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Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent an in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit electrophysiological heterogeneity which hinders their utility in the study of certain cardiac currents. In the healthy adult heart, the current mediated by small conductance, calcium-activated potassium (SK) channels (ISK) is atrial-selective. Functional expression of ISK within atrial-like iPSC-CMs has not been explored thoroughly. The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of ISK. iPSCs were differentiated using retinoic acid (RA) to produce iPSC-CMs which exhibited an atrial-like phenotype (RA-iPSC-CMs). Only 18% of isolated RA-iPSC-CMs responded to SK channel inhibition by UCL1684 and isolated iPSC-CMs exhibited substantial cell-to-cell electrophysiological heterogeneity. This variability was significantly reduced by patch clamp of RA-iPSC-CMs in situ as a monolayer (iPSC-ML). A novel method of electrical stimulation was developed to facilitate recording from iPSC-MLs via In situ Monolayer Patch clamp of Acutely Stimulated iPSC-CMs (IMPASC). Using IMPASC, > 95% of iPSC-MLs could be paced at a 1 Hz. In contrast to isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to UCL1684, with APD50 being prolonged by 16.0 ± 2.0 ms (p < 0.0001; n = 12). These data demonstrate that in conjunction with IMPASC, RA-iPSC-MLs represent an improved model for the study of ISK. IMPASC may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs and in pharmacological studies.
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Células Madre Pluripotentes Inducidas , Humanos , Adulto , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Diferenciación Celular/genética , Atrios CardíacosRESUMEN
Atrial fibrillation (AF) is the most common cardiac arrhythmia and is sustained by spontaneous focal excitations and re-entry. Spontaneous electrical firing in the pulmonary vein (PV) sleeves is implicated in AF generation. The aim of this simulation study was to identify the mechanisms determining the localisation of AF triggers in the PVs and their contribution to the genesis of AF. A novel biophysical model of the canine atria was used that integrates stochastic, spontaneous subcellular Ca2+ release events (SCRE) with regional electrophysiological heterogeneity in ionic properties and a detailed three-dimensional model of atrial anatomy, microarchitecture and patchy fibrosis. Simulations highlighted the importance of the smaller inward rectifier potassium current (IK1 ) in PV cells compared to the surrounding atria, which enabled SCRE more readily to result in delayed-afterdepolarisations that induced triggered activity. There was a leftward shift in the dependence of the probability of triggered activity on sarcoplasmic reticulum Ca2+ load. This feature was accentuated in 3D tissue compared to single cells (Δ half-maximal [Ca2+ ]SR = 58 µM vs. 22 µM). In 3D atria incorporating electrical heterogeneity, excitations preferentially emerged from the PV region. These triggered focal excitations resulted in transient re-entry in the left atrium. Addition of fibrotic patches promoted localised emergence of focal excitations and wavebreaks that had a more substantial impact on generating AF-like patterns than the PVs. Thus, a reduced IK1 , less negative resting membrane potential, and fibrosis-induced changes of the electrotonic load all contribute to the emergence of complex excitation patterns from spontaneous focal triggers. KEY POINTS: Focal excitations in the atria are most commonly associated with the pulmonary veins, but the mechanisms for this localisation are yet to be elucidated. We applied a multi-scale computational modelling approach to elucidate the mechanisms underlying such localisations. Myocytes in the pulmonary vein region of the atria have a less negative resting membrane potential and reduced time-independent potassium current; we demonstrate that both of these factors promote triggered activity in single cells and tissues. The less negative resting membrane potential also contributes to heterogeneous inactivation of the fast sodium current, which can enable re-entrant-like excitation patterns to emerge without traditional conduction block.
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Fibrilación Atrial , Venas Pulmonares , Animales , Perros , Fibrilación Atrial/etiología , Calcio , Atrios Cardíacos , Calcio de la Dieta , Potenciales de Acción , Fibrosis , PotasioRESUMEN
The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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Bases de Datos Farmacéuticas , Farmacología , Humanos , Ligandos , Canales Iónicos/química , Receptores Acoplados a Proteínas G , Receptores Citoplasmáticos y NuclearesRESUMEN
A mutation to serine of a conserved threonine (T634S) in the hERG K+ channel S6 pore region has been identified as a variant of uncertain significance, showing a loss-of-function effect. However, its potential consequences for ventricular excitation and arrhythmogenesis have not been reported. This study evaluated possible functional effects of the T634S-hERG mutation on ventricular excitation and arrhythmogenesis by using multi-scale computer models of the human ventricle. A Markov chain model of the rapid delayed rectifier potassium current (IKr) was reconstructed for wild-type and T634S-hERG mutant conditions and incorporated into the ten Tusscher et al. models of human ventricles at cell and tissue (1D, 2D and 3D) levels. Possible functional impacts of the T634S-hERG mutation were evaluated by its effects on action potential durations (APDs) and their rate-dependence (APDr) at the cell level; and on the QT interval of pseudo-ECGs, tissue vulnerability to unidirectional conduction block (VW), spiral wave dynamics and repolarization dispersion at the tissue level. It was found that the T634S-hERG mutation prolonged cellular APDs, steepened APDr, prolonged the QT interval, increased VW, destablized re-entry and augmented repolarization dispersion across the ventricle. Collectively, these results imply potential pro-arrhythmic effects of the T634S-hERG mutation, consistent with LQT2.
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Synthetic cannabinoid receptor agonists (SCRAs) have been associated with QT interval prolongation. Limited preclinical information on SCRA effects on cardiac electrogenesis results from the rapid emergence of new compounds and restricted research availability. We used two machine-learning-based tools to evaluate seven novel SCRAs' interaction potential with the hERG potassium channel, an important drug antitarget. Five SCRAs were predicted to have the ability to block the hERG channel by both prediction tools; ADB-FUBIATA was predicted to be a strong hERG blocker. ADB-5Br-INACA and ADB-4en-PINACA showed varied predictions. These findings highlight potentially proarrhythmic hERG block by novel SCRAs, necessitating detailed safety evaluations.
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BACKGROUND: The three-ringed polycyclic aromatic hydrocarbon (PAH) phenanthrene (Phe) has been implicated in the cardiotoxicity of petroleum-based pollution in aquatic systems, where it disrupts the contractile and electrical function of the fish heart. Phe is also found adsorbed to particulate matter and in the gas phase of air pollution, but to date, no studies have investigated the impact of Phe on mammalian cardiac function. OBJECTIVES: Our objectives were to determine the arrhythmogenic potential of acute Phe exposure on mammalian cardiac function and define the underlying mechanisms to provide insight into the toxicity risk to humans. METHODS: Ex vivo Langendorff-perfused mouse hearts were used to test the arrhythmogenic potential of Phe on myocardial function, and voltage- and current-clamp recordings were used to define underlying cellular mechanisms in isolated cardiomyocytes. RESULTS: Mouse hearts exposed to â¼8µM Phe for 15-min exhibited a significantly slower heart rate (p=0.0006, N=10 hearts), a prolonged PR interval (p=0.036, N=8 hearts), and a slower conduction velocity (p=0.0143, N=7 hearts). Whole-cell recordings from isolated cardiomyocytes revealed action potential (AP) duration prolongation (at 80% repolarization; p=0.0408, n=9 cells) and inhibition of key murine repolarizing currents-transient outward potassium current (Ito) and ultrarapid potassium current (IKur)-following Phe exposure. A significant reduction in AP upstroke velocity (p=0.0445, n=9 cells) and inhibition of the fast sodium current (INa; p=0.001, n=8 cells) and calcium current (ICa; p=0.0001) were also observed, explaining the slowed conduction velocity in intact hearts. Finally, acute exposure to â¼8µM Phe significantly increased susceptibility to arrhythmias (p=0.0455, N=9 hearts). DISCUSSION: To the best of our knowledge, this is the first evidence of direct inhibitory effects of Phe on mammalian cardiac electrical activity at both the whole-heart and cell levels. This electrical dysfunction manifested as an increase in arrhythmia susceptibility due to impairment of both conduction and repolarization. Similar effects in humans could have serious health consequences, warranting greater regulatory attention and toxicological investigation into this ubiquitous PAH pollutant generated from fossil-fuel combustion. https://doi.org/10.1289/EHP12775.
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Contaminantes Atmosféricos , Fenantrenos , Humanos , Ratones , Animales , Contaminantes Atmosféricos/toxicidad , Arritmias Cardíacas/inducido químicamente , Miocitos Cardíacos , Potenciales de Acción , Modelos Animales de Enfermedad , Fenantrenos/toxicidad , Potasio/farmacología , MamíferosRESUMEN
hERG (human Ether-à-go-go Related Gene)-encoded potassium channels underlie the cardiac rapid delayed rectifier (IKr) potassium current, which is a major target for antiarrhythmic agents and diverse non-cardiac drugs linked to the drug-induced form of long QT syndrome. E-4031 is a high potency hERG channel inhibitor from the methanesulphonanilide drug family. This study utilized a methanesulphonate-lacking E-4031 analogue, "E-4031-17", to evaluate the role of the methanesulphonamide group in E-4031 inhibition of hERG. Whole-cell patch-clamp measurements of the hERG current (IhERG) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT IhERG was inhibited by a half-maximal inhibitory concentration (IC50) of 15.8 nM, whilst the comparable value for E-4031-17 was 40.3 nM. Both compounds exhibited voltage- and time-dependent inhibition, but they differed in their response to successive applications of a long (10 s) depolarisation protocol, consistent with greater dissociation of E-4031-17 than the parent compound between applied commands. Voltage-dependent inactivation was left-ward voltage shifted for E-4031 but not for E-4031-17; however, inhibition by both compounds was strongly reduced by attenuated-inactivation mutations. Mutations of S6 and S5 aromatic residues (F656V, Y652A, F557L) greatly attenuated actions of both drugs. The S624A mutation also reduced IhERG inhibition by both molecules. Overall, these results demonstrate that the lack of a methanesulphonate in E-4031-17 is not an impediment to high potency inhibition of IhERG.
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We report the case of a 13-year-old female patient presenting with presyncope and palpitations. Her electrocardiogram revealed an abbreviation of the rate-corrected QT interval with imaging showing significant left ventricular dysfunction. Carnitine levels were measured as part of her diagnostic workup, discovering a rare, reversible cause of short QT syndrome (SQTS) and associated cardiomyopathy-primary carnitine deficiency (PCD) caused by a homozygous mutation in the SLC22A5 gene, leading to an in-frame deletion mutation (NP_003051.1:p.Phe23del) affecting the organic cation transporter 2 (OCTN2) protein. Following the treatment with oral carnitine supplementation, her QT interval returned to within the normal range with significant improvement in left ventricular function.
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Arritmias Cardíacas , Cardiomiopatías , Carnitina/deficiencia , Hiperamonemia , Enfermedades Musculares , Proteínas de Transporte de Catión Orgánico , Femenino , Humanos , Adolescente , Proteínas de Transporte de Catión Orgánico/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Electrocardiografía , Cardiomiopatías/complicaciones , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/genética , Mutación , Carnitina/uso terapéutico , Carnitina/genética , SíndromeRESUMEN
Pharmacological blockade of the IKr channel (hERG) by diverse drugs in clinical use is associated with the Long QT Syndrome that can lead to life threatening arrhythmia. Various computational tools including machine learning models (MLM) for the prediction of hERG inhibition have been developed to facilitate the throughput screening of drugs in development and optimise thus the prediction of hERG liabilities. The use of MLM relies on large libraries of training compounds for the quantitative structure-activity relationship (QSAR) modelling of hERG inhibition. The focus on inhibition omits potential effects of hERG channel agonist molecules and their associated QT shortening risk. It is instructive, therefore, to consider how known hERG agonists are handled by MLM. Here, two highly developed online computational tools for the prediction of hERG liability, Pred-hERG and HergSPred were probed for their ability to detect hERG activator drug molecules as hERG interactors. In total, 73 hERG blockers were tested with both computational tools giving overall good predictions for hERG blockers with reported IC50s below Pred-hERG and HergSPred cut-off threshold for hERG inhibition. However, for compounds with reported IC50s above this threshold such as disopyramide or sotalol discrepancies were observed. HergSPred identified all 20 hERG agonists selected as interacting with the hERG channel. Further studies are warranted to improve online MLM prediction of hERG related cardiotoxicity, by explicitly taking into account channel agonism as well as inhibition.
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Canales de Potasio Éter-A-Go-Go , Bloqueadores de los Canales de Potasio , Humanos , Bloqueadores de los Canales de Potasio/farmacología , Arritmias Cardíacas , Aprendizaje Automático , InternetRESUMEN
Previous studies have linked dysfunctional Ito arising from mutations to KCND3-encoded Kv4.3 and KCND2-encoded Kv4.2 to atrial fibrillation. Using computational models, this study aimed to investigate the mechanisms underlying pro-arrhythmic effects of the gain-of-function Kv4.3 (T361S, A545P) and Kv4.2 (S447R) mutations. Wild-type and mutant Ito formulations were developed from and validated against experimental data and incorporated into the Colman et al. model of human atrial cells. Single-cell models were incorporated into one- (1D) and two-dimensional (2D) models of atrial tissue, and a three-dimensional (3D) realistic model of the human atria. The three gain-of-function mutations had similar, albeit quantitatively different, effects: shortening of the action potential duration; lowering the plateau membrane potential, abbreviating the effective refractory period (ERP) and the wavelength (WL) of atrial excitation at the tissue level. Restitution curves for the WL, the ERP and the conduction velocity were leftward shifted, facilitating the conduction of atrial excitation waves at high excitation rates. The mutations also increased lifespan and stationarity of re-entry in both 2D and 3D simulations, which further highlighted a mutation-induced increase in spatial dispersion of repolarization. Collectively, these changes account for pro-arrhythmic effects of these Kv4.3 and Kv4.2 mutations in facilitating AF. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.
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Fibrilación Atrial , Humanos , Fibrilación Atrial/genética , Atrios Cardíacos , Potenciales de Acción/fisiología , Mutación , Simulación por ComputadorRESUMEN
The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a â¼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.
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Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.
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Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Humanos , Apamina/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/química , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismoRESUMEN
Loss of function (LOF) mutations of voltage sensitive K+ channel proteins hERG (Kv11.1) and KCNQ1 (Kv7.1) account for the majority of instances of congenital Long QT Syndrome (cLQTS) with the dominant molecular phenotype being a mistrafficking one resulting from protein misfolding. We explored the use of Evolutionary Coupling (EC) analysis, which identifies evolutionarily conserved pairwise amino acid interactions that may contribute to protein structural stability, to identify regions of the channels susceptible to misfolding mutations. Comparison with published experimental trafficking data for hERG and KCNQ1 showed that the method strongly predicts "scaffolding" regions of the channel membrane domains and has useful predictive power for trafficking phenotypes of individual variants. We identified a region in and around the cytoplasmic S2-S3 loop of the hERG Voltage Sensor Domain (VSD) as susceptible to destabilising mutation, and this was confirmed using a quantitative LI-COR ® based trafficking assay that showed severely attenuated trafficking in eight out of 10 natural hERG VSD variants selected using EC analysis. Our analysis highlights an equivalence in the scaffolding structures of the hERG and KCNQ1 membrane domains. Pathogenic variants of ion channels with an underlying mistrafficking phenotype are likely to be located within similar scaffolding structures that are identifiable by EC analysis.
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Rett Syndrome (RTT) is a congenital, X-chromosome-linked developmental disorder characterized by developmental delay, dysautonomia, and breathing irregularities. RTT is also associated with sudden death and QT intervals are prolonged in some RTT patients. Most individuals with RTT have mutations in the MECP2 gene. Whilst there is some evidence for QT prolongation in mouse models of RTT, there is comparatively little information on how loss of Mecp2 function affects ventricular action potentials (APs) and, to-date, none on ventricular APs from female RTT mice. Accordingly, the present study was conducted to determine ECG and ventricular AP characteristics of Mecp2Null/+ female mice. ECG recordings from 12-13 month old female Mecp2Null/+ mice showed prolonged rate corrected QT (QTc) intervals compared to wild-type (WT) controls. Although Mecp2Null/+ animals exhibited longer periods of apnoea than did controls, no correlation between apnoea length and QTc interval was observed. Action potentials (APs) from Mecp2Null/+ myocytes had longer APD90 values than those from WT myocytes and showed augmented triangulation. Application of the investigational INa,Late inhibitor GS-6615 (eleclazine; 10 µM) reduced both APD90 and AP triangulation in Mecp2Null/+ and WT myocytes. These results constitute the first direct demonstration of delayed repolarization in Mecp2Null/+ myocytes and provide further evidence that GS-6615 may have potential as an intervention against QT prolongation in RTT.
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Síndrome de QT Prolongado , Síndrome de Rett , Potenciales de Acción , Animales , Apnea , Modelos Animales de Enfermedad , Femenino , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Noqueados , Síndrome de Rett/genéticaRESUMEN
Loss of ventricular action potential (AP) early phase 1 repolarization may contribute to the impaired Ca2+ release and increased risk of sudden cardiac death in heart failure. Therefore, restoring AP phase 1 by augmenting the fast transient outward K+ current (Itof) might be beneficial, but direct experimental evidence to support this proposition in failing cardiomyocytes is limited. Dynamic clamp was used to selectively modulate the contribution of Itof to the AP and Ca2+ transient in both normal (guinea pig and rabbit) and in failing rabbit cardiac myocytes. Opposing native Itof in non-failing rabbit myocytes increased Ca2+ release heterogeneity, late Ca2+ sparks (LCS) frequency and AP duration. (APD). In contrast, increasing Itof in failing myocytes and guinea pig myocytes (the latter normally lacking Itof) increased Ca2+ transient amplitude, Ca2+ release synchrony, and shortened APD. Computer simulations also showed faster Ca2+ transient decay (mainly due to fewer LCS), decreased inward Na+/Ca2+ exchange current and APD. When the Itof conductance was increased to ~0.2 nS/pF in failing cells (a value slightly greater than seen in typical human epicardial myocytes), Ca2+ release synchrony improved and AP duration decreased slightly. Further increases in Itof can cause Ca2+ release to decrease as the peak of the bell-shaped ICa-voltage relationship is passed and premature AP repolarization develops. These results suggest that there is an optimal range for Itof enhancement that may support Ca2+ release synchrony and improve electrical stability in heart failure with the caveat that uncontrolled Itof enhancement should be avoided.
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Insuficiencia Cardíaca , Miocitos Cardíacos , Humanos , Conejos , Cobayas , Animales , Miocitos Cardíacos/fisiología , Potenciales de Acción/fisiología , Ventrículos Cardíacos , Sodio , CalcioRESUMEN
Heart failure is associated with electrical remodeling of the electrical properties and kinetics of the ion channels and transporters that are responsible for cardiac action potentials. However, it is still unclear whether heart failure-induced ionic remodeling can affect the conduction of excitation waves at the Purkinje fiber-ventricle junction contributing to pro-arrhythmic effects of heart failure, as the complexity of the heart impedes a detailed experimental analysis. The aim of this study was to employ computational models to investigate the pro-arrhythmic effects of heart failure-induced ionic remodeling on the cardiac action potentials and excitation wave conduction at the Purkinje fiber-ventricle junction. Single cell models of canine Purkinje fiber and ventricular myocytes were developed for control and heart failure. These single cell models were then incorporated into one-dimensional strand and three-dimensional wedge models to investigate the effects of heart failure-induced remodeling on propagation of action potentials in Purkinje fiber and ventricular tissue and at the Purkinje fiber-ventricle junction. This revealed that heart failure-induced ionic remodeling of Purkinje fiber and ventricular tissue reduced conduction safety and increased tissue vulnerability to the genesis of the unidirectional conduction block. This was marked at the Purkinje fiber-ventricle junction, forming a potential substrate for the genesis of conduction failure that led to re-entry. This study provides new insights into proarrhythmic consequences of heart failure-induced ionic remodeling.
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Rett syndrome (RTT) is a severe developmental disorder that is strongly linked to mutations in the MECP2 gene. RTT has been associated with sudden unexplained death and ECG QT interval prolongation. There are mixed reports regarding QT prolongation in mouse models of RTT, with some evidence that loss of Mecp2 function enhances cardiac late Na current, INa,Late. The present study was undertaken in order to investigate both ECG and ventricular AP characteristics in the Mecp2Null/Y male murine RTT model and to interrogate both fast INa and INa,Late in myocytes from the model. ECG recordings from 8-10-week-old Mecp2Null/Y male mice revealed prolongation of the QT and rate corrected QT (QTc) intervals and QRS widening compared to wild-type (WT) controls. Action potentials (APs) from Mecp2Null/Y myocytes exhibited longer APD75 and APD90 values, increased triangulation and instability. INa,Late was also significantly larger in Mecp2Null/Y than WT myocytes and was insensitive to the Nav1.8 inhibitor A-803467. Selective recordings of fast INa revealed a decrease in peak current amplitude without significant voltage shifts in activation or inactivation V0.5. Fast INa 'window current' was reduced in RTT myocytes; small but significant alterations of inactivation and reactivation time-courses were detected. Effects of two INa,Late inhibitors, ranolazine and GS-6615 (eleclazine), were investigated. Treatment with 30 µM ranolazine produced similar levels of inhibition of INa,Late in WT and Mecp2Null/Y myocytes, but produced ventricular AP prolongation not abbreviation. In contrast, 10 µM GS-6615 both inhibited INa,Late and shortened ventricular AP duration. The observed changes in INa and INa,Late can account for the corresponding ECG changes in this RTT model. GS-6615 merits further investigation as a potential treatment for QT prolongation in RTT.
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Síndrome de QT Prolongado , Síndrome de Rett , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ranolazina , Síndrome de Rett/genética , Sodio , Canales de SodioRESUMEN
The T618I KCNH2-encoded hERG mutation is the most frequently observed mutation in genotyped cases of the congenital short QT syndrome (SQTS), a cardiac condition associated with ventricular fibrillation and sudden death. Most T618I hERG carriers exhibit a pronounced U wave on the electrocardiogram and appear vulnerable to ventricular, but not atrial fibrillation (AF). The basis for these effects is unclear. This study used the action potential (AP) voltage clamp technique to determine effects of the T618I mutation on hERG current (IhERG) elicited by APs from different cardiac regions. Whole-cell patch-clamp recordings were made at 37 °C of IhERG from hERG-transfected HEK-293 cells. Maximal IhERG during a ventricular AP command was increased â¼4-fold for T618I IhERG and occurred much earlier during AP repolarization. The mutation also increased peak repolarizing currents elicited by Purkinje fibre (PF) APs. Maximal wild-type (WT) IhERG current during the PF waveform was 87.2 ± 4.5% of maximal ventricular repolarizing current whilst for the T618I mutant, the comparable value was 47.7 ± 2.7%. Thus, the T618I mutation exacerbated differences in repolarizing IhERG between PF and ventricular APs; this could contribute to heterogeneity of ventricular-PF repolarization and consequently to the U waves seen in T618I carriers. The comparatively shorter duration and lack of pronounced plateau of the atrial AP led to a smaller effect of the T618I mutation during the atrial AP, which may help account for the lack of reported AF in T618I carriers. Use of a paired ventricular AP protocol revealed an alteration to protective IhERG transients that affect susceptibility to premature excitation late in AP repolarization/early in diastole. These observations may help explain altered arrhythmia susceptibility in this form of the SQTS.
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Potenciales de Acción/genética , Arritmias Cardíacas/genética , Canal de Potasio ERG1/genética , Mutación , Técnicas de Placa-Clamp/métodos , Electrocardiografía/métodos , Células HEK293 , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Humanos , Ramos Subendocárdicos/metabolismoRESUMEN
The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 µmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.
Asunto(s)
Insuficiencia Cardíaca , Canales de Potasio Shal , Potenciales de Acción/fisiología , Animales , Células HEK293 , Humanos , Miocitos Cardíacos/metabolismo , Conejos , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , TransgenesRESUMEN
The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K+ channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (IhERG) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT IhERG1a was inhibited by phenanthrene with an IC50 of 17.6 ± 1.7 µM, whilst IhERG1a/1b exhibited an IC50 of 1.8 ± 0.3 µM. WT IhERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented IhERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit IhERG, as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K+ channel by directly interacting with the channel, binding to a distinct site in the channel pore domain.