An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG.

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Pharmacological corrections of the mutant hERG channels by posaconazole.

Properties of mutant human ether-à-go-go-related gene (hERG) channels can be modified by some antibiotics. However, the pharmacological effects of posaconazole on cardiomyocyte hERG channels remain unclear. Whole-cell patch clamping, western blotting and laser confocal scanning microscopy were used to evaluate the effects of posaconazole on wild-type (WT)-, A561V- and L539 fs/47-hERG channels expressed in human embryonic kidney (HEK) 293 cells. In electrophysiological experiments, HEK 293 cells were transiently co-transfected with equal amounts of WT-hERG, WT+A561 V-hERG and WT+L539 fs/47-hERG plasmids to mimic a heterozygous genotype. Posaconazole (30 µM) increased tail currents in cells expressing WT-hERG, WT+A561 V-hERG and WT+L539 fs/47-hERG by 82.65%, 147.72% and 134.73%, respectively, compared to controls. Posaconazole increased hERG protein expression in cells expressing WT-hERG, WT+A561 V-hERG and WT+L539 fs/47-hERG compared to controls condition as well as their trafficking to the cell membrane. To our knowledge, this is the first study to show that antifungal agent posaconazole rescues the mutant A561 V-hERG and L539 fs/47-hERG channels by altering the gating kinetics, enhancing the expression and trafficking of hERG channels. The results demonstrate that posaconazole could be a promising candidate for the prevention and treatment of long QT syndrome and other arrhythmia-related diseases.

Restoration of mutant hERG stability by inhibition of HDAC6.

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.

An Interdomain KCNH2 Mutation Produces an Intermediate Long QT Syndrome.

Hereditary long QT syndrome is caused by deleterious mutation in one of several genetic loci, including locus LQT2 that contains the KCNH2 gene (or hERG, human ether-a-go-go related gene), causing faulty cardiac repolarization. Here, we describe and characterize a novel mutation, p.Asp219Val in the hERG channel, identified in an 11-year-old male with syncope and prolonged QT interval. Genetic sequencing showed a nonsynonymous variation in KCNH2 (c.656A>T: amino acid p.Asp219Val). p.Asp219Val resides in a region of the channel predicted to be unstructured and flexible, located between the PAS (Per-Arnt-Sim) domain and its interaction sites in the transmembrane domain. The p.Asp219Val hERG channel produced K(+) current that activated with modest changes in voltage dependence. Mutant channels were also slower to inactivate, recovered from inactivation more readily and demonstrated a significantly accelerated deactivation rate compared with the slow deactivation of wild-type channels. The intermediate nature of the biophysical perturbation is consistent with the degree of severity in the clinical phenotype. The findings of this study demonstrate a previously unknown role of the proximal N-terminus in deactivation and support the hypothesis that the proximal N-terminal domain is essential in maintaining slow hERG deactivation.

Provocation testing in congenital long QT syndrome: A practical guide.

Congenital long QT syndrome (LQTS) is a hereditary cardiac channelopathy with an estimated prevalence of 1 in 2500. A prolonged resting QT interval corrected for heart rate (QTc interval) remains a key diagnostic component; however, the QTc value may be normal in up to 40% of patients with genotype-positive LQTS and borderline in a further 30%. Provocation of QTc prolongation and T-wave changes may be pivotal to unmasking the diagnosis and useful in predicting genotype. LQTS provocation testing involves assessment of repolarization during and after exercise, in response to changes in heart rate or autonomic tone, with patients with LQTS exhibiting a maladaptive repolarization response. We review the utility and strengths and limitations of 4 forms of provocation testing-stand-up test, exercise stress test, epinephrine challenge, and mental stress test-in diagnosing LQTS and provide some practical guidance for performing provocation testing. Ultimately, exercise testing, when feasible, is the most useful form of provocation testing when considering diagnostic sensitivity and specificity.

Sex hormones and repolarization dynamics during the menstrual cycle in women with congenital long QT syndrome.

BACKGROUND: Women with congenital long QT syndrome (LQTS) experience increased cardiac event risk after the onset of adolescence, perhaps stemming from the known modulating effects of sex hormones on the cardiac potassium channels. OBJECTIVE: We hypothesized that the effect of sex hormones on cardiac ion channel function may modify electrocardiographic (ECG) parameters associated with the propensity for ventricular tachyarrhythmias during the menstrual cycle in women with LQTS. METHODS: We prospectively enrolled 65 women with congenital LQTS (type 1 LQTS [LQT1], n = 24 [36.9%]; type 2 LQTS [LQT2], n = 20 [30.8%]) and unaffected female relatives (n = 21 [32.3%]). Patients underwent three 7-day ECG recordings during their menstrual cycles. Simultaneous saliva testing of sex hormone levels was conducted on the first day of each 7-day ECG recording cycle. RESULTS: The mean age was 35 ± 8 years, without a significant difference among the groups. In women with LQT2, linear mixed effects models showed significant inverse correlations of the corrected QT interval with progesterone levels (P < .001) and with the progesterone to estradiol ratio (P < .001). Inverse relationships of the R-R interval with estradiol levels (P = .003) and of the T-wave duration with testosterone levels (P = .014) were also observed in women with LQT2. In contrast, no significant associations were observed between ECG parameters and sex hormone levels in women with LQT1 or unaffected relatives. CONCLUSION: This is the first study to prospectively assess correlations between repolarization dynamics and sex hormone levels during the menstrual cycle in women with congenital LQTS. Our findings show genotype-specific unique corrected QT dynamics during the menstrual cycle that may affect the propensity for ventricular tachyarrhythmia in women with LQTS, particularly women with LQT2.

A Novel Mutation in the KCNH2 Gene Associatedwith Long QT Syndrome: A Case Report.

OBJECTIVE: Long QT syndrome is a cardiovascular disease with a prolonged QT interval. CASE REPORT: We report a 22-year-old woman presenting with frequent syncopal episodes two months after childbirth. Electrocardiography showed a sinus rhythm, QT interval prolongation, and Torsade de Pointes. Her mother had experienced an episode of syncope, but her father had not. Genetic analyses revealed that a new mutation in the KCNH2 gene, the c.2108dupA mutation (p.H703Qfs*20, exon8, M_000238), was found in the patient and in her mother and sister. CONCLUSION: The c.2108dupA mutation (p.H703Qfs*20, exon8, M_000238) is the first reported case of a KCNH2 mutation at this site.

Electrophysiological characterization of the modified hERGT potassium channel used to obtain the first cryo-EM hERG structure.

The voltage-gated hERG (human-Ether-à-go-go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss-of-function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain-of-function mutations lead to short QT syndrome. A recent open channel cryo-EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure-function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT ) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full-length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole-cell measurements of hERG current (IhERG ) were made at 37°C from HEK 293 cells expressing wild-type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end-pulse nor tail IhERG density significantly differed between WT and hERGT . However, the IhERG deactivation rate was significantly slower for hERGT . Half-maximal activation voltage (V0.5 ) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT , but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady-state "window" current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain.

hiPSC-Derived Cardiomyocyte Model of LQT2 Syndrome Derived from Asymptomatic and Symptomatic Mutation Carriers Reproduces Clinical Differences in Aggregates but Not in Single Cells.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.

Ten-year experience in atenolol use and exercise evaluation in children with genetically proven long QT syndrome.

BACKGROUND: Due to its availability, atenolol is the primary beta-blocker used in Australia for children with long QT syndrome. There is limited data on long-term follow-up of its use. METHODS: A single-tertiary-center, retrospective, observational study investigating all children and adolescents who had genetically proven long QT syndrome type 1 (LQT1) and type 2 (LQT2) was conducted. Their pretreatment exercise tests were evaluated for QTc intervals into the recovery phase of exercise. RESULTS: Eighty six patients were identified (LQT1, 67, and LQT2, 19) from 2004 to 2014. The majority (86%) of patients were initially referred for family screening. Atenolol was administered at a mean dose of 1.58 ± 0.51 mg/kg/day. During the median follow-up period of 4.29 years, only one proband developed ventricular arrhythmia whilst taking atenolol, No patient had cardiac arrest or aborted cardiac arrest. With respect to side effects of atenolol, only two patients had intolerable side effects necessitating changes of medication. Evaluation of exercise tests (pretreatment) demonstrated that corrected QT (QTc) intervals at 2-3 min into the recovery phase of exercise were significantly prolonged for LQT1 patients. LQT1 patients with transmembrane mutation had longer QTc intervals than their C-terminus mutation counterparts, reaching statistical significance at 3 min into the recovery phase of exercise. CONCLUSIONS: Atenolol is an effective treatment for genetically proven LQT1 and LQT2 children and adolescents, with good tolerability. In LQT1 patients, QTc intervals at 2-3 min into the recovery phase of exercise were significantly prolonged, particularly in patients with transmembrane mutations.

Experience with bisoprolol in long-QT1 and long-QT2 syndrome.

BACKGROUND: The protective effect of beta-blockers in patients with inherited Long-QT syndrome is well established. Recent reports have suggested that beta-blockers are not equally effective in Long-QT (LQT). Bisoprolol is an attractive candidate for use in LQT because of its cardioselective properties and favorable side-effect profile. METHODS: We performed a retrospective cohort study of 114 consecutive patients with gene-positive Long-QT syndrome type 1 (LQT1) or Long-QT syndrome type 2 (LQT2) treated with bisoprolol, nadolol or atenolol with a total of 580 person-years of follow-up. Electrocardiogram (ECG) parameters and cardiac events during follow-up were compared. In addition, exercise treadmill testing was performed in bisoprolol-treated patients. RESULTS: Fifty-nine patients were treated with bisoprolol, 39 with atenolol and 16 with nadolol. Overall, 59 % were females and 62 % had LQT1. Baseline heart rate and corrected QT (QTc) interval were similar between the groups. QTc shortening was observed in individuals on bisoprolol (ΔQTc -5 ± 31 ms; p = 0.049) and nadolol (ΔQTc -13 ± 16 ms; p = 0.02) but not on atenolol (ΔQTc +9 ± 24 ms; p = 0.16). Median follow-up was similar for bisoprolol and nadolol (3 years), but longer for atenolol (6 years; p = 0.03); one cardiac event occurred in the bisoprolol group (1.7 %) and two events occurred in the atenolol group (5.1 %; p = 0.45), whereas none occurred in nadolol-treated patients. Beta-blocker efficacy was not affected by the underlying genotype. The antiadrenergic effect of bisoprolol correlated with the reduction of peak heart rates at exercise testing. CONCLUSIONS: Bisoprolol treatment results in QTc shortening in gene-positive LQT1 and LQT2 patients and is well tolerated during long-term administration. The equivalence of bisoprolol for protection from ventricular arrhythmia in LQT patients compared to established beta-blockers remains unknown. Further large-scale studies are required.

hERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms.

BACKGROUND: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. OBJECTIVE: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. METHODS: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. RESULTS: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. CONCLUSION: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

The link between abnormal calcium handling and electrical instability in acquired long QT syndrome--Does calcium precipitate arrhythmic storms?

Release of Ca(2+) ions from sarcoplasmic reticulum (SR) into myocyte cytoplasm and their binding to troponin C is the final signal form myocardial contraction. Synchronous contraction of ventricular myocytes is necessary for efficient cardiac pumping function. This requires both shuttling of Ca(2+) between SR and cytoplasm in individual myocytes, and organ-level synchronization of this process by means of electrical coupling among ventricular myocytes. Abnormal Ca(2+) release from SR causes arrhythmias in the setting of CPVT (catecholaminergic polymorphic ventricular tachycardia) and digoxin toxicity. Recent optical mapping data indicate that abnormal Ca(2+) handling causes arrhythmias in models of both repolarization impairment and profound bradycardia. The mechanisms involve dynamic spatial heterogeneity of myocardial Ca(2+) handling preceding arrhythmia onset, cell-synchronous systolic secondary Ca(2+) elevation (SSCE), as well as more complex abnormalities of intracellular Ca(2+) handling detected by subcellular optical mapping in Langendorff-perfused hearts. The regional heterogeneities in Ca(2+) handling cause action potential (AP) heterogeneities through sodium-calcium exchange (NCX) activation and eventually overwhelm electrical coupling of the tissue. Divergent Ca(2+) dynamics among different myocardial regions leads to temporal instability of AP duration and - on the patient level - in T wave lability. Although T-wave alternans has been linked to cardiac arrhythmias, non-alternans lability is observed in pre-clinical models of the long QT syndrome (LQTS) and CPVT, and in LQTS patients. Analysis of T wave lability may provide a real-time window on the abnormal Ca(2+) dynamics causing specific arrhythmias such as Torsade de Pointes (TdP).

Prevalence and spectrum of electroencephalogram-identified epileptiform activity among patients with long QT syndrome.

BACKGROUND: Congenital long QT syndrome (LQTS) is a heritable cardiac disease whereby patients are at an increased risk for LQTS-triggered syncope, seizures, and sudden cardiac arrest. Seizure episodes are common in LQTS and most often seen in patients with type 2 LQTS (LQT2). OBJECTIVE: To determine the prevalence of electroencephalogram (EEG)-identified epileptiform activity among patients with LQTS. METHODS: A retrospective electronic medical record review of 610 patients with LQTS (250 [41%] men), evaluated between 2000 and 2012, was performed to identify (1) all patients with LQTS who presented with seizures/seizure-like episodes, (2) patients with LQTS who underwent a subsequent neurologic evaluation and EEG study, and (3) patients with LQTS and abnormal EEG recordings that showed epileptiform activity during sinus rhythm, confirming a seizure independent from cardiac arrhythmia. RESULTS: Overall, seizures/seizure-like episodes were recorded in 68 of 610 (11%) patients with LQTS. Ten patients were diagnosed with a seizure disorder by an epileptologist on the basis of the clinical findings and EEG studies, giving a prevalence of 10 of 610 (1.6%; 95% confidence interval 0.8%-3%) among patients with LQTS. A diagnosis of epilepsy was overrepresented in patients with LQT2 (7 of 190 [3.7%]) in comparison to all other LQT subgroups (3 of 420 [0.7%]; P = .0126). CONCLUSIONS: While the overall prevalence of epilepsy among patients with LQTS is low, 10 of 68 (15%) of the patients who presented with seizures/seizure-like episodes had EEG-identified epileptiform activity. Confirming earlier observational reports, epilepsy is more common in patients with LQT2, further supporting the shared pathogenetic link hypothesis of this KCNH2-encoded potassium channel that is expressed in both the heart and the brain.

Single nucleotide polymorphisms in arrhythmia genes modify the risk of cardiac events and sudden death in long QT syndrome.

BACKGROUND: Disease-modifying single nucleotide polymorphisms (SNPs) can help explain incomplete penetrance and variable expressivity in congenital long QT syndrome (LQTS) by altering susceptibility to arrhythmias. OBJECTIVE: The purpose of this study was to assess multiple arrhythmia SNPs (in 16 genes) in a distinct cohort of LQTS patients to identify modifier SNPs influencing the risk of sudden death. METHODS: This study included 273 patients with LQTS from the New Zealand Cardiac Inherited Disease Registry (154 long QT type 1, 96 long QT type 2, and 23 long QT type 3), including 31 patients who had experienced death or resuscitated sudden cardiac death (RSCD). Patients were genotyped for 29 SNPs and tested for associations with clinical events and QTc length. Caucasian (n = 220) and Pacific Islander/New Zealand Maori (n = 53) ethnic groups were analyzed separately. This subgroup of Polynesian ancestry has not been previously studied for LQTS in either presentation or outcome. RESULTS: In Caucasians, four SNPs at two risk loci (NOS1AP: rs12143842 and rs16847548; and KCNQ1: rs10798 and rs8234) were significantly associated with clinical events after correction for multiple testing. Patients homozygous for the risk allele of rs12143842 had an increased risk of death/RSCD [hazard ratio 10.15, 95% confidence interval (2.38, 43.34), q = 0.045). Several other SNPs showed trends toward association with QTc length and clinical events. CONCLUSION: This study demonstrates that SNPs in NOS1AP and KCNQ1 are associated with an increased risk of cardiac events in LQTS patients, with the hazard ratio suggesting they have significant potential in clinical risk stratification.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

Spatial correlation of action potential duration and diastolic dysfunction in transgenic and drug-induced LQT2 rabbits.

BACKGROUND: Enhanced dispersion of action potential duration (APD) is a major contributor to long QT syndrome (LQTS)-related arrhythmias. OBJECTIVE: To investigate spatial correlations of regional heterogeneities in cardiac repolarization and mechanical function in LQTS. METHODS: Female transgenic LQTS type 2 (LQT2; n = 11) and wild-type littermate control (LMC) rabbits (n = 9 without E4031 and n = 10 with E4031) were subjected to phase contrast magnetic resonance imaging to assess regional myocardial velocities. In the same rabbits' hearts, monophasic APDs were assessed in corresponding segments. RESULTS: In LQT2 and E4031-treated rabbits, APD was longer in all left ventricular segments (P < .01) and APD dispersion was greater than that in LMC rabbits (P < .01). In diastole, peak radial velocities (Vr) were reduced in LQT2 and E4031-treated compared to LMC rabbits in LV base and mid (LQT2: -3.36 ± 0.4 cm/s, P < .01; E4031-treated: -3.24 ± 0.6 cm/s, P < .0001; LMC: -4.42 ± 0.5 cm/s), indicating an impaired diastolic function. Regionally heterogeneous diastolic Vr correlated with APD (LQT2: correlation coefficient [CC] 0.38, P = .01; E4031-treated: CC 0.42, P < .05). Time-to-diastolic peak Vr were prolonged in LQT2 rabbits (LQT2: 196.8 ± 2.9 ms, P < .001; E4031-treated: 199.5 ± 2.2 ms, P < .0001, LMC 183.1 ± 1.5), indicating a prolonged contraction duration. Moreover, in transgenic LQT2 rabbits, diastolic time-to-diastolic peak Vr correlated with APD (CC 0.47, P = .001). In systole, peak Vr were reduced in LQT2 and E4031-treated rabbits (P < .01) but longitudinal velocities or ejection fraction did not differ. Finally, random forest machine learning algorithms enabled a differentiation between LQT2, E4031-treated, and LMC rabbits solely based on "mechanical" magnetic resonance imaging data. CONCLUSIONS: The prolongation of APD led to impaired diastolic and systolic function in transgenic and drug-induced LQT2 rabbits. APD correlated with regional diastolic dysfunction, indicating that LQTS is not purely an electrical but an electromechanical disorder.