Targeted activation of human ether-à-go-go-related gene channels rescues electrical instability induced by the R56Q+/- long QT syndrome variant.

AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243.

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.

The Extrapulmonary Effects of Cystic Fibrosis Transmembrane Conductance Regulator Modulators in Cystic Fibrosis.

The effects of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators on lung function, pulmonary exacerbations, and quality of life have been well documented. However, CF is a multiorgan disease, and therefore an evidence base is emerging on the systemic effects of CFTR modulators beyond the pulmonary system. This is of great clinical importance, as many of these studies provide proof of concept that CFTR modulators might be used one day to prevent or treat extrapulmonary manifestations stemming from CFTR dysfunction. In this concise review of the literature, we summarize the results of key publications that have evaluated the effects of CFTR modulators on weight and growth, pancreatic function, the gastrointestinal and hepatobiliary systems, sinus disease, bone disease, exercise tolerance, fertility, mental health, and immunity. Although many of these studies have reported beneficial extrapulmonary effects related to the use of ivacaftor (IVA) in patients with CF with at least one gating mutation, most of the evidence is low or very low quality, given the limited number of patients evaluated and the lack of control groups. Based on an even smaller number of studies evaluating the extrapulmonary effects of lumacaftor-IVA, the benefits are less clear. Although limited, these studies may provide the basis for future clinical trials to evaluate CFTR modulators on the extrapulmonary manifestations of CF.

Functional characterization of a novel hERG variant in a family with recurrent sudden infant death syndrome: Retracting a genetic diagnosis.

Long QT syndrome (LQTS) is the most common cardiac ion channelopathy and has been found to be responsible for approximately 10% of sudden infant death syndrome (SIDS) cases. Despite increasing use of broad panels and now whole exome sequencing (WES) in the investigation of SIDS, the probability of identifying a pathogenic mutation in a SIDS victim is low. We report a family-based study who are afflicted by recurrent SIDS in which several members harbor a variant, p.Pro963Thr, in the C-terminal region of the human-ether-a-go-go (hERG) gene, published to be responsible for cases of LQTS type 2. Functional characterization was undertaken due to the variable phenotype in carriers, the discrepancy with published cases, and the importance of identifying a cause for recurrent deaths in a single family. Studies of the mutated ion channel in in vitro heterologous expression systems revealed that the mutation has no detectable impact on membrane surface expression, biophysical gating properties such as activation, deactivation and inactivation, or the amplitude of the protective current conducted by hERG channels during early repolarization. These observations suggest that the p.Pro963Thr mutation is not a monogenic disease-causing LQTS mutation despite evidence of co-segregation in two siblings affected by SIDS. Our findings demonstrate some of the potential pitfalls in post-mortem molecular testing and the importance of functional testing of gene variants in determining disease-causation, especially where the impacts of cascade screening can affect multiple generations.

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker.

Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.