ABSTRACT
BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.
ABSTRACT
Background: Long QT syndrome (LQTS) is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant classification is difficult, often owing to lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here, we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. Methods: We quantified cell-surface trafficking of 18,796 variants in KCNH2 using a Multiplexed Assay of Variant Effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping (APC). We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1,458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian LQTS penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. Results: Variant MAVE trafficking scores and APC peak tail currents were highly correlated (Spearman Rank-order ρ = 0.69). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian LQTS penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and adjusted QT interval (QTc); however, MAVE data became non-significant when peak-tail current and penetrance estimates were also available. The area under the ROC for 20-year event outcomes based on patient-specific sex and QTc (AUC 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (AUC 0.86 [0.83-0.89]) or attainable APC peak tail current data (AUC 0.84 [0.81-0.88]). Conclusion: High throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale while LQTS penetrance estimates and APC peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.
ABSTRACT
Long QT syndrome (LQTS), caused by the dysfunction of cardiac ion channels, increases the risk of sudden death in otherwise healthy young people. For many variants in LQTS genes, there is insufficient evidence to make a definitive genetic diagnosis. We have established a robust functional patch-clamp assay to facilitate classification of missense variants in KCNH2, one of the key LQTS genes. A curated set of 30 benign and 30 pathogenic missense variants were used to establish the range of normal and abnormal function. The extent to which variants reduced protein function was quantified using Z scores, the number of standard deviations from the mean of the normalized current density of the set of benign variant controls. A Z score of -2 defined the threshold for abnormal loss of function, which corresponds to 55% wild-type function. More extreme Z scores were observed for variants with a greater loss-of-function effect. We propose that the Z score for each variant can be used to inform the application and weighting of abnormal and normal functional evidence criteria (PS3 and BS3) within the American College of Medical Genetics and Genomics variant classification framework. The validity of this approach was demonstrated using a series of 18 KCNH2 missense variants detected in a childhood onset LQTS cohort, where the level of function assessed using our assay correlated to the Schwartz score (a scoring system used to quantify the probability of a clinical diagnosis of LQTS) and the length of the corrected QT (QTc) interval.
Subject(s)
Long QT Syndrome , Mutation, Missense , Child , Humans , Death, Sudden , ERG1 Potassium Channel/genetics , Heart , Long QT Syndrome/diagnosisABSTRACT
Many classes of drugs can induce fatal cardiac arrhythmias by disrupting the electrophysiology of cardiomyocytes. Safety guidelines thus require all new drugs to be assessed for pro-arrhythmic risk prior to conducting human trials. The standard safety protocols primarily focus on drug blockade of the delayed-rectifier potassium current (IKr). Yet the risk is better assessed using four key ion currents (IKr, ICaL, INaL, IKs). We simulated 100,000 phenotypically diverse cardiomyocytes to identify the underlying relationship between the blockade of those currents and the emergence of ectopic beats in the action potential. We call that relationship the axis of arrhythmia. It serves as a yardstick for quantifying the arrhythmogenic risk of any drug from its profile of multi-channel block alone. We tested it on 109 drugs and found that it predicted the clinical risk labels with an accuracy of 88.1-90.8%. Pharmacologists can use our method to assess the safety of novel drugs without resorting to animal testing or unwieldy computer simulations.
Subject(s)
Arrhythmias, Cardiac , Myocytes, Cardiac , Animals , Humans , Arrhythmias, Cardiac/chemically induced , Action PotentialsABSTRACT
Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.
ABSTRACT
Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.
Subject(s)
Organic Cation Transport Proteins , Organic Cation Transporter 1 , Humans , Organic Cation Transporter 1/genetics , Organic Cation Transporter 1/chemistry , Organic Cation Transporter 1/metabolism , Organic Cation Transport Proteins/chemistry , Biological Transport , Organic Cation Transporter 2/metabolismABSTRACT
BACKGROUND: Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells. METHODS: A novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing. RESULTS: Application of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active Ikach currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s-1, and form rotor style arrhythmia in response to burst pacing. CONCLUSIONS: This method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.
Subject(s)
Atrial Fibrillation , Induced Pluripotent Stem Cells , Animals , Female , Male , Humans , Induced Pluripotent Stem Cells/metabolism , Atrial Fibrillation/metabolism , Myocytes, Cardiac/metabolism , Carbachol/metabolism , Carbachol/pharmacology , Cell Differentiation , Action Potentials/physiology , Tretinoin/pharmacologyABSTRACT
BACKGROUND: Drug-induced or acquired long QT syndrome occurs as a result of the unintended disruption of cardiac repolarization due to drugs that block cardiac ion channels. These side effects have been responsible for the withdrawal of a range of drugs from market and are a common reason for termination of the development of new drugs in the preclinical stage. Existing approaches to risk prediction are expensive and overly sensitive meaning that recently there have been renewed efforts, largely driven by the comprehensive proarrhythmic assay initiative, to develop more accurate methods for allocation of proarrhythmic risk. METHODS: In this study, we aimed to quantify changes in the morphology of the repolarization phase of the cardiac action potential as an indicator of proarrhythmia, supposing that these shape changes might precede the emergence of ectopic depolarizations that trigger arrhythmia. To do this, we describe a new method of quantifying action potential morphology by measuring the radius of curvature of the repolarization phase both in simulated action potentials, as well as in action potentials measured from induced pluripotent stem cell-derived cardiomyocytes. Features derived from the curvature signal were used as inputs for logistic regressions to predict proarrhythmic risk. RESULTS: Optimal risk classifiers based on morphology were able to correctly classify risk to drugs in the comprehensive proarrhythmic assay initiative panels with very high accuracy (0.9375) and outperformed conventional metrics based on action potential duration at 90% repolarization, triangulation, and charge movement (qNet). CONCLUSIONS: Analysis of action potential morphology in response to proarrhythmic drugs improves prediction of torsadogenic risk. Furthermore, morphology metrics can be measured directly from the action potential, potentially eliminating the burden of undertaking complex screens of potency and drug-binding kinetics against multiple cardiac ion channels. As such, this method has the potential to improve and streamline regulatory assessment of proarrhythmia in preclinical drug development.
Subject(s)
Action Potentials , Arrhythmias, Cardiac , Humans , Arrhythmias, Cardiac/chemically induced , Electrocardiography , Myocytes, Cardiac , Torsades de Pointes/chemically inducedABSTRACT
BACKGROUND: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. METHODS: A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. RESULTS: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. CONCLUSION: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.
Subject(s)
Long QT Syndrome , Humans , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Ether-A-Go-Go Potassium Channels/genetics , HEK293 Cells , Penetrance , Heart , ERG1 Potassium Channel/geneticsABSTRACT
INTRODUCTION: QTc prolongation is key in diagnosing long QT syndrome (LQTS), however 25%-50% with congenital LQTS (cLQTS) demonstrate a normal resting QTc. T wave morphology (TWM) can distinguish cLQTS subtypes but its role in acquired LQTS (aLQTS) is unclear. METHODS: Electronic databases were searched using the terms "LQTS," "long QT syndrome," "QTc prolongation," "prolonged QT," and "T wave," "T wave morphology," "T wave pattern," "T wave biomarkers." Whole text articles assessing TWM, independent of QTc, were included. RESULTS: Seventeen studies met criteria. TWM measurements included T-wave amplitude, duration, magnitude, Tpeak-Tend, QTpeak, left and right slope, center of gravity (COG), sigmoidal and polynomial classifiers, repolarizing integral, morphology combination score (MCS) and principal component analysis (PCA); and vectorcardiographic biomarkers. cLQTS were distinguished from controls by sigmoidal and polynomial classifiers, MCS, QTpeak, Tpeak-Tend, left slope; and COG x axis. MCS detected aLQTS more significantly than QTc. Flatness, asymmetry and notching, J-Tpeak; and Tpeak-Tend correlated with QTc in aLQTS. Multichannel block in aLQTS was identified by early repolarization (ERD30% ) and late repolarization (LRD30% ), with ERD reflecting hERG-specific blockade. Cardiac events were predicted in cLQTS by T wave flatness, notching, and inversion in leads II and V5 , left slope in lead V6 ; and COG last 25% in lead I. T wave right slope in lead I and T-roundness achieved this in aLQTS. CONCLUSION: Numerous TWM biomarkers which supplement QTc assessment were identified. Their diagnostic capabilities include differentiation of genotypes, identification of concealed LQTS, differentiating aLQTS from cLQTS; and determining multichannel versus hERG channel blockade.
Subject(s)
Electrocardiography , Long QT Syndrome , Humans , Long QT Syndrome/genetics , Genotype , BiomarkersABSTRACT
Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.
ABSTRACT
Despite significant advances in interventional and therapeutic approaches, cardiovascular disease (CVD) remains the leading cause of death and mortality. To lower this health burden, cardiovascular discovery scientists need to play an integral part in the solution. Successful clinical translation is achieved when built upon a strong foundational understanding of the disease mechanisms involved. Changes in the Australian funding landscape, to place greater emphasis on translation, however, have increased job insecurity for discovery science researchers and especially early-mid career researchers. To highlight the importance of discovery science in cardiovascular research, this review compiles six science stories in which fundamental discoveries, often involving Australian researchers, has led to or is advancing to clinical translation. These stories demonstrate the importance of the role of discovery scientists and the need for their work to be prioritised now and in the future. Australia needs to keep discovery scientists supported and fully engaged within the broader cardiovascular research ecosystem so they can help realise the next game-changing therapy or diagnostic approach that diminishes the burden of CVD on society.
Subject(s)
Cardiovascular Diseases , Ecosystem , Australia/epidemiology , Cardiovascular Diseases/therapy , Humans , Research PersonnelABSTRACT
The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.
Subject(s)
Ether-A-Go-Go Potassium Channels , Terfenadine , Bepridil , Cisapride/pharmacology , Kinetics , Terfenadine/pharmacology , Verapamil/pharmacologyABSTRACT
Many genes, including KCNH2, contain "hotspot" domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this "hotspot" domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.
Subject(s)
ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Long QT Syndrome , Alleles , Death, Sudden, Cardiac , ERG1 Potassium Channel/genetics , ERG1 Potassium Channel/metabolism , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Protein Transport/geneticsABSTRACT
Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.
Subject(s)
Long QT Syndrome , ERG1 Potassium Channel/genetics , HEK293 Cells , Humans , Long QT Syndrome/diagnosis , Long QT Syndrome/genetics , Mutation, Missense/geneticsABSTRACT
BACKGROUND AND PURPOSE: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat coronavirus disease 2019 (COVID-19). While concern already existed around their proarrhythmic potential, there are little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles. EXPERIMENTAL APPROACH: Potency of human ether-à-go-go related gene (hERG) block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals. KEY RESULTS: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47 ± 0.07 and 3.78 ± 0.17 µM, respectively, indicating proarrhythmic risk at concentrations effective against severe acute respiratory syndrome-coronovirus-2 (SARS-CoV-2) in vitro. Hypokalaemia and hypermagnesaemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure and identified specific genetic backgrounds associated with emergence of arrhythmia. CONCLUSION AND IMPLICATIONS: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations, can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation.
Subject(s)
Acidosis , COVID-19 Drug Treatment , Hypokalemia , Induced Pluripotent Stem Cells , Acidosis/chemically induced , Acidosis/drug therapy , Azithromycin/adverse effects , Chloroquine/adverse effects , Female , Humans , Hydroxychloroquine/adverse effects , Hypokalemia/chemically induced , SARS-CoV-2ABSTRACT
BACKGROUND: Variants in the SCN5A gene, that encodes the cardiac sodium channel, Nav1.5, are associated with a highly arrhythmogenic form of dilated cardiomyopathy (DCM). Our aim was to review the phenotypes, natural history, functional effects, and treatment outcomes of DCM-associated rare SCN5A variants. METHODS: A systematic review of reported DCM-associated rare SCN5A variants was undertaken using PubMed and Embase. RESULTS: Eighteen SCN5A rare variants in 29 families with DCM (173 affected individuals) were identified. Eleven variants had undergone experimental evaluation, with 7 of these resulting in increased sustained current flow during the action potential (eg, increased window current) and at resting membrane potentials (eg, creation of a new gating pore current). These variants were located in transmembrane voltage-sensing domains and had a consistent phenotype characterized by frequent multifocal narrow and broad complex ventricular premature beats (VPB; 72% of affected relatives), ventricular arrhythmias (33%), atrial arrhythmias (32%), sudden cardiac death (13%), and DCM (56%). This VPB-predominant phenotype was not seen with 1 variant that increased late sodium current, or with variants that reduced peak current density or had mixed effects. In the latter groups, affected individuals mainly showed sinus node dysfunction, conduction defects, and atrial arrhythmias, with infrequent VPB and ventricular arrhythmias. DCM did not occur in the absence of arrhythmias for any variant. Twelve studies (23 total patients) reported treatment success in the VPB-predominant cardiomyopathy using sodium channel-blocking drug therapy. CONCLUSIONS: SCN5A variants can present with a diverse spectrum of primary arrhythmic features. A majority of DCM-associated variants cause a multifocal VPB-predominant cardiomyopathy that is reversible with sodium channel blocking drug therapy. Early recognition of the distinctive phenotype and prompt genetic testing to identify variant carriers are needed. Our findings have implications for interpretation and management of SCN5A variants found in DCM patients with and without arrhythmias.