ORCID Profile
0000-0002-3859-3716
Current Organisations
Victor Chang Cardiac Research Institute
,
University of New South Wales
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biophysics | Biochemistry and Cell Biology | Receptors and Membrane Biology | Other Physical Sciences | Membrane Biology | Applied Mathematics | Structural Biology (incl. Macromolecular Modelling) | Instruments And Techniques | Biological Mathematics | Pharmaceutical Sciences | Human Biophysics | Cell Physiology | Biological Physics | Biological Mathematics | Protein Trafficking | Basic Pharmacology | Analytical Biochemistry | Mathematical Software
ANZSRC Socio-Economic Objective (SEO)
Biological sciences | Expanding Knowledge in the Biological Sciences | Chemical sciences | Physical sciences | Cancer and related disorders | Expanding Knowledge in the Medical and Health Sciences | Infectious diseases | Human Diagnostics | Cardiovascular system and diseases | Human Pharmaceutical Treatments (e.g. Antibiotics) | Cardiovascular System and Diseases | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Physical Sciences | Disease distribution and transmission |
Related Links
Publications
Voltage-Gated Potassium Channels (Kv10–Kv12)☆
Publisher: Elsevier
Date: 2015
Tryptophan scanning mutagenesis of the HERG K+ channel: the S4 domain is loosely packed and likely to be lipid exposed
Publisher: Wiley
Date: 24-11-2005
High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants
Publisher: Elsevier BV
Date: 03-2020
DOI: 10.1016/J.HRTHM.2019.09.020
Abstract: KCNH2 encodes the human ether-à-go-go-related gene potassium channel, which passes the rapid delayed rectifier potassium current. Loss-of-function variants in KCNH2 cause long QT syndrome type 2, which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants, however, are likely to be benign. The purpose of this study was to develop a high-throughput assay for discriminating pathogenic from benign KCNH2 variants. Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines were phenotyped using an automated patch-cl platform and a cell surface enzyme-linked immunosorbent assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines using a bicistronic vector was performed using an automated patch-cl platform. In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured using an enzyme-linked immunosorbent assay can be explained by changes in gating properties of the variant channels. For the 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of the 13 pore-domain variants studied, 11 had a dominant-negative expression defect while the remaining 2 had enhanced inactivation gating, resulting in a dominant-negative phenotype. High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants.
The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating
Publisher: Public Library of Science (PLoS)
Date: 16-02-2012
Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels
Publisher: Elsevier BV
Date: 05-2014
Heritability of ECG Biomarkers in the Netherlands Twin Registry Measured from Holter ECGs
Publisher: Frontiers Media SA
Date: 29-04-2016
Action potential shortening through the putative β4‐adrenoceptor in ferret ventricle: comparison with β1‐ and β2‐adrenoceptor‐mediated effects
Publisher: Wiley
Date: 08-1998
The diagnostic role of T wave morphology biomarkers in congenital and acquired long QT syndrome: A systematic review
Publisher: Wiley
Date: 07-11-2022
DOI: 10.1111/ANEC.13015
Abstract: 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. 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. Seventeen studies met criteria. TWM measurements included T‐wave litude, 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 (ERD 30% ) and late repolarization (LRD 30% ), with ERD reflecting hERG‐specific blockade. Cardiac events were predicted in cLQTS by T wave flatness, notching, and inversion in leads II and V 5 , left slope in lead V 6 and COG last 25% in lead I. T wave right slope in lead I and T‐roundness achieved this in aLQTS. 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.
Effects of premature stimulation on HERG K+ channels
Publisher: Wiley
Date: 15-12-2001
Electrogram prolongation and nifedipine-suppressible ventricular arrhythmias in mice following targeted disruption of KCNE1
Publisher: Wiley
Date: 15-10-2003
Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization
Publisher: Oxford University Press (OUP)
Date: 19-10-2020
DOI: 10.1093/CVR/CVZ247
Abstract: Current treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by Kv11.1 channels. Small molecule activators of Kv11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of Kv11.1 that impairs transition to the inactivated state, to restore function to heterozygous Kv11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. ICA-105574 effectively restored Kv11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective Kv11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The Kv11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. Kv11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the caveat that there may be a risk of overcorrection that could itself be pro-arrhythmic.
The sodium channel -subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart
Publisher: Wiley
Date: 15-12-2001
Slow conduction and spatial dispersion of repolarization are intrinsic properties of cardiomyocyte electrophysiology that contribute to proarrhythmia in an iPSC model of hypertrophic cardiomyopathy
Publisher: Cold Spring Harbor Laboratory
Date: 23-07-2023
DOI: 10.1101/2023.07.20.549952
Abstract: Hypertrophic cardiomyopathy (HCM) is an inherited heart muscle disease characterised by left ventricular wall thickening, cardiomyocyte disarray, and fibrosis, and is associated with arrhythmias, heart failure and sudden death. However, it is unclear to what extent the electrophysiological disturbances that lead to sudden death occur secondary to the structural changes in the myocardium, or as a result of intrinsic properties of the HCM cardiomyocyte. In this study, we used an induced pluripotent stem cell model of the Arg403Gln variant in myosin heavy chain 7 ( MYH7 ) to study ‘tissue level’ electrophysiological properties of HCM cardiomyocytes. For the first time, we show significant slowing of conduction velocity and an increase in local spatial dispersion of repolarisation - both well-established substrates for arrhythmia - in monolayers of HCM cardiomyocytes. Analysis of rhythmonome protein expression in R403Q cardiomyocytes revealed dramatically reduced connexin-43, sodium channels, and inward rectifier channels – a three-way hit that combines to reduce electrotonic coupling between HCM cardiomyocytes and slow cardiac conduction. Our data therefore represent a novel, biophysical basis for arrhythmia in HCM, that is intrinsic to cardiomyocyte electrophysiology. Later in the progression of the disease, these proarrhythmic electrical phenotypes may be accentuated by fibrosis and myocyte disarray to contribute to sudden death in HCM patients.
Co-expression of calcium channels and delayed rectifier potassium channels protects the heart from proarrhythmic events
Publisher: Cold Spring Harbor Laboratory
Date: 05-06-2019
DOI: 10.1101/659821
Abstract: Cardiac electrical activity is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. Yet, there is considerable variability in cardiac ion channel expression levels both within and between subjects. In this study we tested the hypothesis that variations in ion channel expression between in iduals are not random but rather there are modules of co-expressed genes and that these modules make electrical signaling in the heart more robust. Meta-analysis of 3653 public RNA-Seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, I CaL ) and KCNH2 (rapid delayed rectifier K + current, I Kr ), which was verified in mRNA extracted from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). In silico modeling, validated with functional measurements in hiPSC-CM, indicates that the co-expression of CACNA1C and KCNH2 limits the variability in action potential duration and reduces susceptibility to early afterdepolarizations, a surrogate marker for pro-arrhythmia. Coexpressed levels of potassium and calcium ion channel genes in the heart encode more robust cardiac electrophysiology and provide insights into genetic basis of arrhythmic risk
General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy
Publisher: Wiley
Date: 10-11-2019
DOI: 10.1002/CPT.1647
HeterozygousKCNH2variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
Publisher: Cold Spring Harbor Laboratory
Date: 02-02-2021
DOI: 10.1101/2021.02.02.427891
Abstract: KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-cl electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.
Oxidative stress fine-tunes the dance of hERG K+ channels
Publisher: Wiley
Date: 13-08-2010
Atrial Fibrillation—A New Cardiac Channelopathy
Publisher: Elsevier BV
Date: 10-2007
DOI: 10.1016/J.HLC.2007.07.003
Abstract: Atrial fibrillation (AF) is a common complication of a wide range of cardiac and systemic diseases and is regarded generally as a sporadic, acquired disorder. Familial clustering of AF has been reported but definitive links of genetic factors with AF pathogenesis have been lacking. Genome-wide linkage studies and the discovery of mutations in families with AF have provided compelling evidence that genetic factors can have a role in the development of AF. Although relatively few disease genes have been identified, current data indicate that inherited defects in cardiac ion channel genes can predispose to AF by altering ion channel activation and atrial conduction properties. Mutations in the reported disease genes account for only a minority of all familial AF cases and further gene discovery studies are required. Characterisation of the genetic variants that cause AF in families provides a framework for elucidation of key disease pathways that underlie the more commonly-occurring complex forms of AF. A better understanding of the molecular and electrophysiological defects that promote AF in families and in the general population will facilitate new approaches to the diagnosis, prevention and treatment of AF.
The assimilation of tri- and tetrapeptides by human erythrocytes
Publisher: Elsevier BV
Date: 07-1985
DOI: 10.1016/0167-4889(85)90118-1
Abstract: Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F. and Kuchel, P.W. (1985) Biochem. J. 227, 833-842) but that the transmembrane flux rates for tetrapeptides are considerably less. 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo. Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave Km = 2.11 +/- 0.08 and 23.4 +/- 0.9 mmol/l and Vmax = 307 +/- 3 and 905 +/- 22 mmol/h per 1 packed cells, respectively. In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide. Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport the values were Km = 11.6 +/- 1.1 and 56 +/- 18 mmol/l and Vmax = 12.9 +/- 3.0 and 36.4 +/- 3.2 mmol/h per 1 packed cells, respectively, for the previously mentioned peptides. The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides. The above mentioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo.
In silico assessment of kinetics and state dependent binding properties of drugs causing acquired LQTS
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.PBIOMOLBIO.2015.12.005
Abstract: The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development.
Is medroxyprogesterone safe in women with long QT syndrome?
Publisher: Elsevier BV
Date: 07-2012
The Pore Domain Outer Helix Contributes to Both Activation and Inactivation of the hERG K+ Channel
Publisher: Elsevier BV
Date: 2009
Cell swelling and ion transport pathways in cardiac myocytes
Publisher: Oxford University Press (OUP)
Date: 07-1996
Ion channels — a plethora of pharmaceutical targets
Publisher: Elsevier BV
Date: 11-2000
Getting to the heart of hERG K+ channel gating
Publisher: Wiley
Date: 15-06-2015
DOI: 10.1113/JP270095
Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics
Publisher: Cold Spring Harbor Laboratory
Date: 13-03-2017
DOI: 10.1101/100677
Abstract: Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics — the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an ex le, with the aim of generating a quantitatively predictive representation of the ion current. We fit a mathematical model to currents evoked by a novel 8 second sinusoidal voltage cl in CHO cells over-expressing hERG1a. The model is then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage cl s, and also a novel voltage cl comprised of a collection of physiologically-relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches. Techniques for Physiology 1 Ion current kinetics are commonly represented by current-voltage relationships, time-constant voltage relationships, and subsequently mathematical models fitted to these. These experiments take substantial time which means they are rarely performed in the same cell. Rather than traditional square-wave voltage cl s, we fit a model to the current evoked by a novel sum-of-sinusoids voltage cl that is only 8 seconds long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically-relevant series of action potential cl s is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to in idual cells, allowing us to examine cell-cell variability in current kinetics for the first time.
'Shooting gallery' for membrane proteins provides new insights into complexities of their function and structural dynamics
Publisher: Wiley
Date: 15-01-2015
Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels
Publisher: Rockefeller University Press
Date: 13-08-2012
Abstract: Human ether-a-go-go–related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an hipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-cl fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.
Differential Response to Risperidone in Schizophrenia Patients byKCNH2Genotype and Drug Metabolizer Status
Publisher: American Psychiatric Association Publishing
Date: 2016
DOI: 10.1176/APPI.AJP.2015.14050653
Abstract: Antipsychotic drugs target dopamine and serotonin receptors as well as Kv11.1 potassium channels encoded by KCNH2. Variable patient responses and a wide range of side effects, however, limit their efficacy. Slow metabolizer status and gene variants in KCNH2 associated with increased expression of Kv11.1-3.1, an alternatively spliced isoform of Kv11.1, are correlated with improved responses to antipsychotic medications. Here, the authors test the hypothesis that these effects may be influenced by differential drug binding to Kv11.1 channel isoforms. Drug block of Kv11.1 isoforms was tested in cellular electrophysiology assays. The effects of drug metabolism and KCNH2 genotypes on clinical responses were assessed in patients enrolled in the multicenter Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE). Risperidone caused greater in vitro block of the alternatively spliced Kv11.1-3.1 isoform than full-length Kv11.1-1A channels, whereas its metabolite paliperidone and other atypical antipsychotics have similar potencies for the two isoforms. In the CATIE study (N=362), patients with genotypes associated with increased Kv11.1-3.1 expression (N=52) showed a better treatment response to risperidone compared with other drugs, but this association was dependent on metabolism status. Patients with KCNH2 risk genotypes and slow metabolizer status (approximately 7% of patients) showed marked improvement in symptoms when treated with risperidone compared with patients with fast metabolizer status or without the KCNH2 risk genotypes. These data support the hypothesis that Kv11.1 channels play a role in the therapeutic action of antipsychotic drugs, particularly risperidone, and further highlight the promise of optimizing response with genotype-guided therapy for schizophrenia patients.
Long QT syndrome type 1 in an Australian indigenous patient
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2020
Associations between greenspace and mortality vary across contexts of community change: a longitudinal ecological study
Publisher: BMJ
Date: 04-03-2020
Abstract: Concerns about loss of greenspace with urbanisation motivate much research on nature and health however, contingency of greenspace-health associations on the character of community change remains understudied. With aggregate data from governmental sources for 1432 Swedish parishes, we used negative binomial regression to estimate incidence rate ratios (IRRs) for all-cause and cardiovascular disease (CVD) mortality during 2000–2008 in relation to percentage area (in 2000) of urban residential greenspace, urban parks and rural greenspace, looking across parishes with decrease, stability or increase in population density. We also assessed interactions between land use and population change. Parishes with 1 decile increase in population density had lower incidence of all-cause (IRR=0.91, 95% CI 0.87 to 0.95) and CVD mortality (IRR=0.89, 95% CI 0.84 to 0.94) compared with parishes with stable populations. In stable parishes, all-cause mortality was lower with higher percentages of urban green (IRR=0.998, 95% CI 0.996 to 1.000) and rural green land uses (IRR=0.997, 95% CI 0.996 to 0.999). These results were inverted in densifying parishes higher all-cause mortality attended higher initial percentages of urban (IRR=1.081, 95% CI 1.037 to 1.127) and rural greenspace (IRR=1.042, 95% CI 1.007 to 1.079) as measured in 2000. Similar associations held for CVD mortality. More greenspace was associated with lower all-cause and CVD mortality in communities with relatively stable populations. In densifying communities, population growth per se may reduce mortality, but it may also entail harm through reductions in amount per capita and/or quality of greenspace.
High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KV11.1
Publisher: Elsevier BV
Date: 12-2020
Developmental regulation of the gradient of cftr expression in the rabbit heart
Publisher: Elsevier BV
Date: 06-2000
DOI: 10.1016/S0925-4773(00)00312-9
Abstract: Gradients of ion channels across the left ventricular free wall of the heart have been found for a number of repolarizing ion channels. Amongst these are the cAMP-activated chloride channels encoded by cftr. In this report, we show that the epicardial (higher) to endocardial (lower) gradient of cftr mRNA found in adult rabbit hearts is not present in embryonic hearts. The gradient starts to develop shortly after birth, and over a period of 5-6 weeks increases to the levels found in the adult. This is the first report of the developmental regulation of any cardiac ion channel mRNA gradient.
Measuring kinetics and potency of hERG block for CiPA
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.VASCN.2017.02.017
Abstract: The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol. Data were acquired using a 'step depolarisation' protocol using manual patch-cl at ambient temperature. Potency, kinetics and trapping characteristics of hERG block for the CiPA training panel of twelve drugs were measured. Timecourse of block and trapping characteristics could be reliably measured if the time constant for onset of block was between ~500ms and ~15s. Seven drugs, however had time courses of block faster than this cut-off. Here we describe the implementation of the standardized protocol for measurement of kinetics and potency of hERG block for CiPA. The results highlight the challenges in identifying a single protocol to measure hERG block over a range of kinetics. The dataset from this study is being used by the In Silico Working Group to develop models of drug binding for risk prediction and is freely available as a 'gold standard' ambient temperature dataset to evaluate variability across high throughput platforms.
BIOPHYSCHEM2011: A joint meeting of the Australian society for biophysics and the RACI physical chemistry division
Publisher: CSIRO Publishing
Date: 2012
DOI: 10.1071/CH12213
Genes and Atrial Fibrillation
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 14-08-2007
Recent advances in understanding and prevention of sudden cardiac death
Publisher: F1000 Research Ltd
Date: 31-08-2017
DOI: 10.12688/F1000RESEARCH.11855.1
Abstract: There have been tremendous advances in the diagnosis and treatment of heart disease over the last 50 years. Nevertheless, it remains the number one cause of death. About half of heart-related deaths occur suddenly, and in about half of these cases the person was unaware that they had underlying heart disease. Genetic heart disease accounts for only approximately 2% of sudden cardiac deaths, but as it typically occurs in younger people it has been a particular focus of activity in our quest to not only understand the underlying mechanisms of cardiac arrhythmogenesis but also develop better strategies for earlier detection and prevention. In this brief review, we will highlight trends in the recent literature focused on sudden cardiac death in genetic heart diseases and how these studies are contributing to a broader understanding of sudden death in the community.
Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance
Publisher: Cold Spring Harbor Laboratory
Date: 05-08-2022
DOI: 10.1101/2022.08.05.502917
Abstract: Variants in KCNH2 , encoding the hERG channel which is responsible for the rapid component of the cardiac delayed rectifier K + current (I Kr ), 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. A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed by manual whole-cell patch-cl using 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). A calibrated automated patch-cl assay using Flp-In HEK293 was used to follow up on the functional data. 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-cl 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 to KCNH2 -WT. Assessment of KCNH2-p.S906L-Hz, by calibrated automatic patch-cl showed a reduction in current density by 35.6%. 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.
Drug Binding to the Inactivated State Is Necessary but Not Sufficient for High-Affinity Binding to Human Ether-à-go-go-Related Gene Channels
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 13-08-2008
Abstract: Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an ex le of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.
Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin
Publisher: Wiley
Date: 06-03-2003
DOI: 10.1016/S0014-5793(03)00216-3
Abstract: The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an alpha-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin.
Temperature effects on kinetics of KV 11.1 drug block have important consequences for in silico proarrhythmic risk prediction
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 12-05-2016
Abstract: Drug block of voltage-gated potassium channel subtype 11.1 human ether-a-go-go related gene (Kv11.1) (hERG) channels, encoded by the KCNH2 gene, is associated with reduced repolarization of the cardiac action potential and is the predominant cause of acquired long QT syndrome that can lead to fatal cardiac arrhythmias. Current safety guidelines require that potency of KV11.1 block is assessed in the preclinical phase of drug development. However, not all drugs that block KV11.1 are proarrhythmic, meaning that screening on the basis of equilibrium measures of block can result in high attrition of potentially low-risk drugs. The basis of the next generation of drug-screening approaches is set to be in silico risk prediction, informed by in vitro mechanistic descriptions of drug binding, including measures of the kinetics of block. A critical issue in this regard is characterizing the temperature dependence of drug binding. Specifically, it is important to address whether kinetics relevant to physiologic temperatures can be inferred or extrapolated from in vitro data gathered at room temperature in high-throughout systems. Here we present the first complete study of the temperature-dependent kinetics of block and unblock of a proarrhythmic drug, cisapride, to KV11.1. Our data highlight a complexity to binding that manifests at higher temperatures and can be explained by accumulation of an intermediate, non-blocking encounter-complex. These results suggest that for cisapride, physiologically relevant kinetic parameters cannot be simply extrapolated from those measured at lower temperatures rather, data gathered at physiologic temperatures should be used to constrain in silico models that may be used for proarrhythmic risk prediction.
An improved curvilinear gradient method for parameter optimization in complex biological models
Publisher: Springer Science and Business Media LLC
Date: 30-07-2011
DOI: 10.1007/S11517-010-0667-1
Abstract: Mathematical modeling is an often used approach in biological science which, given some understanding of a system, is employed as a means of predicting future behavior and quantitative hypothesis testing. However, as our understanding of processes becomes more in depth, the models we use to describe them become correspondingly more complex. There is a paucity of effective methods available for s ling the vast objective surfaces associated with complex multiparameter models while at the same time maintaining the accuracy needed for local evaluation of minima-all in a practical time period. We have developed a series of modifications to the curvilinear gradient method for parameter optimization. We demonstrate the power and efficiency of our routine through fitting of a 22 parameter Markov state model to an electrophysiological recording of a cardiac ion channel. Our method efficiently and accurately locates parameter minima which would not be easily identified using the currently available means. While the computational overhead involved in implementing the curvilinear gradient method may have contributed to resistance to adopting this technique, the performance improvements allowed by our modifications make this an extremely valuable tool in development of models of complex biological systems.
Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy.
Publisher: Elsevier BV
Date: 05-1986
DOI: 10.1016/0003-2697(86)90221-6
Abstract: The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque s les. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.
A calibrated functional patch clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome
Publisher: Cold Spring Harbor Laboratory
Date: 15-12-2021
DOI: 10.1101/2021.12.13.472492
Abstract: Modern sequencing technologies have revolutionised our detection of gene variants. In most genes, including KCNH2 , the majority of missense variants are currently classified as variants of uncertain significance (VUS). The aim of this study is to investigate the utility of an automated patch-cl assay for aiding clinical variant classification in the KCNH2 gene. The assay was designed according to recommendations of the ClinGen sequence variant interpretation framework. Thirty-one control variants of known clinical significance (17 pathogenic/likely pathogenic, 14 benign/likely benign) were heterozygously expressed in Flp-In HEK293 cells. Variants were analysed for effects on current density and channel gating. A panel of 44 VUS was then assessed for reclassification. All 17 pathogenic variant controls had reduced current density and 13/14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying moderate or supporting evidence strength for variant classification. Inclusion of KCNH2 functional assay evidence enabled us to reclassify 6 out of 44 VUS as likely pathogenic. The high-throughput patch cl assay can provide moderate strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value proposition for developing automated patch cl assays for other ion channel genes.
Enkephalin degradation by human erythrocytes and hemolysates studied using 1H NMR spectroscopy
Publisher: Elsevier BV
Date: 11-1985
DOI: 10.1016/0003-9861(85)90238-3
Abstract: High resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to monitor the degradation of leucine-enkephalin, and peptide fragments of it, by human erythrocytes and hemolysates. We showed that leucine-enkephalin is rapidly degraded by the cytosolic peptidases of the human erythrocyte, and we have elucidated the most probable pathway of degradation. Computer simulations of the proposed pathway, using a model incorporating the experimentally derived steady-state kinetic parameters obtained for the in idual enzyme steps, showed close agreement with the experimental results. From a methodological perspective, the work demonstrates the value of 1H spin-echo NMR spectroscopy for rapidly elucidating, both qualitatively and quantitatively, an entire peptide-degradation pathway as it operates in situ.
Structural Basis for Rapid Voltage Dependent Inactivation of HERG Potassium Channels
Publisher: Research Square Platform LLC
Date: 08-12-2021
DOI: 10.21203/RS.3.RS-1105661/V1
Abstract: The exquisite fine tuning of biological electrical signalling is mediated by variations in the rates of opening and closing of different ion channels(1). In addition to open and closed conformations, ion channels can exist in an inactivated state, which prevents conduction in the presence of a prolonged activating stimulus(2). Human ether-a-go-go related gene (HERG) K+ channels undergo uniquely rapid and voltage dependent inactivation(3-5), which confers upon them a critical role in protecting against cardiac arrhythmias and sudden death(6). Previous structural studies have captured only the open state of the HERG channel(7,8). Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both the conductive state and the elusive inactivated state of HERG. We show that hERG inactivation is facilitated by two competing networks of hydrogen bonds behind the selectivity filter that enable rapid and voltage dependent flipping of the valine carbonyls in the centre of the selectivity filter. Our data also explains how changes in extracellular K+ affects the distribution between conductive and inactivated states(9,10) and thereby explains why hypokalaemia reduces HERG channel activity thereby increasing the risk of cardiac arrhythmias(11).
The HERG K + channel: progress in understanding the molecular basis of its unusual gating kinetics
Publisher: Springer Science and Business Media LLC
Date: 04-2004
DOI: 10.1007/S00249-003-0338-3
Abstract: The HERG K+ channel has very unusual kinetic behaviour that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarisation as well as in preventing lethal ventricular arrhythmias. Extensive mutagenesis of the HERG K+ channel has allowed identification of which regions of the channel are important for the unusual kinetic behaviour of the channel. Furthermore, structural studies on scorpion toxins that potently inhibit HERG are beginning to provide clues as to the structural differences between HERG and other voltage-gated K+ channels.
Action Potential Morphology Accurately Predicts Proarrhythmic Risk for Drugs With Potential to Prolong Cardiac Repolarization
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2023
DOI: 10.1161/CIRCEP.122.011574
Abstract: 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. 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. 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). 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.
Development of a robust induced pluripotent stem cell atrial cardiomyocyte differentiation protocol to model atrial arrhythmia
Publisher: Springer Science and Business Media LLC
Date: 27-07-2023
DOI: 10.1186/S13287-023-03405-5
Abstract: 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. 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. 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 I kach 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. 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.
Trafficking defects in PAS domain mutant Kv11.1 channels: roles of reduced domain stability and altered domain–domain interactions
Publisher: Portland Press Ltd.
Date: 26-07-2013
DOI: 10.1042/BJ20130328
Abstract: Loss of Kv11.1 potassium channel function is the underlying cause of pathology in long-QT syndrome type 2, one of the commonest causes of sudden cardiac death in the young. Previous studies have identified the cytosolic PAS (Per/Arnt/Sim) domain as a hotspot for mutations that cause Kv11.1 trafficking defects. To investigate the underlying basis of this observation, we have quantified the effect of mutants on domain folding as well as interactions between the PAS domain and the remainder of the channel. Apart from R56Q, all mutants impaired the thermostability of the isolated PAS domain. Six mutants, located in the vicinity of a hydrophobic patch on the PAS domain surface, also affected binding of the isolated PAS domain to an N-terminal truncated hERG (human ether-a-go-go-related gene) channel. Conversely, four other surface mutants (C64Y, T65P, A78P and I96T) and one buried mutant (L86R) did not prevent the isolated PAS domain binding to the truncated channels. Our results highlight a critical role for interactions between the PAS domain and the remainder of the channel in the hERG assembly and that mutants that affect PAS domain interactions with the remainder of the channel have a more severe trafficking defect than that caused by domain unfolding alone.
Arrhythmogenesis as the failure of repolarization
Publisher: Cold Spring Harbor Laboratory
Date: 03-07-2020
DOI: 10.1101/2020.07.02.185694
Abstract: Contemporary theories of cardiac fibrillation typically rely on the emergence of rotors to explain the transition from regular sinus rhythm to disordered electrophysiological activity. How those rotors spontaneously arise in the absence of re-entrant anatomical circuits is not fully understood. Here we propose a novel mechanism where arrhythmias are initiated by cardiac cells that fail to repolarize following a normal heartbeat. Those cells subsequently act as a focal ectopic source that drive the ensuing fibrillation. We used a simple computational model to investigate the impact of such cells in both homogeneous and heterogeneous excitable media. We found that heterogeneous media can tolerate a surprisingly large number of abnormal cells and still support normal rhythmic activity. At a critical limit the medium becomes chronically arrhythmogenic. Numerical analysis revealed that the critical threshold for arrhythmogenesis depends on both the strength of the coupling between cells and the extent to which the abnormal cells resist repolarization. Arrhythmogenesis was also found to emerge first at tissue boundaries where cells naturally have fewer neighbors to influence their behavior. These findings may explain why atrial fibrillation typically originates from the cuff of the pulmonary vein. Cardiac fibrillation is a medical condition where normal heart function is compromised as electrical activity becomes disordered. How fibrillation arises spontaneously is not fully understood. It is generally thought to be triggered by premature depolarization of the cardiac action potential in one or more cells. Those premature beats, known as early-afterdepolarizations, subsequently initiate a self-sustaining rotor in the otherwise normal heart tissue. In this study, we propose an alternative mechanism whereby arrhythmias are initiated by cardiac cells that fail to repolarize of their own accord but still operate normally when embedded in functional heart tissue. We find that such cells can act as focal ectopic sources under appropriate conditions of inter-cellular coupling. Moreover, cells on natural tissue boundaries are more susceptible to arrhythmia because they are coupled to fewer cells. This may explain why the pulmonary vein is often implicated as a source of atrial fibrillation.
Epistatic Effects of Potassium Channel Variation on Cardiac Repolarization and Atrial Fibrillation Risk
Publisher: Elsevier BV
Date: 03-2012
DOI: 10.1016/J.JACC.2011.11.039
Abstract: The aim of this study was to evaluate the role of cardiac K(+) channel gene variants in families with atrial fibrillation (AF). The K(+) channels play a major role in atrial repolarization but single mutations in cardiac K(+) channel genes are infrequently present in AF families. The collective effect of background K(+) channel variants of varying prevalence and effect size on the atrial substrate for AF is largely unexplored. Genes encoding the major cardiac K(+) channels were resequenced in 80 AF probands. Nonsynonymous coding sequence variants identified in AF probands were evaluated in 240 control subjects. Novel variants were characterized using patch-cl techniques and in silico modeling was performed using the Courtemanche atrial cell model. Nineteen nonsynonymous variants in 9 genes were found, including 11 rare variants. Rare variants were more frequent in AF probands (18.8% vs. 4.2%, p < 0.001), and the mean number of variants was greater (0.21 vs. 0.04, p 30 ms) shortening or lengthening of action potential duration as well as increased dispersion of repolarization. Families with AF show an excess of rare functional K(+) channel gene variants of varying phenotypic effect size that may contribute to an atrial arrhythmogenic substrate. Atrial cell modeling is a useful tool to assess epistatic interactions between multiple variants.
Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1
Publisher: Elsevier BV
Date: 06-2007
Molecular basis of slow activation of the human ether-a-go-go related gene potassium channel.
Publisher: Wiley
Date: 07-2004
Arrhythmogenic effects of ultra-long and bistable cardiac action potentials
Publisher: Public Library of Science (PLoS)
Date: 16-02-2021
DOI: 10.1371/JOURNAL.PCBI.1008683
Abstract: Contemporary accounts of the initiation of cardiac arrhythmias typically rely on after-depolarizations as the trigger for reentrant activity. The after-depolarizations are usually triggered by calcium entry or spontaneous release within the cells of the myocardium or the conduction system. Here we propose an alternative mechanism whereby arrhythmias are triggered autonomously by cardiac cells that fail to repolarize after a normal heartbeat. We investigated the proposal by representing the heart as an excitable medium of FitzHugh-Nagumo cells where a proportion of cells were capable of remaining depolarized indefinitely. As such, those cells exhibit bistable membrane dynamics. We found that heterogeneous media can tolerate a surprisingly large number of bistable cells and still support normal rhythmic activity. Yet there is a critical limit beyond which the medium is persistently arrhythmogenic. Numerical analysis revealed that the critical threshold for arrhythmogenesis depends on both the strength of the coupling between cells and the extent to which the abnormal cells resist repolarization. Moreover, arrhythmogenesis was found to emerge preferentially at tissue boundaries where cells naturally have fewer neighbors to influence their behavior. These findings may explain why atrial fibrillation typically originates from tissue boundaries such as the cuff of the pulmonary vein.
The M1P1 Loop of TASK3 K2P Channels Apposes the Selectivity Filter and Influences Channel Function
Publisher: Elsevier BV
Date: 06-2008
A massively parallel assay accurately discriminates between functionally normal and abnormal variants in a hotspot domain of KCNH2
Publisher: Elsevier BV
Date: 07-2022
Sensitivity limits for voltage control of P2Y receptor‐evoked Ca2+ mobilization in the rat megakaryocyte
Publisher: Wiley
Date: 15-02-2004
A calibrated functional patch-clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome
Publisher: Elsevier BV
Date: 07-2022
High throughput measurement of hERG drug block kinetics using the CiPA dynamic protocol.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.VASCN.2022.107192
Abstract: 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-cl . The application of this approach at scale requires the development of a high-throughput, automated patch-cl (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 IC
Substrate specificity of platypus venom L-to-D-peptide isomerase
Publisher: Elsevier BV
Date: 04-2008
The effect of heptanol on the electrical and contractile function of the isolated, perfused rabbit heart
Publisher: Springer Science and Business Media LLC
Date: 2000
An ‘alternans’ way to quantify arrhythmogenic substrates
Publisher: Wiley
Date: 28-04-2016
DOI: 10.1113/JP271838
Genetic Engineering and Cardiac Ion Channels
Publisher: Springer Netherlands
Date: 1999
Pore Helices Play a Dynamic Role as Integrators of Domain Motion during Kv11.1 Channel Inactivation Gating
Publisher: Elsevier BV
Date: 04-2013
Multiscale cardiac modelling reveals the origins of notched T waves in long QT syndrome type 2
Publisher: Springer Science and Business Media LLC
Date: 25-09-2014
DOI: 10.1038/NCOMMS6069
Abstract: The heart rhythm disorder long QT syndrome (LQTS) can result in sudden death in the young or remain asymptomatic into adulthood. The features of the surface electrocardiogram (ECG), a measure of the electrical activity of the heart, can be equally variable in LQTS patients, posing well-described diagnostic dilemmas. Here we report a correlation between QT interval prolongation and T-wave notching in LQTS2 patients and use a novel computational framework to investigate how in idual ionic currents, as well as cellular and tissue level factors, contribute to notched T waves. Furthermore, we show that variable expressivity of ECG features observed in LQTS2 patients can be explained by as little as 20% variation in the levels of ionic conductances that contribute to repolarization reserve. This has significant implications for interpretation of whole-genome sequencing data and underlies the importance of interpreting the entire molecular signature of disease in any given in idual.
Don’t Turn Off the Tap! The Importance of Discovery Science to the Australian Cardiovascular Sector and Improving Clinical Outcomes Into the Future
Publisher: Elsevier BV
Date: 10-2022
DOI: 10.1016/J.HLC.2022.06.669
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.
The S631A Mutation Causes a Mechanistic Switch in the Block of hERG Channels by CnErg1
Publisher: Elsevier BV
Date: 09-2007
HERG K+ channels: friend and foe
Publisher: Elsevier BV
Date: 05-2001
DOI: 10.1016/S0165-6147(00)01662-X
Abstract: The K+ channel encoded by the human ether-à-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.
Extracellular Acidosis Modulates Drug Block of Kv4.3 Currents by Flecainide and Quinidine
Publisher: Wiley
Date: 06-2003
DOI: 10.1046/J.1540-8167.2003.03026.X
Abstract: As a molecular model of the effect of ischemia on drug block of the transient outward potassium current, the effect of acidosis on the blocking properties of flecainide and quinidine on Kv4.3 currents was studied. Kv4.3 channels were stably expressed in Chinese hamster ovary cells. Whole-cell, voltage cl techniques were used to measure the effect of flecainide and quinidine on Kv4.3 currents in solutions of pH 7.4 and 6.0. Extracellular acidosis attenuated flecainide block of Kv4.3 currents, with the IC50 for flecainide (based on current-time integrals) increasing from 7.8 +/- 1.1 microM at pH 7.4 to 125.1 +/- 1.1 microM at pH 6.0. Similar effects were observed for quinidine (IC50 5.2 +/- 1.1 microM at pH 7.4 and 22.1 +/- 1.3 microM at pH 6.0). Following block by either drug, Kv4.3 channels showed a hyperpolarizing shift in the voltage sensitivity of inactivation and a slowing in the time to recover from inactivation/block that was unaffected by acidosis. In contrast, acidosis attenuated the effects on the time course of inactivation and the degree of tonic- and frequency-dependent block for both drugs. Extracellular acidosis significantly decreases the potency of blockade of Kv4.3 by both flecainide and quinidine. This change in potency may be due to allosteric changes in the channel, changes in the proportion of uncharged drug, and/or changes in the kinetics of drug binding or unbinding. These findings are in contrast to the effects of extracellular acidosis on block of the fast sodium channel by these agents and provide a molecular mechanism for ergent modulation of drug block potentially leading to ischemia-associated proarrhythmia.
Stretch-Sensitive KCNQ1Mutation
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.JACC.2006.09.044
Abstract: This study sought to evaluate mutations in genes encoding the slow component of the cardiac delayed rectifier K+ current (I(Ks)) channel in familial atrial fibrillation (AF). Although AF can have a genetic etiology, links between inherited gene defects and acquired factors such as atrial stretch have not been explored. Mutation screening of the KCNQ1, KCNE1, KCNE2, and KCNE3 genes was performed in 50 families with AF. The effects of mutant protein on cardiac I(Ks) activation were evaluated using electrophysiological studies and human atrial action potential modeling. One missense KCNQ1 mutation, R14C, was identified in 1 family with a high prevalence of hypertension. Atrial fibrillation was present only in older in iduals who had developed atrial dilation and who were genotype positive. Patch-cl studies of wild-type or R14C KCNQ1 expressed with KCNE1 in CHO cells showed no statistically significant differences between wild-type and mutant channel kinetics at baseline, or after activation of adenylate cyclase with forskolin. After exposure to hypotonic solution to elicit cell swelling/stretch, mutant channels showed a marked increase in current, a leftward shift in the voltage dependence of activation, altered channel kinetics, and shortening of the modeled atrial action potential duration. These data suggest that the R14C KCNQ1 mutation alone is insufficient to cause AF. Rather, we suggest a model in which a "second hit", such as an environmental factor like hypertension, which promotes atrial stretch and thereby unmasks an inherited defect in ion channel kinetics (the "first hit"), is required for AF to be manifested. Such a model would also account for the age-related increase in AF development.
Cortisol influences the ontogeny of both α- and β-subunits of the cardiac sodium channel in fetal sheep
Publisher: Bioscientifica
Date: 03-2004
Abstract: During development, the heart has to adapt to changes in shape, size and, at birth, to significant changes in arterial pressure. The orderly contraction of the heart is dependent on the coordinated expression of ion channels at appropriate densities in in idual cardiac myocytes. The present study demonstrated that the expression of the alpha-subunit of the cardiac sodium channel, SCN5a, was high at mid gestation but then decreased until 10 days before birth before increasing again. Whereas the beta-subunit, SCN1b, gradually increased in expression towards partum, there was no detectable expression of SCN3b at any gestational time point. Fetal adrenalectomy prior to the normal prepartum surge in cortisol caused a reduction in expression of SCN1b and a 7.0 kb transcript of SCN5a, but not the major 8.5 kb transcript. Conversely, cortisol infusion into immature fetuses precociously increased expression levels of SCN1b and the SCN5a 7.0 kb transcript. The results show that cortisol regulates cardiac SCN gene expression in fetal sheep during late gestation. These findings could have implications for the aetiology of sudden infant death syndrome and for the intrauterine programming of adult cardiovascular disease.
Ion channelopathies: what have they taught us about arrhythmias and anti‐arrhythmic therapy
Publisher: Wiley
Date: 07-2005
Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics
Publisher: Wiley
Date: 05-01-2017
DOI: 10.1113/JP272883
When it takes two to get one into trouble.
Publisher: Elsevier BV
Date: 02-2021
The temperature dependence of kinetics associated with drug block of hERG channels is compound-specific and an important factor for proarrhythmic risk prediction
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 04-05-2018
Abstract: Current mandated preclinical tests for drug-induced proarrhythmia are very sensitive, but not sufficiently specific. This has led to concern that there is a high attrition rate of potentially safe drugs that could have been beneficial to patients. The comprehensive in vitro proarrhythmia initiative has proposed new metrics based around in silico risk predictions, which are informed, among other things, by measures of human ether-à-go-go-related gene channel (hERG) block kinetics. However, high-throughput patch-cl systems set to collect these data largely operate at ambient temperature, whereas the simulations for risk prediction are carried out at physiologic temperature. The aims of this study were to: 1) determine to what degree kinetics of drug block of hERG are temperature-dependent, 2) assess the impact of any temperature dependence of drug binding kinetics on repolarization in silico, and 3) identify whether a common set of Q
Cell swelling has differential effects on the rapid and slow components of delayed rectifier potassium current in guinea pig cardiac myocytes.
Publisher: Rockefeller University Press
Date: 12-1995
Abstract: Cell swelling has been shown to cause activation of a variety of cardiac sarcolemmal ionic conductances including potassium channels. The aim of this study was to investigate the effect of swelling on the two subtypes of delayed rectifier potassium current (IKr and IKs) in single guinea pig myocytes using the whole-cell configuration of the patch cl technique. When the holding potential was set at -40 mV and stepped to +40 mV for 1 s under isoosmotic conditions (300 mOsm) a delayed rectifier current (IK) was activated (0.86 +/- 0.05 nA n = 43). Switching to a hypoosmotic solution (200 mOsm) caused a rapid increase in IK to a mean value of 1.43 +/- 0.10 nA (p & 0.05 n = 43). The effect of swelling on the two subtypes of IK was studied by analysis of deactivating tail currents using an envelope of tails protocol (stepping from -40 to +40 mV for 18 different pulse durations between 50 ms and 2.9 s n = 16). Swelling caused a decrease in current litude measured at the end of the pulse (and IKtail) at short durations (& or = 150 ms) however, when the pulse duration was & 1 s swelling caused a significant increase in current. Using a pulse protocol to measure IKr with minimal contamination by IKs (voltage step from -40 to -10 mV for 250 ms) a 50-100 pA current was elicited which could be completely blocked by dofetilide (0.2 microM n = 3). Introduction of hypoosmotic solution caused a significant decrease in IKr and when dofetilide (0.2 or 1.0 microM) was introduced the current remaining was decreased further (p & 0.05 n = 5), but was not completely blocked, thus suggesting that swelling had decreased the ability of dofetilide to block IKr. Similar results were obtained over a range of dofetilide concentrations and with a second IKr blocker, La3+. In Ca(2+)-free external solutions, pulsing to -10 mV for 500 ms to measure IKr in the absence of IKs, and to +60 mV for 5 s (with 0.2 microM dofetilide) to evoke only IKs, it was clear that swelling significantly increased IKs (pulse and tail currents) and decreased IKr. In addition, when measured using the perforated patch method, swelling modulated IKt and IKs in a similar fashion. We conclude that swelling has differential effects on the subtypes of the classical cardiac IK, which may have important implications in our understanding of the mechanisms underlying ischaemia- and reperfusion-induced arrhythmogenesis.
Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events
Publisher: Oxford University Press (OUP)
Date: 10-2021
DOI: 10.1093/CVR/CVAA280
Abstract: Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between in iduals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue s les. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr. Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C–KCNH2 co-expression increases the robustness of cardiac electrical activity.
Post-transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy
Publisher: Elsevier BV
Date: 06-2004
Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation
Publisher: Elsevier BV
Date: 10-2003
C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels
Publisher: Public Library of Science (PLoS)
Date: 25-10-2013
R222Q SCN5A Mutation Is Associated With Reversible Ventricular Ectopy and Dilated Cardiomyopathy
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.JACC.2012.05.050
Abstract: The goal of this study was to characterize a variant in the SCN5A gene that encodes the alpha-subunit of the cardiac sodium channel, Nav1.5, which was identified in 1 large kindred with dilated cardiomyopathy (DCM) and multiple arrhythmias, including premature ventricular complexes (PVCs). Treatment guidelines for familial DCM are based on conventional heart failure therapies, and no gene-based interventions have been established. Family members underwent clinical evaluation and screening of the SCN5A and LMNA genes. Cellular electrophysiology and computational modeling were used to determine the functional consequences of the mutant Nav1.5 protein. An R222Q missense variant located in a Nav1.5 voltage-sensing domain was identified in affected family members. Patch-cl studies showed that R222Q Nav1.5 did not alter sodium channel current density, but did left shift steady-state parameters of activation and inactivation. Using a voltage r protocol, normalized current responses of R222Q channels were of earlier onset and greater magnitude than wild-type channels. Action potential modeling using Purkinje fiber and ventricular cell models suggested that rate-dependent ectopy of Purkinje fiber origin is the predominant ventricular effect of the R222Q variant and a potential cause of DCM. In R222Q carriers, there were only modest responses to heart failure therapies, but PVCs and DCM were substantially reduced by amiodarone or flecainide, which are drugs that have sodium channel-blocking properties. The R222Q SCN5A variant has an activating effect on sodium channel function and is associated with reversible ventricular ectopy and DCM. Elucidation of the genetic basis of familial DCM can enable effective gene-targeted therapy to be implemented.
Assessing drug safety by identifying the axis of arrhythmia in cardiomyocyte electrophysiology
Publisher: Cold Spring Harbor Laboratory
Date: 20-04-2023
DOI: 10.1101/2023.04.19.537441
Abstract: 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 erse 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 to an accuracy of 88.1% to 90.8%. Pharmacologists can use our method to assess the safety of novel drugs without resort to animal testing nor unwieldy computer simulations. Many classes of drugs interfere with the electrical signaling of the heart, leading to arrhythmias and cardiac arrest. Newly developed drugs must therefore undergo mandatory safety testing in animals prior to human trials. Computational models of cardiac electrophysiology offer an ethical alternative but the current methods are difficult to apply beyond specialist computing laboratories. This study uses such models to identify the underlying relationship between drugs and cardiac arrhythmias. Those findings are then translated into a compact metric that can be applied using simple pen and paper calculations. The new metric allows pharmacology laboratories to assess the safety of novel drugs without using animals nor unwieldy computer simulations.
Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a
Publisher: Wiley
Date: 30-08-2005
The Schizophrenia-Associated Kv11.1-3.1 Isoform Results in Reduced Current Accumulation during Repetitive Brief Depolarizations
Publisher: Public Library of Science (PLoS)
Date: 24-09-2012
Kinetics of drug interaction with the Kv 11.1 potassium channel
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 28-02-2014
Abstract: The Kv11.1 potassium channel is the molecular target for the majority of drugs implicated in acquired long QT syndrome, the most common cause of drug-induced sudden cardiac death, and a common reason for drug restriction or withdrawal from the market. While the IC50 for block of Kv11.1 is commonly used to estimate the risk of acquired long QT syndrome, this approach is crude, and it is widely accepted that the kinetics of drug interactions with the channel are a critical component in understanding their mechanism of action and risk profiles. In this study we report the first directly measured kinetics of block and unblock of Kv11.1 by a QT prolonging drug: the antipsychotic clozapine. Our data show that clozapine binding to Kv11.1 is complex. There are at least two kinetically distinct components to both block and unblock, while the kinetics of unblock are dependent on the dose or duration of drug application. Based on these observations, we have proposed a model incorporating kinetically distinct binding to the open and inactivated states of Kv11.1 that can describe the observed kinetic features of clozapine block and correctly predict the overall affinity and apparent nonstate-dependent interaction of clozapine with Kv11.1. Mechanistic insights into drug block of Kv11.1 gained though detailed kinetic analyses such as this have a potential role in development of drugs targeted to specific channel states to reduce unwanted side effects, as well as in the design of better high-throughput preclinical tests for assessing the proarrhythmic effects of QT prolonging drugs.
Molecular dynamics and continuum electrostatics studies of inactivation in the HERG potassium channel
Publisher: American Chemical Society (ACS)
Date: 18-01-2007
DOI: 10.1021/JP066294D
Insights into hERG K+ channel structure and function from NMR studies
Publisher: Springer Science and Business Media LLC
Date: 03-05-2013
DOI: 10.1007/S00249-012-0808-6
Abstract: The unique gating kinetics of hERG K(+) channels are critical for normal cardiac repolarization, and patients with mutations in hERG have a markedly increased risk of cardiac arrhythmias and sudden cardiac arrest. HERG K(+) channels are also remarkably promiscuous with respect to drug binding, which has been a very significant problem for the pharmaceutical industry. Here, we review the progress that has been made in understanding the structure and function of hERG K(+) channels with a particular focus on nuclear magnetic resonance studies of the domains of the hERG K(+) channel.
Temperature dependence of human ether-à-go-go-related gene K+ currents
Publisher: American Physiological Society
Date: 07-2006
DOI: 10.1152/AJPCELL.00596.2005
Abstract: The function of voltage-gated human ether-à-go-gorelated gene ( hERG) K + channels is critical for both normal cardiac repolarization and suppression of arrhythmias initiated by premature excitation. These important functions are facilitated by their unusual kinetics that combine relatively slow activation and deactivation with rapid and voltage-dependent inactivation and recovery from inactivation. The thermodynamics of these unusual features were examined by exploring the effect of temperature on the activation and inactivation processes of hERG channels expressed in Chinese hamster ovary cells. Increased temperature shifted the voltage dependence of activation in the hyperpolarizing direction but that of inactivation in the depolarizing direction. This increases the relative occupancy of the open state and contributes to the marked temperature sensitivity of hERG current magnitude observed during action potential voltage cl s. The rates of activation and deactivation also increase with higher temperatures, but less markedly than do the rates of inactivation and recovery from inactivation. Our results also emphasize that one cannot extrapolate results obtained at room temperature to 37°C by using a single temperature scale factor.
T-wave morphology can distinguish healthy controls from LQTS patients
Publisher: IOP Publishing
Date: 11-08-2016
DOI: 10.1088/0967-3334/37/9/1456
Abstract: Long QT syndrome (LQTS) is an inherited disorder associated with prolongation of the QT/QTc interval on the surface electrocardiogram (ECG) and a markedly increased risk of sudden cardiac death due to cardiac arrhythmias. Up to 25% of genotype-positive LQTS patients have QT/QTc intervals in the normal range. These patients are, however, still at increased risk of life-threatening events compared to their genotype-negative siblings. Previous studies have shown that analysis of T-wave morphology may enhance discrimination between control and LQTS patients. In this study we tested the hypothesis that automated analysis of T-wave morphology from Holter ECG recordings could distinguish between control and LQTS patients with QTc values in the range 400-450 ms. Holter ECGs were obtained from the Telemetric and Holter ECG Warehouse (THEW) database. Frequency binned averaged ECG waveforms were obtained and extracted T-waves were fitted with a combination of 3 sigmoid functions (upslope, downslope and switch) or two 9th order polynomial functions (upslope and downslope). Neural network classifiers, based on parameters obtained from the sigmoid or polynomial fits to the 1 Hz and 1.3 Hz ECG waveforms, were able to achieve up to 92% discrimination between control and LQTS patients and 88% discrimination between LQTS1 and LQTS2 patients. When we analysed a subgroup of subjects with normal QT intervals (400-450 ms, 67 controls and 61 LQTS), T-wave morphology based parameters enabled 90% discrimination between control and LQTS patients, compared to only 71% when the groups were classified based on QTc alone. In summary, our Holter ECG analysis algorithms demonstrate the feasibility of using automated analysis of T-wave morphology to distinguish LQTS patients, even those with normal QTc, from healthy controls.
Proteins, membranes and cells: the structure–function nexus—ASB 2008
Publisher: Springer Science and Business Media LLC
Date: 19-09-2009
The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation
Publisher: Public Library of Science (PLoS)
Date: 13-01-2011
Pathophysiological metabolic changes associated with disease modify the proarrhythmic risk profile of drugs with potential to prolong repolarisation
Publisher: Wiley
Date: 27-01-2022
DOI: 10.1111/BPH.15757
Abstract: 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. 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 in iduals. Chloroquine and hydroxychloroquine blocked hERG with IC 50 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. 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.
Not All hERG Pore Domain Mutations Have a Severe Phenotype: G584S Has an Inactivation Gating Defect with Mild Phenotype Compared to G572S, Which Has a Dominant Negative Trafficking Defect and a Severe
Publisher: Wiley
Date: 28-07-2009
DOI: 10.1111/J.1540-8167.2009.01468.X
Abstract: Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of in idual mutations can assist with risk stratification. We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584S-hERG channels. hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.
Structure of the pore-helix of the hERG K+ channel
Publisher: Springer Science and Business Media LLC
Date: 21-03-2009
DOI: 10.1007/S00249-009-0433-1
Abstract: The hERG K(+) channel undergoes rapid inactivation that is mediated by 'collapse' of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600-642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two-four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K(+) channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.
From kinetics to imaging: An NMR odyssey - A festschrift symposium in honour of Philip William Kuchel
Publisher: Springer Science and Business Media LLC
Date: 21-12-2013
Human ether-a-go-go related gene (hERG) K+ channels: Function and dysfunction
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.PBIOMOLBIO.2008.10.006
Abstract: The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.
Domain reorientation and rotation of an intracellular assembly regulate conduction in kir potassium channels
Publisher: Elsevier BV
Date: 06-2010
DOI: 10.1016/J.CELL.2010.05.003
Abstract: Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.
Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo
Publisher: Springer Science and Business Media LLC
Date: 09-2002
DOI: 10.1007/S00395-002-0360-0
Abstract: Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non- iding cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes - E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field.
Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a
Publisher: Proceedings of the National Academy of Sciences
Date: 23-04-2002
Abstract: Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A , have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a , causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch cl analyses of isolated ventricular myocytes from adult Scn5a +/− mice demonstrate a ≈50% reduction in sodium conductance. Scn5a +/− hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently erse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.
The yin and yang ofTbx5variant effects on sodium channel function
Publisher: Oxford University Press (OUP)
Date: 22-02-2022
DOI: 10.1093/CVR/CVAC015
The S1 helix critically regulates the finely tuned gating of Kv11.1 channels
Publisher: Elsevier BV
Date: 03-2017
A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells
Publisher: Oxford University Press (OUP)
Date: 28-10-2015
Abstract: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.
Assessing drug safety by identifying the axis of arrhythmia in cardiomyocyte electrophysiology
Publisher: eLife Sciences Publications, Ltd
Date: 21-08-2023
Abstract: 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 erse 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 to an accuracy of 88.1% to 90.8%. Pharmacologists can use our method to assess the safety of novel drugs without resort to animal testing nor unwieldy computer simulations. Many classes of drugs interfere with the electrical signaling of the heart, leading to arrhythmias and cardiac arrest. Newly developed drugs must therefore undergo mandatory safety testing in animals prior to human trials. Computational models of cardiac electrophysiology offer an ethical alternative but the current methods are difficult to apply beyond specialist computing laboratories. This study uses such models to identify the underlying relationship between drugs and cardiac arrhythmias. Those findings are then translated into a compact metric that can be applied using simple pen and paper calculations. The new metric allows pharmacology laboratories to assess the safety of novel drugs without using animals nor unwieldy computer simulations.
In vitro and in silico risk assessment in acquired long QT syndrome: The devil is in the details
Publisher: Frontiers Media SA
Date: 16-11-2017
Structural basis of promiscuous substrate transport by Organic Cation Transporter 1
Publisher: Springer Science and Business Media LLC
Date: 11-10-2023
Reply from Jamie I. Vandenberg, Adam P. Hill, Terence J. Campbell, Catherine E. Clarke [2]
Publisher: Wiley
Date: 08-11-2006
Mutant MiRP1 subunits modulate HERG K+ channel gating: a mechanism for pro-arrhythmia in long QT syndrome type 6
Publisher: Wiley
Date: 14-08-2003
Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics
Publisher: Wiley
Date: 17-04-2018
DOI: 10.1113/JP275733
Clinical interpretation ofKCNH2variants using a robust PS3/BS3 functional patch clamp assay
Publisher: Cold Spring Harbor Laboratory
Date: 09-10-2023
Assessing drug safety by identifying the axis of arrhythmia in cardiomyocyte electrophysiology
Publisher: eLife Sciences Publications, Ltd
Date: 21-08-2023
DOI: 10.7554/ELIFE.90027
Abstract: 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 erse 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 to an accuracy of 88.1% to 90.8%. Pharmacologists can use our method to assess the safety of novel drugs without resort to animal testing nor unwieldy computer simulations. Many classes of drugs interfere with the electrical signaling of the heart, leading to arrhythmias and cardiac arrest. Newly developed drugs must therefore undergo mandatory safety testing in animals prior to human trials. Computational models of cardiac electrophysiology offer an ethical alternative but the current methods are difficult to apply beyond specialist computing laboratories. This study uses such models to identify the underlying relationship between drugs and cardiac arrhythmias. Those findings are then translated into a compact metric that can be applied using simple pen and paper calculations. The new metric allows pharmacology laboratories to assess the safety of novel drugs without using animals nor unwieldy computer simulations.
Experimentally Validated Pharmacoinformatics Approach to Predict hERG Inhibition Potential of New Chemical Entities
Publisher: Frontiers Media SA
Date: 19-09-2018
Never at rest: insights into the conformational dynamics of ion channels from cryo‐electron microscopy
Publisher: Wiley
Date: 05-03-2018
DOI: 10.1113/JP274888
Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation
Publisher: Elsevier BV
Date: 08-2016
Potassium channels in the heart: structure, function and regulation
Publisher: Wiley
Date: 13-11-2016
DOI: 10.1113/JP272864
Arrhythmic Phenotypes Are a Defining Feature of Dilated Cardiomyopathy-Associated SCN5A Variants: A Systematic Review
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2022
DOI: 10.1161/CIRCGEN.121.003432
Abstract: 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. A systematic review of reported DCM-associated rare SCN5A variants was undertaken using PubMed and Embase. Eighteen SCN5A rare variants in 29 families with DCM (173 affected in iduals) 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 in iduals 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. SCN5A variants can present with a erse 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.
Extracellular osmotic pressure modulates sodium‐calcium exchange in isolated guinea‐pig ventricular myocytes.
Publisher: Wiley
Date: 15-10-1995
DOI: 10.1113/JPHYSIOL.1995.SP020967
Abstract: 1. The sensitivity of the cardiac Na(+)-Ca2+ exchange current to changes in osmotic pressure was investigated in guinea-pig ventricular myocytes, using the whole-cell patch-cl technique. 2. A hyposmotic challenge applied by removal of sucrose from the standard bathing solution reduced exchanger current, measured as the Ni(2+)-sensitive component of whole-cell transsarcolemmal current. These changes were fully reversible. 3. No response of whole-cell current to hyposmosis was observed when Ca2+ was removed from the bathing solution by chelation with 1 mM EGTA. 4. Inclusion of 25 microM exchanger inhibitory peptide (XIP) in the pipette solution caused a marked reduction in the Ni(2+)-sensitive component of membrane current, but the percentage change in Ni(2+)-sensitive membrane slope conductance evoked by hyposmosis was the same as when XIP was omitted from the pipette solution. 5. Exposure of cells to hyperosmotic solutions produced variable responses. In a majority of cells, solutions 30% hyperosmotic compared with control evoked a persistent increase in exchanger current, whereas for solutions 50% hyperosmotic, a larger but transient increase in current was observed. 6. Over a wide range of osmolalities (50-130% of isosmotic) the changes in Ni(2+)-sensitive membrane slope conductance were linearly related to the changes in extracellular osmotic pressure. 7. We propose that one consequence of exposing ventricular myocytes to anisosmotic solutions is modulation of Na(+)-Ca2+ exchange current.
Effect of S5P α‐helix charge mutants on inactivation of hERG K+ channels
Publisher: Wiley
Date: 31-05-2006
Cardiac Expression of the Cystic Fibrosis Transmembrane Conductance Regulator Involves Novel Exon 1 Usage to Produce a Unique Amino-terminal Protein
Publisher: Elsevier BV
Date: 04-2004
Complex patterns of schist tor exposure and surface uplift, Otago (New Zealand)
Publisher: Elsevier BV
Date: 09-2021
Rescue of protein expression defects may not be enough to abolish the pro‐arrhythmic phenotype of long QT type 2 mutations
Publisher: Wiley
Date: 27-05-2016
DOI: 10.1113/JP271805
Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology.
Publisher: Rockefeller University Press
Date: 12-1994
Abstract: We have used the whole-cell patch cl recording technique to characterize a swelling-activated chloride current in guinea pig atrial and ventricular myocytes and to compare the electrophysiological and pharmacological properties of this current with the isoprenaline-activated chloride current in the same cell types. Osmotic swelling of guinea pig cardiac myocytes caused activation of an outwardly rectifying, anion-selective current with a conductance and permeability sequence of I- approximately NO3- & Br- & Cl- & Asp-. This current was inhibited by tamoxifen, 4,4'-diisothiocyano-stilbene-2,2'-disulphonate and anthracene-9-carboxylic acid, in decreasing order of potency. The isoprenaline-activated anion current, like the swelling-activated current, had a higher permeability to I- relative to Cl-, but it had a markedly reduced conductance for I- compared to Cl-. The isoprenaline-activated current was insensitive to inhibition by tamoxifen, 4,4'-diisothiocyanostilbene-2,2'-disulphonate and anthracene-9-carboxylic acid. The swelling-activated current could be elicited in & 90% atrial myocytes studied but only 34% ventricular myocytes. Conversely, the isoprenaline-activated current was elicited in & 10% atrial myocytes and & 90% ventricular myocytes. In those ventricular myocytes where it was possible to elicit swelling-activated and isoprenaline-activated currents simultaneously, the currents retained the same distinguishing characteristics as when they were elicited in isolation. Thus, while guinea pig atrial cells appear to preferentially express swelling-activated chloride channels and guinea pig ventricular myocytes preferentially express isoprenaline-activated chloride channels, the presence of these two channel types are not necessarily mutually exclusive. This raises the possibility that there may be coordinated regulation of the expression of different Cl- channels within the heart.
Towards a Structural View of Drug Binding to hERG K + Channels
Publisher: Elsevier BV
Date: 10-2017
Inherited cardiac arrhythmia syndromes: What have they taught us about arrhythmias and anti‐arrhythmic therapy?
Publisher: Wiley
Date: 12-2004
DOI: 10.1111/J.1440-1681.2004.04132.X
Abstract: 1. In recent years, the identification of the gene defects in a vast array of monogenic disorders has revolutionized our understanding of the basic mechanisms underlying numerous disease processes. 2. Mutations in cardiac ion channels have been identified as the basis of a wide range of inherited arrhythmia syndromes, including the congenital long QT syndromes, Brugada syndrome, Lenegre syndrome, Andersen's disease and familial atrial fibrillation. 3. Identification of mutations in the human-ether-a-go-go-related gene (HERG) K(+) channel as the molecular basis of congenital long QT syndrome type 2 also led to the discovery that HERG is the molecular target for the vast majority of drugs (both cardiac and non-cardiac) that cause drug-induced arrhythmias. This has had profound implications not only for the development of anti-arrhythmic agents, but also for drug development in general. 4. The sequencing of the human genome in a sense represents the pinnacle of the reductionist era of molecular medicine. The great challenge now is to re-integrate the information gathered during the 'reductionist era' to provide a better understanding of the intact organism. Computer modelling is likely to be a key component of that re-integration process.
Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels
Publisher: Rockefeller University Press
Date: 26-08-2013
Abstract: Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed “activation” and “inactivation,” located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 “voltage-sensor” helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4–S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.
Development of a robust induced pluripotent stem cell atrial cardiomyocyte differentiation protocol to model atrial arrhythmia
Publisher: Research Square Platform LLC
Date: 24-04-2023
DOI: 10.21203/RS.3.RS-2744099/V1
Abstract: Background Atrial fibrillation (AF) 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 I kach 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, cm/s, 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, whilst being economically viable for larger anti-arrhythmic drug screens.
Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv 11.1 potassium channel
Publisher: Springer Science and Business Media LLC
Date: 19-12-2011
DOI: 10.1038/NSMB.1966
Abstract: The potassium channel selectivity filter both discriminates between K(+) and sodium ions and contributes to gating of ion flow. Static structures of conducting (open) and nonconducting (inactivated) conformations of this filter are known however, the sequence of protein rearrangements that connect these two states is not. We show that closure of the selectivity filter gate in the human K(v)11.1 K(+) channel (also known as hERG, for ether-a-go-go-related gene), a key regulator of the rhythm of the heartbeat, is initiated by K(+) exit, followed in sequence by conformational rearrangements of the pore domain outer helix, extracellular turret region, voltage sensor domain, intracellular domains and pore domain inner helix. In contrast to the simple wave-like sequence of events proposed for opening of ligand-gated ion channels, a complex spatial and temporal sequence of widespread domain motions connect the open and inactivated states of the K(v)11.1 K(+) channel.
Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance
Publisher: Oxford University Press (OUP)
Date: 21-10-2022
DOI: 10.1093/HMG/DDAC261
Abstract: 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 cl . 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 cl 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 cl 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 cl 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.
Convergence of models of human ventricular myocyte electrophysiology after global optimization to recapitulate clinical long QT phenotypes
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.YJMCC.2016.09.011
Abstract: In-silico models of human cardiac electrophysiology are now being considered for prediction of cardiotoxicity as part of the preclinical assessment phase of all new drugs. We ask the question whether any of the available models are actually fit for this purpose. We tested three models of the human ventricular action potential, the O'hara-Rudy (ORD11), the Grandi-Bers (GB10) and the Ten Tusscher (TT06) models. We extracted clinical QT data for LQTS1 and LQTS2 patients with nonsense mutations that would be predicted to cause 50% loss of function in I
Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels
Publisher: Elsevier BV
Date: 09-2014
Protocol-Dependent Differences in IC50 Values Measured in Human Ether-Á-Go-Go–Related Gene Assays Occur in a Predictable Way and Can Be Used to Quantify State Preference of Drug Binding
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 15-02-2019
Abstract: Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of voltage-gated potassium channel subtype 11.1 (K
Hodgkin and Huxley and the basis for electrical signalling: A remarkable legacy still going strong
Publisher: Wiley
Date: 31-05-2012
Can many subunits make light work of ion channel inactivation?
Publisher: Wiley
Date: 15-10-2014
A Massively Parallel Trafficking Assay Accurately Predicts Loss of Channel Function inKCNH2Variants
Publisher: Cold Spring Harbor Laboratory
Date: 11-07-2021
DOI: 10.1101/2021.07.10.451881
Abstract: High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch cl electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.
TECRL: connecting sequence to consequence for a new sudden cardiac death gene
Publisher: EMBO
Date: 26-10-2016
Abstract: The sudden unexpected death of a child is a devastating event. One of the first questions a family will ask is “Why did this happen?” In some cases, the answer may become obvious during a postmortem examination, but in up to 40% of cases, the postmortem is negative (Bagnall et al , ). In the last 1–2 decades, an improved understanding of the genetic basis of the primary arrhythmia syndromes, the major cause of sudden unexplained death in children with structurally normal hearts, has greatly enhanced our ability to make a postmortem diagnosis (Van Norstrand & Ackerman, ). Establishing an accurate genetic diagnosis can not only answer the parents' question as to why did this happen to my child, but is invaluable for cascade screening of all family members to identify other in iduals harbouring the same mutation and who therefore may be at risk of sudden cardiac death. However, even after screening for all of the established genes associated with primary arrhythmia syndromes, up to two thirds of unexplained cardiac deaths will remain unsolved. Such was the case for a family of Sudanese origin with a highly malignant form of exercise‐induced arrhythmias, originally reported by Bhuiyan et al ( ).
Loss of the Normal Epicardial to Endocardial Gradient of cftr mRNA Expression in the Hypertrophied Rabbit Left Ventricle
Publisher: Elsevier BV
Date: 11-2000
Normal conduction of surface action potentials in detubulated amphibian skeletal muscle fibres
Publisher: Wiley
Date: 09-2001
DOI: 10.1111/J.1469-7793.2001.T01-1-00579.X
Abstract: 1. The influence of the transverse (T) tubules on surface action potential conduction was investigated by comparing electrophysiological and confocal microscopic assessments of tubular changes in osmotically shocked and control fibres from frog sartorius muscle. 2. The membrane-impermeant fluorescent dye, di-8-ANEPPs spread readily from the bathing extracellular solution into the tubular membranes in control, intact fibres. Prior exposure of muscles to a hypertonic glycerol-Ringer solution, its replacement by an isotonic Ca(2+)-Mg(2+) Ringer solution and cooling sharply reduced such access. In contrast, dye application in the course of this osmotic shock procedure stained the large tubular vacuoles hitherto associated with successful muscle detubulation. 3. Conduction velocities in intact, control fibres (1.91 +/- 0.048 m s(-1), mean +/- S.E.M., n = 32 fibres) agreed with earlier values reported at room temperature (18-21 degrees C) and were unaffected by prior episodes of steady cooling to 8-10 degrees C (1.91 +/- 0.043 m s(-1), n = 30). 4. Cooling to 11.5 degrees C reduced these velocities (1.47 +/- 0.081 m s(-1), n = 25) but action potential waveforms still included early overshoots and the delayed after-depolarizations associated with tubular electrical activity. 5. In contrast, action potentials from cooled, superficial fibres in osmotically shocked muscles lacked after-depolarization phases implying tubular detachment. Their mean conduction velocities (1.62 +/- 0.169 m s(-1), n = 25) were not significantly altered from values obtained in untreated controls or in intact fibres in muscle similarly treated with glycerol, in direct contrast to earlier results. 6. Cooling produced similar reductions in maximum rates of voltage change dV/dt in action potentials from all fibre groups with lower rates of change shown by detubulated fibres. 7. Use of an antibody to a conserved epitope of the alpha-subunit of voltage-gated sodium channels suggested a concentration of sodium channels close to the mouths of the T tubules. 8. These electrophysiological and anatomical findings are consistent with a partial independence of electrical events in the transverse tubules from those responsible for the rapid conduction of surface regenerative activity. 9. The findings are discussed in terms of a partial separation of the electrical activity propagated over the surface membrane, from the initiation of propagated activity within the T tubules, by the triggering of the sodium channels clustered selectively around the mouths of the T tubules.
Related Organisations
University Of Cambridge
Location: United Kingdom of Great Britain and Northern Ireland
University Of Oxford
Location: United Kingdom of Great Britain and Northern Ireland
Related Funding Activities
Development Of A New High Throughput Screen For Drug Binding To HERG K+ Channels
Start Date: 2010
End Date: 2012
Funder: National Health and Medical Research Council
View Funded ActivityCharacterisation Of Two-pore Domain Potassium Channels: Structure-function Studies Of The M1-P1 Loops Of TASK Channels.
Start Date: 2005
End Date: 2007
Funder: Australian Research Council
View Funded ActivityElectro-active And Migratory Peptides In Lipid Bilayers: NMR And Biophysical Studies
Start Date: 2009
End Date: 2011
Funder: Australian Research Council
View Funded Activity800 MHz NMR Spectrometer For Biomolecular Structure-function Analysis
Start Date: 2008
End Date: 2008
Funder: Australian Research Council
View Funded ActivityMathematical Biosciences Network
Start Date: 2003
End Date: 2003
Funder: Australian Research Council
View Funded ActivityThe Structural Basis For Promiscuity Of Drug Binding To HERG K+ Channels
Start Date: 2018
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivitySinusoidal Voltage Protocols For Characterisation Of Ion Channel Kinetics
Start Date: 2019
End Date: 2021
Funder: Australian Research Council
View Funded ActivityElectrophysiology Facility For Cell Phenotyping And Drug Discovery
Start Date: 2018
End Date: 2018
Funder: Australian Research Council
View Funded ActivityProject DP150101929
Start Date: 2015
End Date: 2017
Funder: Australian Research Council
View Funded ActivityThe Structural Basis For Promiscuity Of Drug Binding To HERG K+ Channels
Start Date: 2018
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivitySudden Cardiac Arrest: Improving Detection Of Patients At Risk
Start Date: 2017
End Date: 2021
Funder: National Health and Medical Research Council
View Funded ActivityNMR Studies Of Membrane Proteins And Peptides In Novel Amphiphilic Mesophases
Start Date: 2004
End Date: 2008
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP200102540
Start Date: 2020
End Date: 12-2022
Amount: $477,513.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP190101758
Start Date: 2019
End Date: 06-2023
Amount: $409,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0557701
Start Date: 2005
End Date: 12-2008
Amount: $277,000.00
Funder: Australian Research Council
View Funded ActivitySpecial Research Initiatives - Grant ID: SR0354592
Start Date: 02-2004
End Date: 11-2004
Amount: $20,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP150101929
Start Date: 2015
End Date: 01-2018
Amount: $420,236.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0450808
Start Date: 02-2004
End Date: 01-2009
Amount: $1,250,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0986316
Start Date: 05-2009
End Date: 12-2013
Amount: $480,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE180100066
Start Date: 2018
End Date: 12-2018
Amount: $443,311.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE0883032
Start Date: 02-2009
End Date: 12-2010
Amount: $1,300,000.00
Funder: Australian Research Council
View Funded Activity