ± 56 msec, respectively; P=0.003) and had a higher rate of life threatening cardiac events during follow-up (23% vs. 11%, respectively; p=0.031). Consistently, multivariate analysis demonstrated that patients with multiple mutations had a 2.3-fold (P=0.015) increased risk for life threatening cardiac events as compared to patients with a single mutation. The presence of multiple mutations in a single LQTS gene was associated with a 3.2-fold increased risk for life threatening cardiac events (P=0.010) whereas the risk associated with multiple mutation status involving >1 LQTS gene was not significantly different from the risk associated with a single mutation (HR 1.7, P=0.26). CONCLUSIONS: LQTS patients with multiple mutations have a greater risk for life-threatening cardiac events as compared to patients with a single mutation.

In silico cardiac risk assessment in patients with long QT syndrome: type 1: clinical predictability of cardiac models.

OBJECTIVES: The study was designed to assess the ability of computer-simulated electrocardiography parameters to predict clinical outcomes and to risk-stratify patients with long QT syndrome type 1 (LQT1). BACKGROUND: Although attempts have been made to correlate mutation-specific ion channel dysfunction with patient phenotype in long QT syndrome, these have been largely unsuccessful. Systems-level computational models can be used to predict consequences of complex changes in channel function to the overall heart rhythm. METHODS: A total of 633 LQT1-genotyped subjects with 34 mutations from multinational long QT syndrome registries were studied. Cellular electrophysiology function was determined for the mutations and introduced in a 1-dimensional transmural electrocardiography computer model. The mutation effect on transmural repolarization was determined for each mutation and related to the risk for cardiac events (syncope, aborted cardiac arrest, and sudden cardiac death) among patients. RESULTS: Multivariate analysis showed that mutation-specific transmural repolarization prolongation (TRP) was associated with an increased risk for cardiac events (35% per 10-ms increment [p < 0.0001]; ≥upper quartile hazard ratio: 2.80 [p < 0.0001]) and life-threatening events (aborted cardiac arrest/sudden cardiac death: 27% per 10-ms increment [p = 0.03]; ≥upper quartile hazard ratio: 2.24 [p = 0.002]) independently of patients' individual QT interval corrected for heart rate (QTc). Subgroup analysis showed that among patients with mild to moderate QTc duration (<500 ms), the risk associated with TRP was maintained (36% per 10 ms [p < 0.0001]), whereas the patient's individual QTc was not associated with a significant risk increase after adjustment for TRP. CONCLUSIONS: These findings suggest that simulated repolarization can be used to predict clinical outcomes and to improve risk stratification in patients with LQT1, with a more pronounced effect among patients with a lower-range QTc, in whom a patient's individual QTc may provide less incremental prognostic information.

Genotype- and Sex-Specific QT-RR Relationship in the Type-1 Long-QT Syndrome.

BACKGROUND: Genotype-phenotype investigations have revealed significantly larger risk for cardiac events in patients with type 1 long-QT syndrome (LQT-1), particularly in adult females, with missense mutation in the cytoplasmic loop (C-loop) regions of the α subunit of the KCNQ1 gene associated with an impaired ion channel activation by adrenergic stimulus. We hypothesize that the impaired response to increases in heart rate leads to abnormal QT-RR dynamic profiles and is responsible for the increased cardiac risk for these patients. METHODS AND RESULTS: We measured the QT-RR slope in 24-hour Holter ECGs from LQT-1 patients with the mutations associated with impaired adrenergic stimulus (C-loop, n=18) and compared to LQT-1 patients with other mutations (non-C-loop, n=48), and to a healthy control group (n=195). The diurnal QT-RR slope was less steep in C-loop mutation patients (0.10±0.05) than in the ECGs from non-C-loop mutation patients (0.17±0.09, P=0.002). For female patients, slower heart rates were associated with prolonged QT and increased QT-RR slope. Male patients with C-loop mutations showed an impaired repolarization for shorter range of heart rates than in females, which is consistent with gender differences in triggers for events in this syndrome. CONCLUSIONS: Our observations suggest that the C-loop LQT-1 patients have specific impaired adrenergic regulation of the ventricular repolarization. This response to heart rate increases may be useful in identification of high-risk patients with inherited prolonged QT and may help select an optimal antiarrhythmic therapeutic strategy. (J Am Heart Assoc. 2012;1:e000570 doi: 10.1161/JAHA.112.000570.).

Mutations in cytoplasmic loops of the KCNQ1 channel and the risk of life-threatening events: implications for mutation-specific response to ß-blocker therapy in type 1 long-QT syndrome.

BACKGROUND: ß-Adrenergic stimulation is the main trigger for cardiac events in type 1 long-QT syndrome (LQT1). We evaluated a possible association between ion channel response to ß-adrenergic stimulation and clinical response to ß-blocker therapy according to mutation location. METHODS AND RESULTS: The study sample comprised 860 patients with genetically confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C loops), membrane-spanning domain, C/N terminus, and nonmissense mutations. There were 27 aborted cardiac arrest and 78 sudden cardiac death events from birth through 40 years of age. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for aborted cardiac arrest or sudden cardiac death (hazard ratio versus nonmissense mutations=2.75; 95% confidence interval, 1.29-5.86; P=0.009). ß-Blocker therapy was associated with a significantly greater reduction in the risk of aborted cardiac arrest or sudden cardiac death among patients with C-loop mutations than among all other patients (hazard ratio=0.12; 95% confidence interval, 0.02-0.73; P=0.02; and hazard ratio=0.82; 95% confidence interval, 0.31-2.13; P=0.68, respectively; P for interaction=0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to ß-adrenergic stimulation. CONCLUSIONS: Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high risk for life-threatening events and derive a pronounced benefit from treatment with ß-blockers. Reduced channel activation after sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.

Combined assessment of sex- and mutation-specific information for risk stratification in type 1 long QT syndrome.

BACKGROUND: Men and women with type 1 long QT syndrome (LQT1) exhibit time-dependent differences in the risk for cardiac events. OBJECTIVE: We hypothesized that sex-specific risk for LQT1 is related to the location and function of the disease-causing mutation in the KCNQ1 gene. METHODS: The risk for life-threatening cardiac events (comprising aborted cardiac arrest [ACA] or sudden cardiac death [SCD]) from birth through age 40 years was assessed among 1051 individuals with LQT1 (450 men and 601 women) by the location and function of the LQT1-causing mutation (prespecified as mutations in the intracellular domains linking the membrane-spanning segments [ie, S2-S3 and S4-S5 cytoplasmic loops] involved in adrenergic channel regulation vs other mutations). RESULTS: Multivariate analysis showed that during childhood (age group: 0-13 years) men had >2-fold (P < .003) increased risk for ACA/SCD than did women, whereas after the onset of adolescence the risk for ACA/SCD was similar between men and women (hazard ratio = 0.89 [P = .64]). The presence of cytoplasmic-loop mutations was associated with a 2.7-fold (P < .001) increased risk for ACA/SCD among women, but it did not affect the risk among men (hazard ratio 1.37; P = .26). Time-dependent syncope was associated with a more pronounced risk-increase among men than among women (hazard ratio 4.73 [P < .001] and 2.43 [P = .02], respectively), whereas a prolonged corrected QT interval (≥ 500 ms) was associated with a higher risk among women than among men. CONCLUSION: Our findings suggest that the combined assessment of clinical and mutation location/functional data can be used to identify sex-specific risk factors for life-threatening events for patients with LQT1.

Trigger-specific ion-channel mechanisms, risk factors, and response to therapy in type 1 long QT syndrome.

BACKGROUND: Arrhythmic events in long-QT syndrome type 1 (LQT1) may be associated with exercise, acute arousal, or rest/sleep. OBJECTIVES: We aimed to identify trigger-specific risk factors for cardiac events in patients with LQT1. METHODS: The study population comprised 721 genetically confirmed patients with LQT1 from the US portion of the International LQTS Registry. Multivariate analysis was used to assess the independent contribution of prespecified clinical and mutation-specific factors to the development of a first reported triggered event, associated with exercise, arousal, or sleep/rest. RESULTS: Cardiac events occurred in 221 study patients, of whom 121 (55%) were associated with exercise, 30 (14%) with arousal, 47 (21%) with sleep/rest, and 23 (10%) with other triggers. Multivariate analysis showed that males <13 years had a 2.8-fold (P < .001) increase in the risk for exercise-triggered events whereas females ≥13 years showed a 3.5-fold (P = .002) increase in the risk for sleep/rest nonarousal events. Cytoplasmic-loop mutations within the transmembrane region, involved in adrenergic channel regulation, were associated with the increased risk for both exercise- and arousal-triggered events (hazard ratio = 6.19 [P < .001] and 4.99 [P < .001], respectively) but were not associated with events during sleep/rest (hazard ratio = 0.72; P = .46). Beta-blocker therapy was associated with a pronounced 78% (P < .001) reduction in the risk for exercise-triggered events but did not have a significant effect on events associated with arousal or sleep/rest. CONCLUSIONS: In patients with LQT1, cardiac events triggered by exercise, arousal, or rest/sleep are associated with distinctive risk factors and response to medical therapy. These findings can be used for improved recommendations for lifestyle modifications and therapeutic management in this population.

Adrenergic signaling controls RGK-dependent trafficking of cardiac voltage-gated L-type Ca2+ channels through PKD1.

RATIONALE: The Rad-Gem/Kir-related family (RGKs) consists of small GTP-binding proteins that strongly inhibit the activity of voltage-gated calcium channels. Among RGKs, Rem1 is strongly and specifically expressed in cardiac tissue. However, the physiological role and regulation of RGKs, and Rem1 in particular, are largely unknown. OBJECTIVE: To determine if Rem1 function is physiologically regulated by adrenergic signaling and thus impacts voltage-gated L-type calcium channel (VLCC) activity in the heart. METHODS AND RESULTS: We found that activation of protein kinase D1, a protein kinase downstream of α(1)-adrenergic signaling, leads to direct phosphorylation of Rem1 at Ser18. This results in an increase of the channel activity and plasma membrane expression observed by using a combination of electrophysiology, live cell confocal microscopy, and immunohistochemistry in heterologous expression system and neonatal cardiomyocytes. In addition, we show that stimulation of α(1)-adrenergic receptor-protein kinase D1-Rem1 signaling increases transverse-tubule VLCC expression that results in increased L-type Ca(2+) current density in adult ventricular myocytes. CONCLUSION: The α(1)-adrenergic stimulation releases Rem1 inhibition of VLCCs through direct phosphorylation of Rem1 at Ser18 by protein kinase D1, resulting in an increase of the channel activity and transverse-tubule expression. Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function in the heart and provide the first demonstration of physiological regulation of RGK function.

Risk of syncope in family members who are genotype-negative for a family-associated long-QT syndrome mutation.

BACKGROUND: Current clinical diagnosis of long-QT syndrome (LQTS) includes genetic testing of family members of mutation-positive patients. The present study was designed to assess the clinical course of individuals who are found negative for the LQTS-causing mutation in their families. METHODS AND RESULTS: Multivariate Cox proportional hazards model was used to assess the risk for cardiac events (comprising syncope, aborted cardiac arrest [ACA], or sudden cardiac death [SCD]) from birth through age 40 years among 1828 subjects from the LQTS Registry who were found negative for their family LQTS-causing mutation. The median QTc of study subjects was 423 ms (interquartile range, 402-442 ms). The cumulative probability of a first syncope through age 40 years was 15%. However, only 2 patients (0.1%) had ACA, and none died suddenly during follow-up. Independent risk factors for syncope in genotype-negative subjects included female sex (hazard ratio [HR], 1.60; P=0.002), prolonged QTc (HR=1.63 per 100 ms increment, P=0.02), family history of ACA or SCD (HR=1.89, P=0.002), and LQT2 versus LQT1 family mutation (HR=1.41, P=0.03). Subgroup analysis showed that the presence of the K897T polymorphism in the LQT2 gene in an affected family was associated with an 11-fold (P=0.001) increase in the risk of recurrent syncope in genotype-negative subjects. CONCLUSIONS: Our findings suggest that cardiac events among genotype-negative family members of LQTS patients are dominated by nonfatal syncopal episodes without occurrence of sudden cardiac death. The risk for nonfatal events in this population may be mediated by the presence of common polymorphisms in LQTS genes.