RESUMO
Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.
Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Canal de Potássio ERG1/agonistas , Canais de Potássio Éter-A-Go-Go/agonistas , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Piperidinas/farmacologia , Quinolinas/farmacologia , Proteínas de Peixe-Zebra/agonistas , Potenciais de Ação , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Cinética , Miócitos Cardíacos/metabolismo , Oócitos , Período Refratário Eletrofisiológico , Transdução de Sinais , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The human ether-à-go-go-related gene (KCNH2)-encoded protein hERG constitutes the α subunit of the Kv11.1 channel and contributes to the I kr current, which plays an important role in the cardiac action potential. Genetically and xenobiotically triggered malfunctions of hERG can cause arrhythmia. The expression of hERG in various study systems was assessed mainly as the fold change relative to the corresponding control. Here, we developed a simple and sensitive quantitation method using targeted mass spectrometry, i.e., the parallel reaction monitoring approach, to measure the absolute quantity of hERG in copy number. Such measurements do not require controls, and the obtained values can be compared with similar results for any other protein. To effectively avoid matrix effects, we used the heavy-match-light (HML) in-sample calibration approach that requires only a single isotopologue to achieve copy-number quantitation. No significant difference was observed in the results obtained by HML and by the classic standard addition in-sample calibration approach. Using four proteotypic peptides, we quantified the average number of copies of hERG in the HEK293T heterologous expression system as 3.6 ± 0.5 × 106 copies/cell, i.e., 1 million copies/cell for the fully assembled Kv11.1 channel.
RESUMO
Marfan syndrome (MFS) is a hereditary condition caused by mutations in the FBN1 gene. Genetic mutations in the FBN1 locus impact the function of the encoded protein, Fibrillin 1, a structural molecule forming microfibrils found in the connective tissue. MFS patients develop severe cardiovascular complications including thoracic aortic aneurysm and aortic dissection, which predispose them to an enhanced risk of premature death. Here, we generated two induced pluripotent stem cell (iPSC) lines harboring mutations in the FBN1 gene (p.C1942C>A and c.1954 T>C), directly derived from MFS patients. We have shown that both iPSC lines displayed expression of pluripotency markers, normal karyotype and ability of trilineage differentiation, representing a valuable tool for the identification of new therapeutic strategies for intervening in this disease.
Assuntos
Fibrilina-1 , Células-Tronco Pluripotentes Induzidas , Síndrome de Marfan , Mutação , Síndrome de Marfan/genética , Síndrome de Marfan/patologia , Fibrilina-1/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Diferenciação Celular , Linhagem Celular , Masculino , AdipocinasRESUMO
We generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of breast cancer patients carrying germline ATM mutations, a gene associated with a 7% prevalence in breast cancer. These iPSC lines displayed typical morphology, expressed pluripotency markers, maintained a stable karyotype, and retained the ability to differentiate into the three germ layers. These patient-specific iPSC lines hold great potential for mechanistic investigations and the development of drug screening strategies aimed at addressing ATM-related cancer.
Assuntos
Neoplasias da Mama , Células-Tronco Pluripotentes Induzidas , Humanos , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Neoplasias da Mama/genética , Leucócitos Mononucleares/metabolismo , Mutação/genética , Mutação em Linhagem Germinativa , Proteínas Mutadas de Ataxia Telangiectasia/genéticaRESUMO
AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.
Assuntos
Canais de Potássio Éter-A-Go-Go , Síndrome do QT Longo , Humanos , Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevenção & controle , Miócitos Cardíacos , Potenciais de Ação , Éteres , Canal de Potássio ERG1/genéticaRESUMO
Mathematical models of voltage-gated ion channels are used in basic research, industrial and clinical settings. These models range in complexity, but typically contain numerous variables representing the proportion of channels in a given state, and parameters describing the voltage-dependent rates of transition between states. An open problem is selecting the appropriate degree of complexity and structure for an ion channel model given data availability. Here, we simplify a model of the cardiac human Ether-à-go-go related gene (hERG) potassium ion channel, which carries cardiac IKr, using the manifold boundary approximation method (MBAM). The MBAM approximates high-dimensional model-output manifolds by reduced models describing their boundaries, resulting in models with fewer parameters (and often variables). We produced a series of models of reducing complexity starting from an established five-state hERG model with 15 parameters. Models with up to three fewer states and eight fewer parameters were shown to retain much of the predictive capability of the full model and were validated using experimental hERG1a data collected in HEK293 cells at 37°C. The method provides a way to simplify complex models of ion channels that improves parameter identifiability and will aid in future model development.
Assuntos
Canais de Potássio Éter-A-Go-Go , Canais Iônicos , Canal de Potássio ERG1/genética , Células HEK293 , Coração , HumanosRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.
Assuntos
Sistemas CRISPR-Cas , Pequeno RNA não Traduzido , Peixe-Zebra , Animais , Edição de Genes , Genoma , Peixe-Zebra/genética , Pequeno RNA não Traduzido/genéticaRESUMO
Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.
Assuntos
Canais de Potássio Éter-A-Go-Go , Síndrome do QT Longo , Arritmias Cardíacas , Canal de Potássio ERG1/genética , Éter , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/genética , Piperidinas , QuinolinasRESUMO
Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for â¼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.