long-QT Type Research Articles

Impaired CaV1.2 inactivation reduces the efficacy of calcium channel blockers in the treatment of LQT8

Mutations in the CaV1.2 L-type calcium channel can cause a profound form of long-QT syndrome known as long-QT type 8 (LQT8), which results in cardiac arrhythmias that are often fatal in early childhood. A growing number of such pathogenic mutations in CaV1.2 have been identified, increasing the need for targeted therapies. As many of these mutations reduce channel inactivation; resulting in excess Ca2+ entry during the action potential, calcium channel blockers (CCBs) would seem to represent a promising treatment option. Yet CCBs have been unsuccessful in the treatment of LQT8. Here, we demonstrate that this lack of efficacy likely stems from the impact of the mutations on CaV1.2 channel inactivation. As CCBs are known to preferentially bind to the inactivated state of the channel, mutation-dependent deficits in inactivation result in a decrease in use-dependent block of the mutant channel. Further, application of the CCB verapamil to induced pluripotent stem cell (iPSC) derived cardiomyocytes from an LQT8 patient demonstrates that this loss of use-dependent block translates to a lack of efficacy in correcting the LQT phenotype. As a growing number of channelopathic mutations demonstrate effects on channel inactivation, reliance on state-dependent blockers may leave a growing population of patients without a viable treatment option. This biophysical understanding of the interplay between inactivation deficits and state-dependent block may provide a new avenue to guide the development of improved therapies.

NaV1.5 knockout in iPSCs: a novel approach to study NaV1.5 variants in a human cardiomyocyte environment

Cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs) successfully reproduce the mechanisms of several channelopathies. However, this approach involve cell reprogramming from somatic tissue biopsies or genomic editing in healthy iPSCs for every mutation found and to be investigated. We aim to knockout (KO) NaV1.5, the cardiac sodium channel, in a healthy human iPSC line, characterize the model and then, use it to express variants of NaV1.5. We develop a homozygous NaV1.5 KO iPSC line able to differentiate into cardiomyocytes with CRISPR/Cas9 tool. The NaV1.5 KO iPSC-CMs exhibited an organized contractile apparatus, spontaneous contractile activity, and electrophysiological recordings confirmed the major reduction in total Na+ currents. The action potentials (APs) exhibited a reduction in their amplitude and in their maximal rate of rise. Voltage optical mapping recordings revealed that the conduction velocity Ca2+ transient waves propagation velocities were slow. A wild-type (WT) NaV1.5 channel expressed by transient transfection in the KO iPSC-CMs restored Na+ channel expression and AP properties. The expression of NaV1.5/delQKP, a long QT type 3 (LQT3) variant, in the NaV1.5 KO iPSC-CMs showed that dysfunctional Na+ channels exhibited a persistent Na+ current and caused prolonged AP duration that led to arrhythmic events, characteristics of LQT3.

Age-Associated Ion Channel Localization and Expression Changes Regulate Cardiac Sodium Gain-of-Function

Madison B. Nowak, Steven Poelzing, and Seth H. Weinberg Age-Associated Ion Channel Localization and Expression Changes Regulate Cardiac SodiumGain-of-Function Action potentials in the heart are initiated by sodium (Na+) ion influx via voltage-gated Na+channels. Na+ channel gain-of-function (GoF) mutations associated with the long QT type-3(LQT3) syndrome can produce a late Na+ current, triggering pro-arrhythmic early after-depolarizations (EADs) that prolong the action potential duration (APD). Recently, we showedthat preferential localization of Na+ channels at the intercalated disk and narrow intercellular cleftssuppress EADs and APD prolongation in LQT3-associated models (Nowak, et al., Biophysical J, 2020).

First episode of ventricular fibrillation in an 84-year-old man with long-QT type 2 syndrome: A case report

Congenital long QT syndrome (LQTS) is associated with ventricular arrhythmia and an increased risk of sudden cardiac death in young people. However, it is extremely rare for an elderly man to experience ventricular fibrillation (VF) due to congenital LQTS as a first episode. We describe the case of an 84-year-old man who experienced syncope after urination. He had a medical history of hypertension and asthma, but no history of syncope. Electrocardiographic findings in 2017 showed QT prolongation (corrected QT = 505 ms). No medication that could induce QT prolongation was administered. Blood test results on admission showed no electrolyte abnormalities, and there were no abnormal findings on echocardiography. The second episode of loss of consciousness occurred during hospitalization, and electrocardiography revealed incessant torsade de pointes, caused by R-on-T with short-long-short (SLS) sequences due to bradyarrhythmia. Coronary angiography did not detect myocardial ischemia, and an implantable cardioverter-defibrillator was implanted for secondary prevention. Genetic testing revealed a mutation of the KCNH2 gene, indicating LQTS type 2. In summary, we report a rare case of prolonged QT interval with SLS sequences due to sick sinus syndrome triggering VF as the first attack in an elderly patient with LQTS type 2.<Learning objective: Physicians should be aware of the prolongation of QT as a cause of syncope in elderly patients and should pay attention to QT duration. Furthermore, patients with elderly-onset QT prolongation may have a genetic background associated with congenital long QT syndrome (LQTS); therefore, we should not hesitate to perform genetic testing in cases where LQTS is suspected in elderly patients.>

Abstract 431: Probing the Pathogenic Mechanisms Underlying Ca V 1.2 Channelopathies

The Ca V 1.2 channel is essential to cardiac function. It is the sentinel that translates the electrical signal on the surface of the cell to the intracellular calcium cascade that leads to contraction. To allow the precise tuning of that is essential for cardiac function, Ca V 1.2 is subject to two forms of feedback regulation, voltage dependent inactivation (VDI) and calcium dependent inactivation (CDI). The first mutations described in Ca V 1.2 were causative of Timothy Syndrome (TS), a multisystem disorder with neurological, developmental and life-threatening cardiac symptoms, defined as long-QT type 8 (LQT8). These single point mutations occur in the S6, a region commonly associated with channel activation. In fact, the original TS mutations were shown to perturb channel activation as well as inactivation. Since this first description of TS, multiple additional mutations have been discovered in Ca V 1.2 and are associated with either TS-like or cardiac specific phenotypes. This phenotypic variance suggests that these mutations disrupt channel mechanisms in divergent ways. Moreover, for patients harboring mutations in Ca V 1.2, the cardiac symptoms have proven recalcitrant to conventional therapy including calcium channel blockers (CCBs). This therapeutic inadequacy reinforces the need for a better understanding of how mutations in the S6 disrupt channel function and how this affects therapeutic options for patients that carry them. Here we show that LQT8 mutations that cluster in the S6 of Ca V 1.2 do indeed have a variety of mechanistic underpinnings. This may account for the gradient of symptoms seen in patients with these mutations. In addition, we demonstrate that many of these mutant channels are resistant to block by CCBs, and this resistance strongly correlates to specific mechanistic perturbations of the channel. Thus, reliance on a CCB based therapeutic strategy is likely to leave many in a growing population of patients without a viable treatment option.

The role and mechanism of chaperones Calnexin/Calreticulin in which ALLN selectively rescues the trafficking defective of HERG-A561V mutation.

Long QT (LQT) type 2 (LQT2) is caused by HERG mutation. L539fs/47 encodes a truncated protein, and its mechanisms in HERG mutation are unknown. HERG mutation plasmids were overexpressed in HEK293T cells, respectively, followed by analyzing lysates with Western blot. Transfected HEK293T cells were treated with or without N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) and Propranolol (Prop) at 24 or 48 h. HERG-WT, HERG-A561V, WT/A561V, HERG-L539fs/47, WT/L539fs/47, and Calnexin (CNX)/Calreticulin (CRT) protein expression and their interactions were detected by Western blot and immunoprecipitation. Each group with HERG currents (Ikr) were detected by Patch-clamp technique. Treated with ALLN, the expression of mature HERG protein and the CNX/CRT protein increased. The interaction of HERG-A561V and WT/A561V protein with the chaperone CNX/CRT increased significantly. The maximum peak currents and tail currents density increased by 70% and 73%, respectively, while maximal peak currents density (24%) and tail currents density (19%) were slight increased in WT-HERG cells. Treated with Prop, the expression and interaction of mature HERG and chaperones CNX/CRT had no difference in each group. The maximal currents and tail currents density were slight increased. CNX/CRT might play a crucial role in the trafficking-deficient process and degradation of HERG-A561V mutant protein, however they had no effect on L539fs/47 HERG due to protein transport deletion. ALLN can restore HERG-A561V mutant protein trafficking process and rescue the dominant negative suppression of WT-HERG.

A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome

Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

Revealing the Concealed Nature of Long-QT Type 3 Syndrome.

Gain-of-function mutations in the voltage-gated sodium channel (Nav1.5) are associated with the long-QT-3 (LQT3) syndrome. Nav1.5 is densely expressed at the intercalated disk, and narrow intercellular separation can modulate cell-to-cell coupling via extracellular electric fields and depletion of local sodium ion nanodomains. Models predict that significantly decreasing intercellular cleft widths slows conduction because of reduced sodium current driving force, termed "self-attenuation." We tested the novel hypothesis that self-attenuation can "mask" the LQT3 phenotype by reducing the driving force and late sodium current that produces early afterdepolarizations (EADs). Acute interstitial edema was used to increase intercellular cleft width in isolated guinea pig heart experiments. In a drug-induced LQT3 model, acute interstitial edema exacerbated action potential duration prolongation and produced EADs, in particular, at slow pacing rates. In a computational cardiac tissue model incorporating extracellular electric field coupling, intercellular cleft sodium nanodomains, and LQT3-associated mutant channels, myocytes produced EADs for wide intercellular clefts, whereas for narrow clefts, EADs were suppressed. For both wide and narrow clefts, mutant channels were incompletely inactivated. However, for narrow clefts, late sodium current was reduced via self-attenuation, a protective negative feedback mechanism, masking EADs. We demonstrated a novel mechanism leading to the concealing and revealing of EADs in LQT3 models. Simulations predict that this mechanism may operate independent of the specific mutation, suggesting that future therapies could target intercellular cleft separation as a compliment or alternative to sodium channels.

Mutation-Specific Cardiac Risk Assessment in LQT3

Long QT type 3 (LQT3) is a common form of LQT syndrome caused by mutations in the cardiac sodium channel. These mutations lead to an increase in the cardiac sodium current during the late phases of the cardiac action potential (AP), leading to cardiac arrhythmias. We attempted to predict mutation-specific cardiac event risk for LQT3 mutations using a combination of in vitro and in silico risk assessment. For that, we first characterized, using patch clamp, eight common mutations associated with LQT3. Using our data as an input to a cardiomyocyte computer model, we predicted action potential duration (APD) and early after-depolarization (EAD) susceptibility for these mutants, at different heart rates. Our risk predictions correlated well to the cardiac event risk observed in patients with these mutations. Second, we measured the effect of the late sodium blocker ranolazine for each of the mutants tested. We used the model to predict drug treatment efficacy. Interestingly, ranolazine showed mutation-specific effects, with some mutations showing strongly shortened APDs and decreased predicted risk, while little effect was observed in other mutants. Most importantly, for one of the mutations assessed, risk was predicted to be increased, despite shortening of APD. Thus, our results suggest that drug treatment efficacy is mutation-specific. In summary, our results show that mutation-specific in silico models may be an important new tool for clinical risk assessment in LQT3.

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

BackgroundRemodeling 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 I ks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.Methods and ResultsLQT1 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.ConclusionsCompared 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.

Mutation Specific Drug Response and Cardiac Risk in Long QT Type 3

Long QT type 3 (LQT3) is caused by mutations that cause an increase in the cardiac sodium current during late phases of the cardiac action potential. For a number of LQT3 mutants this is caused by a failure to inactivate fully, and a consequent increase in late sodium current. For several mutations, shifts in voltage dependence of activation and inactivation cause increased sodium channel contribution to more depolarized voltages, an effect referred as increase in window current. The goal of this study was to perform a study in a large number of LQT3 associated mutations and compare the biophysical effect of the mutation to cardiac risk in LQT3 patients and response to treatment. We measured the function of eight common mutations associated with LQT3 with different mechanism underlying channel dysfunction. We compared the functional effects of the channel with the clinical course of these patients and observed that for the two mutations tested with increased sustained current but without increase in current availability (window current), D1784K and D1790G, the patients had significant lower risk of cardiac events, suggesting an increase in current availability may be associated with increased risk for these patients. In addition, we measured mutation specific effects of ranolazine for these mutants. For all mutants tested, ranolazine preferentially blocked late sodium currents. Ranolazine shifts steady-state availability of the inactivation was dependent on the specific mutant tested. Our results suggest that ranolazine may have a mutation dependent effect. Mutations dysfunction may affect drug binding and drug actions in the channel and may alter treatment effectiveness in patients.

Modulation of Action Potential Alternans by IKs in Myocardial Infarction

Background: Patients with post myocardial infarction (MI) have increased incidence of ventricular arrhythmias. The slowly activating K+ channels (IKs) has been reported to be downregulated consistently but in different degrees in the post-MI hearts. The role of downregulation of IKs in arrhythmogenesis in post MI remains to be defined.Hypothesis: We hypothesize that significant suppression of IKs in post MI impairs the rate-dependent and Ca2+-dependent augmentation of repolarization reserve resulting in increased alternans and thereby arrhythmias.Methods: We evaluated the effect of IKs on alternans in post MI using transgenic rabbit model of Long QT Type 1 (LQT1) lacking IKs. LQT1 and littermate control (LMC) rabbits underwent MI procedures and optical mapping was performed to record Vm and Ca2+ simultaneously from the 3 week post-MI hearts (n=9 LQT1-MI, 4 LQT-MI Sham, 10 LMC-MI, 9 LMC-Sham hearts).Results: The dispersion of APD was enhanced in LQT1-MI compared to LQT1-Sham and LMC-MI (αAPDmax-min = 28±4 ms vs. 20±3 ms and 27±6 ms). APD alternans was more pronounced in LQT1-MI, accompanied with large amplitude Ca2+ alternans, and often spatially discordant (see traces and restitution curves in panel A&B). Spatially discordant alternans in LQT1-MI was highly dynamic, preceded by sudden appearance of Ca2+ nodal lines near the MI border zone (panel C, dark lines), associated with high VF induction (n=9/9 LQT1-MI vs. 1/4 LQT1-sham and 7/10 LMC-MI).Conclusion: Our results demonstrate the importance of IKs as a repolarization reserve in MI and that significant downregulation of IKs may promote spatially discordant Ca2+ and APD alternans and VF induction.

Abstract 18946: A Complex Cardiac Rhythm and Conduction Abnormalities in Three Families Carrying a SCN5A Missense Mutation May Involve Single Nucleotide Polymorphisms in SCN5A and RYR2 genes

Background: Mutations in the gene encoding the cardiac sodium channel (SCN5A) have been implicated in arrhythmia syndromes including Brugada (BrS) and long-QT type 3 syndromes. Also, wide spectrums of mixed phenotypes, known as “overlap syndromes”, have been associated to SCN5A mutations. The contributions of single nucleotide polymorphisms (SNPs) add more complexity to the disease phenotype. Here we report a missense mutation in SCN5A (T1708N) in three Hispanic families with history of sudden cardiac death (SCD) and extensive variability and severity of symptoms, including syncope, atrial and ventricular tachycardia, and conduction abnormalities. Methods and Results: We used the Ion Torrent platform and AmpliSeq panel to sequence 19 genes associated with arrhythmia syndromes. In addition to SCN5A-T1708N mutation, the patients carry two common SNPs, SCN5A-H558R and the cardiac ryanodine receptor RYR2-Q2958R, which we previously reported to be highly prevalent (~30%) in Hispanics and to increase the risk of SCD in homozygous individuals. To study the functional properties of SCN5A-T1708N, the mutation was engineered by site-directed mutagenesis into SCN5A with or without the SCN5A-H558R and expressed in HEK cells for voltage clamp experiments. SCN5A-T1708N showed a significant increase of late INa compared to WT. Also, the kinetic properties of SCN5A-T1708N showed slower time constants of recovery and increase in slow inactivation, which would tend to reduce INa; these effects were not modified by the presence of SCN5A-H558R. On the other hand, INa density for SCN5A-T1708N was reduced compared to WT (289± 63 and 387± 62 pA/pF, respectively), yet, for the double mutant H558R-T1708N, INa was more severely depressed (145± 32 pA/pF). Conclusion: The profound biophysical phenotype of T1708N with loss and gain of function could account for the variability and the severity of the clinical phenotype in these families. The RYR2-Q2958R polymorphism may function as a pro-arrhythmic genetic modifier and it is conceivable that it may predispose to SCD in some members of these families. The study of these polymorphisms in large cohorts may identify common haplotypes associated to cardiac arrhythmias and facilitate identification of patients at high risk for SCD.

16 * Ethnicity and phenotype in the SCN5A E1784K mutation

Background: Long QT (LQTS) and Brugada syndromes (BrS) are inherited arrhythmia syndromes characterised by risk of sudden cardiac death (SCD) and highly variable penetrance. Mutations in SCN5A, which encodes the Nav1.5 cardiac ion channel responsible for the inward cardiac sodium current, are responsible for 5-10% of LQTS and associated with 20% of BrS. The E1784K mutation in SCN5A is the most commonly identified SCN5A mutation in both LQTS and BrS, and causes the Long QT type 3 (LQT3)/BrS overlap disease with variable phenotypic expression. Carriers within the same pedigree may exhibit LQT3 phenotype, BrS phenotype or both. The aim of this international project was to determine if there are any associations between phenotype and ethnicity within a unique cohort of E1784K mutation carriers. Methods: 93 E1784K mutation carriers belonging to 33 families from 6 different countries including Japan were included. Data was collected on age, gender, symptoms, family history and the ECG. Non-digital and digital ECG data were analysed using proprietary software. Automatic readings and measurements from 2 observers were taken. A 3rd observer reviewed the ECGs where there was inter-observer discrepancy between the measurements. Statistical significance was determined using either the 2-tailed t-test or Chi-squared test as appropriate. Results: QTc intervals were significantly longer amongst Japanese E1784K mutation carriers, who were more symptomatic albeit with a lower incidence of SCD. Results are summarised in Table [1][1]. The spontaneous type 1 BrS ECG pattern also appeared to be more prevalent in the Japanese compared to Caucasian mutation carriers although the numbers were small. Mean QRS duration was longer amongst Caucasian mutation carriers. Drug-induced BrS phenotype was unmasked in 63% of Caucasians (34/54) who underwent a test. A family history of SCD was present in 50% of Caucasian families compared to only 13.6% of Japanese families (p=0.015). | | Total (n=93) | Caucasian (n=64) | Japanese (n=29) | p-value | | ------------------------- | ------------ | ---------------- | --------------- | ------- | | Age (years, mean, range) | 28, 1-67 | 30, 1-67 | 23, 6-62 | 0.0826 | | Sex-male (n,%) | 47, 51 | 32, 50 | 15, 52 | 0.8776 | | Spontaneous BrS (n,%) | 4, 4 | 1, 2 | 3, 10 | 0.0531 | | QTc (ms, mean, range) | 480, 404-579 | 472, 404-533 | 499, 430-579 | 0.0001 | | PR (ms, mean, range) | 167, 116-302 | 167, 116-302 | 166, 149-180 | 0.8952 | | QRS (ms, mean, range) | 96, 60-139 | 99, 68-139 | 90, 60-120 | 0.0145 | | Pre-syncope/syncope (n,%) | 17, 18 | 8, 13 | 9, 31 | 0.0741 | | Aborted SCD (n,%) | 6, 6 | 6, 9 | 0, 0 | 0.0882 | Table 1 Clinical characteristics Conclusions: There is variation in expression of E1784K between ethnicities with ethnic differences in severity and risk of sudden death. These results support a strong role for genetic modifiers of phenotype and risk which deserves further study. [1]: #T1

Pronounced effects of HERG-blockers E-4031 and erythromycin on APD, spatial APD dispersion and triangulation in transgenic long-QT type 1 rabbits.

BackgroundProlongation of action potential duration (APD), increased spatial APD dispersion, and triangulation are major factors promoting drug-induced ventricular arrhythmia. Preclinical identification of HERG/IKr-blocking drugs and their pro-arrhythmic potential, however, remains a challenge. We hypothesize that transgenic long-QT type 1 (LQT1) rabbits lacking repolarizing IKs current may help to sensitively detect HERG/IKr-blocking properties of drugs.MethodsHearts of adult female transgenic LQT1 and wild type littermate control (LMC) rabbits were Langendorff-perfused with increasing concentrations of HERG/IKr-blockers E-4031 (0.001–0.1 µM, n = 9/7) or erythromycin (1–300 µM, n = 9/7) and APD, APD dispersion, and triangulation were analyzed.ResultsAt baseline, APD was longer in LQT1 than in LMC rabbits in LV apex and RV mid. Erythromycin and E-4031 prolonged APD in LQT1 and LMC rabbits in all positions. However, erythromycin-induced percentaged APD prolongation related to baseline (%APD) was more pronounced in LQT1 at LV base-lateral and RV mid positions (100 µM, LQT1, +40.6±9.7% vs. LMC, +24.1±10.0%, p<0.05) and E-4031-induced %APD prolongation was more pronounced in LQT1 at LV base-lateral (0.01 µM, LQT1, +29.6±10.6% vs. LMC, +19.1±3.8%, p<0.05) and LV base-septal positions. Moreover, erythromycin significantly increased spatial APD dispersion only in LQT1 and increased triangulation only in LQT1 in LV base-septal and RV mid positions. Similarly, E-4031 increased triangulation only in LQT1 in LV apex and base-septal positions.ConclusionsE-4031 and erythromycin prolonged APD and increased triangulation more pronouncedly in LQT1 than in LMC rabbits. Moreover, erythromycin increased APD dispersion only in LQT1, indicating that transgenic LQT1 rabbits could serve as sensitive model to detect HERG/IKr-blocking properties of drugs.

Flecainide Provocation Reveals Concealed Brugada Syndrome in a Long QT Syndrome Family With a Novel L1786Q Mutation in SCN5A

Mutations in SCN5A can result in both long QT type 3 (LQT3) and Brugada syndrome (BrS), and a few mutations have been found to have an overlapping phenotype. Long QT syndrome is characterized by prolonged QT interval, and a prerequisite for a BrS diagnosis is ST elevation in the right precordial leads of the electrocardiogram. In a Danish family suffering from long QT syndrome, a novel missense mutation in SCN5A, changing a leucine residue into a glutamine residue at position 1786 (L1786Q), was found to be present in heterozygous form co-segregating with prolonged QT interval. The proband presented with an aborted cardiac arrest, and his mother died suddenly and unexpectedly at the age of 65. Flecainide treatment revealed coved ST elevation in all mutation carriers. Electrophysiological investigations of the mutant in HEK293 cells indicated a reduced peak current, a negative shift in inactivation properties and a positive shift in activation properties, compatible with BrS. Furthermore, the sustained (I(Na,late)) tetrodotoxin-sensitive sodium current was found to be drastically increased, explaining the association between the mutation and LQT syndrome. The L1786Q mutation is associated with a combined LQT3 and concealed BrS phenotype explained by gating characteristics of the mutated ion channel protein. Hence, sodium channel blockade should be considered in clinical evaluation of apparent LQT3 patients.

Sex(y) hormone a‐go‐go: Estrogen–dependent stimulation of hERG potassium channel via G Protein Estrogen Receptor (GPER)

Female gender is an independent risk factor for several channelopathies. Malfunction of the potassium channel encoded by the human ether a‐go‐go related gene 1 (hERG1) which associates with Long QT type 2 (LQT2) syndromes is more prominent in women. In addition, drug‐induced LQT2 appears to fluctuate during different phases of the menstrual cycle. This raised the question that the steroid sex hormone estrogen (E2) could play a major role in regulating the hERG channel. E2 effects on cell targets have been explained by activation of genomic mechanism; however, a non transcriptional regulation of ion channels might be responsible for sex‐dependent susceptibility to LQT. Interestingly, recent studies have demonstrated that some of the E2's effects on breast cancer cells can be mediated by the newly characterized G protein‐coupled E2 receptor, GPER.In our present study we have characterized a non‐genomic biochemical cascade through which E2 stimulates hERG channel activity. This effect was mediated by GPER and the ser/thr protein kinase PKCalpha via direct phosphorylation of the channel. Ultimately, channel phosphorylation increased hERG membrane stability which determined an augmentation of its current activity. Understanding E2‐dependent regulation of hERG potassium channel can lead to a better understanding of complex physiological mechanisms including heart function and will help to develop better therapeutical strategies for channelopathies.

Ranolazine stabilizes cardiac ryanodine receptors: A novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2

Ranolazine (Ran) is known to inhibit multiple targets, including the late Na(+)current, the rapid delayed rectifying K(+)current, the L-type Ca(2+)current, and fatty acid metabolism. Functionally, Ran suppresses early afterdepolarization (EADs) and torsades de pointes (TdP) in drug-induced long QT type 2 (LQT2) presumably by decreasing intracellular [Na(+)](i) and Ca(2+)overload. However, simulations of EADs in LQT2 failed to predict their suppression by Ran. To elucidate the mechanism(s) whereby Ran alters cardiac action potentials (APs) and cytosolic Ca(2+)transients and suppresses EADs and TdP in LQT2. The known effects of Ran were included in simulations (Shannon and Mahajan models) of rabbit ventricular APs and Ca(2+)transients in control and LQT2 models and compared with experimental optical mapping data from Langendorff rabbit hearts treated with E4031 (0.5 μM) to block the rapid delayed rectifying K(+)current. Direct effects of Ran on cardiac ryanodine receptors (RyR2) were investigated in single channels and changes in Ca(2+)-dependent high-affinity ryanodine binding. Ran (10 μM) alone prolonged action potential durations (206 ± 4.6 to 240 ± 7.8 ms; P <0.05); E4031 prolonged action potential durations (204 ± 6 to 546 ± 35 ms; P <0.05) and elicited EADs and TdP that were suppressed by Ran (10 μM; n = 7 of 7 hearts). Simulations (Shannon but not Mahajan model) closely reproduced experimental data except for EAD suppression by Ran. Ran reduced open probability (P(o)) of RyR2 (half maximal inhibitory concentration = 10 ± 3 μM; n = 7) in bilayers and shifted half maximal effective concentration for Ca(2+)-dependent ryanodine binding from 0.42 ± 0.02 to 0.64 ± 0.02 μM with 30 μM Ran. Ran reduces P(o) of RyR2, desensitizes Ca(2+)-dependent RyR2 activation, and inhibits Ca(i) oscillations, which represents a novel mechanism for its suppression of EADs and TdP.

Sex-differences in sodium/calcium exchange expression is a determinant of the arrhythmia phenotype in pre-pubertal rabbit hearts with Long QT type 2

Background Adult females are more prone to Torsade de Pointes (TdP) than males in long QT type 2 (LQT2) elicited by inhibiting the K + current, I Kr. Sex-differences in the propensity to early afterdepolarizations (EADs) and TdP are reversed in adolescence and correlate with higher levels of L-type Ca 2+ channels and current, I Ca,L at the base of the epicardium in adolescent male rabbit hearts, even though female hearts have longer action potential (AP) durations. Higher Ca 2+-influx via I Ca,L should be balanced by higher Ca 2+-efflux. Objectives Sex-differences in Na +-Ca 2+ exchanger and its current, I NCX are investigated in adolescent rabbit hearts to determine if they parallel sex-differences in I Ca,L. Methods and Results Optical APs and intracellular Ca 2+ (Ca i) were mapped in pre-pubertal Langendorff rabbit hearts. E4031 (0.5μM) blocked I Kr, elicited EADs and TdP in male (n=10) but not female (n=18) hearts. KB-R4379 (I NCX inhibitor, 1μM) reversed (n=5/5) or protected from EADs if perfused before E4031 (n=6/6). I NCX density was 20-30% greater in base compared to apex myocytes of pre-pubertal male (p<0.02) or to apex and base myocytes of female hearts (p<0.02). In Western blots, NCX was 22% higher at the base of male versus female hearts and 25% higher than the apex (n=4, each gender). Immuno-histochemistry of ventricular sections revealed higher NCX levels in male versus female hearts. Conclusions Sex and regional differences in NCX correlate with L-type Ca 2+ channels and NCX inhibition suppresses EADs and TdP in LQT2. These data provide compelling evidence that sex-differences in Ca 2+ handling determine the arrhythmia phenotype.

Novel Chemical Suppressors of Long QT Syndrome Identified by an In Vivo Functional Screen

Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor-mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.