RESUMEN
The collar of the pulmonary vein (PV) is the focal point for the initiation of atrial arrhythmias, but the mechanisms underlying how PV cells differ from neighboring left atrial tissue are unclear. We examined the biophysical and molecular properties of INa in cells isolated from the canine pulmonary sleeve and compared the properties to left atrial tissue. PV and left atrial myocytes were isolated and patch clamp techniques were used to record INa. Action potential recordings from either tissue type were made using high-resistance electrodes. mRNA was determined using quantitative RT-PCR and proteins were determined by Western blot. Analysis of the action potential characteristics showed that PV tissue had a lower Vmax compared with left atrial tissue. Fast INa showed that current density was slightly lower in PV cells compared with LA cells (-96 ± 18.7 pA/pF vs. -120 ± 6.7 pA/pF, respectively, p < 0.05). The recovery from inactivation of INa in PV cells was slightly slower but no marked difference in steady-state inactivation was noted. Analysis of late INa during a 225-ms pulse showed that late INa was significantly smaller in PV cells compared to LA cells at all measured time points into the pulse. These results suggest PV cells have lower density of both peak and late INa. Molecular analysis of Nav1.5 and the four beta subunits showed lower levels of Nav1.5 as well as Navß1 subunits, confirming the biophysical findings. These data show that a lower density of INa may lead to depression of excitability and predispose the PV collar to re-entrant circuits under pathophysiological conditions.
Asunto(s)
Potenciales de Acción , Atrios Cardíacos/citología , Miocitos Cardíacos/fisiología , Miocitos del Músculo Liso/fisiología , Venas Pulmonares/citología , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Células Cultivadas , Perros , Femenino , Masculino , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/metabolismo , Sodio/metabolismoRESUMEN
BACKGROUND: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). METHOD: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. RESULTS: BCs exhibited spontaneous beating (60.5±2.6 bpm) and maximum-diastolic-potential (MDP) of 67.8±0.8 mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7±1.9 pA/pF at +40 mV; n=14). Recovery of Ito from inactivation (at -80 mV) showed slow kinetics (τ1=200±110 ms (12%) and τ2=2380±240 ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. CONCLUSION: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Potenciales de la Membrana/fisiología , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Proteínas de Interacción con los Canales Kv/metabolismo , Canal de Potasio Kv1.4/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Miocitos Cardíacos/citología , Canales de Potasio Shal/metabolismoRESUMEN
We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1-2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 µM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.
Asunto(s)
Corazón/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Perros , Ganglios/efectos de los fármacos , Ganglios/metabolismo , Ganglios/fisiología , Corazón/efectos de los fármacos , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Tetraetilamonio/farmacologíaRESUMEN
INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. It is unclear whether adult myocytes or hiPSC-CMs are the better platform for cardiac safety pharmacology. We examined the biophysical and molecular properties of INa in adult myocytes and hiPSC-CMs. METHODS: hiPSC-CMs were plated at low density. Atrial and ventricular cells were obtained from dog hearts. Whole cell patch clamp was used to record INa. RESULTS: Voltage clamp recordings showed a large INa in all three cell types but different densities. Small differences in steady-state inactivation and recovery from inactivation were noted in the three cell types. Application of lidocaine to the three cell types showed a similar pattern of block of INa under voltage clamp; however, lidocaine produced different effects on AP waveform under current clamp. AP clamp experiments showed that application of ventricular or atrial cell waveforms to the same hiPSC-CM elicited a large INa while application of a sinoatrial node waveform elicited no INa. Molecular analysis of Na+ channel subunits showed SCN5A and SCN1B-4B were expressed in adult cells and iPSC-CMs. However, iPSC-CMs express both fetal (exon 6A) and adult (exon 6) isoforms of SCN5A. DISCUSSION: There are major differences in INa density and smaller differences in other biophysical properties of INa in adult atrial, ventricular, and hiPSC-CMs. The depolarized maximum diastolic potential coupled with the presence of phase 4 depolarization limits the contribution of INa in hiPSC-CM action potentials. Our results suggest that hiPSC-CMs may be useful for drug screening of Na+ channel inhibitors under voltage clamp but not current clamp.
Asunto(s)
Potenciales de Acción/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Sodio/metabolismo , Adulto , Ventrículos Cardíacos/metabolismo , Humanos , Técnicas de Placa-Clamp/métodosRESUMEN
INTRODUCTION: Long QT Syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and life-threatening polymorphic ventricular tachyarrhythmias. LQT1 caused by KCNQ1 mutations is the most common form of LQTS. METHODS AND RESULTS: Patients diagnosed with LQTS were screened for disease-associated mutations in KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, and SCN5A. A novel mutation was identified in KCNQ1 caused by a three-base deletion at the position 824-826, predicting a deletion of phenylalanine at codon 275 in segment 5 of KCNQ1 (DeltaF275). Wild-type (WT) and DeltaF275-KCNQ1 constructs were generated and transiently transfected together with a KCNE1 construct in CHO-K1 cells to characterize the properties of the slowly activating delayed rectifier current (IKs) using conventional whole-cell patch-clamp techniques. Cells transfected with WT-KCNQ1 and KCNE1 (1:1.3 molar ratio) produced slowly activating outward current with the characteristics of IKs. Tail current density measured at -40 mV following a two-second step to +60 mV was 381.3 +/- 62.6 pA/pF (n = 11). Cells transfected with DeltaF275-KCNQ1 and KCNE1 exhibited essentially no current. (Tail current density: 0.8 +/- 2.1 pA/pF, n = 11, P = 0.00001 vs WT). Cotransfection of WT- and DeltaF275- KCNQ1 (50/50), along with KCNE1, produced little to no current (tail current density: 10.3 +/- 3.5 pA/pF, n = 11, P = 0.00001 vs WT alone), suggesting a potent dominant negative effect. Immunohistochemistry showed normal membrane trafficking of DeltaF275-KCNQ1. CONCLUSION: Our data suggest that a DeltaF275 mutation in KCNQ1 is associated with a very potent dominant negative effect leading to an almost complete loss of function of IKs and that this defect underlies a LQT1 form of LQTS.
Asunto(s)
Genes Dominantes/genética , Predisposición Genética a la Enfermedad/genética , Sistema de Conducción Cardíaco/fisiopatología , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/clasificación , Síndrome de QT Prolongado/fisiopatología , Adolescente , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Humanos , Síndrome de QT Prolongado/genéticaRESUMEN
BACKGROUND: The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases. OBJECTIVES: The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons. METHODS: Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia. RESULTS: APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Na(v)1.5 channels. TTX sensitivity (IC(50) = 1.17 microM) of the TTX-R component was in the range reported for Na(v)1.5 channels. Expression of Na(v)1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing. CONCLUSION: Our results suggest that canine intracardiac neurons functionally express Na(v)1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Na(v)1.5 channels, including Brugada syndrome and LQT3.
Asunto(s)
Ganglios Autónomos/metabolismo , Expresión Génica , Corazón/inervación , Proteínas Musculares/genética , ARN/genética , Canales de Sodio/genética , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Perros , Ganglios Autónomos/citología , Ganglios Autónomos/efectos de los fármacos , Corazón/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Proteínas Musculares/biosíntesis , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Canales de Sodio/biosíntesis , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND: Ganglionated plexus have been developed as additional ablation targets to improve the outcome of atrial fibrillation (AF) besides pulmonary vein isolation. Recent studies implicated an intimate relationship between neuronal sodium channel Nav1.8 (encoded by SCN10A) and AF. The underlying mechanism between Nav1.8 and AF remains unclear. This study aimed to determine the role of Nav1.8 in cardiac electrophysiology in an acute AF model and explore possible therapeutic targets. METHODS AND RESULTS: Immunohistochemical study was used on canine cardiac ganglionated plexus. Both Nav1.5 and Nav1.8 were expressed in ganglionated plexus with canonical neuronal markers. Sixteen canines were randomly administered either saline or the Nav1.8 blocker A-803467. Electrophysiological study was compared between the 2 groups before and after 6-hour rapid atrial pacing. Compared with the control group, administration of A-803467 decreased the incidence of AF (87.5% versus 25.0%, P<0.05), shortened AF duration, and prolonged AF cycle length. A-803467 also significantly suppressed the decrease in the effective refractory period and the increase in effective refractory period dispersion and cumulative window of vulnerability caused by rapid atrial pacing in all recording sites. Patch clamp study was performed under 100 nmol/L A-803467 in TSA201 cells cotransfected with SCN10A-WT, SCN5A-WT, and SCN3B-WT. INa,P was reduced by 45.34% at -35 mV, and INa,L by 68.57% at -20 mV. Evident fast inactivation, slow recovery, and use-dependent block were also discovered after applying the drug. CONCLUSIONS: Our study demonstrates that Nav1.8 could exert its effect on electrophysiological characteristics through cardiac ganglionated plexus. It indicates that Nav1.8 is a novel target in understanding cardiac electrophysiology and SCN10A-related arrhythmias.
Asunto(s)
Fibrilación Atrial/metabolismo , Corazón/inervación , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Neuronas/metabolismo , Enfermedad Aguda , Compuestos de Anilina/farmacología , Animales , Fibrilación Atrial/terapia , Perros , Furanos/farmacología , Corazón/efectos de los fármacos , Inmunohistoquímica , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.8/genética , Técnicas de Placa-Clamp , Factores de Tiempo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Subunidad beta-3 de Canal de Sodio Activado por Voltaje/genéticaRESUMEN
Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the ß1 and ß4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular ß subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory ß subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.
Asunto(s)
Ganglios/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Miocardio/citología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Perros , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Datos de Secuencia Molecular , Neuronas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (nâ=â103; 37°C) and to examine the response to 5 µM E-4031 (nâ=â21) or BaCl(2) (nâ=â22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (nâ=â49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; nâ=â21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2), at concentrations that selectively block I(K1) (50-100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied, but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells, but robust expression of I(Kr.) In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.
Asunto(s)
Potenciales de Acción , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potenciales de Acción/efectos de los fármacos , Compuestos de Bario/farmacología , Diferenciación Celular/efectos de los fármacos , Cloruros/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Piperidinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/genética , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Mutations in KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1 (IK1 or IKir2.1), have been identified in Andersen-Tawil syndrome. Andersen-Tawil syndrome is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features at times mimicking catecholaminergic polymorphic ventricular tachycardia. METHODS AND RESULTS: Our proband displayed dysmorphic features including micrognathia, clinodactyly, and syndactyly and exhibited multiform extrasystoles and bidirectional ventricular tachycardia both at rest and during exercise testing. The patient's symptoms continued after administration of nadolol but subsided after treatment with flecainide. Molecular genetic screening revealed a novel heterozygous mutation (c.779G>C/p.R260P) in KCNJ2. Whole-cell patch-clamp studies conducted in TSA201 cells transfected with wild-type human KCNJ2 cDNA (WT-KCNJ2) yielded robust IKir2.1 but no measurable current in cells expressing the R260P mutant. Coexpression of WT and R260P-KCNJ2 (heterozygous expression) yielded a markedly reduced inward IKir2.1 compared with WT alone (-36.5±9.8 pA/pF versus -143.5±11.4 pA/pF, n=8 for both, P<0.001, respectively, at -90 mV), indicating a strong dominant negative effect of the mutant. The outward component of IKir2.1 measured at -50 mV was also markedly reduced with the heterozygous expression versus WT (0.52±5.5 pA/pF versus 23.4±6.7 pA/pF, n=8 for both, P<0.001, respectively). Immunocytochemical analysis indicates that impaired trafficking of R260P-KCNJ2 channels. CONCLUSIONS: We report a novel de novo KCNJ2 mutation associated with classic phenotypic features of Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia mimicry. The R260P mutation produces a strong dominant negative effect leading to marked suppression of IK1 secondary to a trafficking defect.
Asunto(s)
Síndrome de Andersen/complicaciones , Síndrome de Andersen/genética , Fenómenos Biofísicos , Mutación/genética , Canales de Potasio de Rectificación Interna/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Síndrome de Andersen/diagnóstico por imagen , Síndrome de Andersen/fisiopatología , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/diagnóstico por imagen , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Secuencia de Bases , Línea Celular , Niño , Análisis Mutacional de ADN , Femenino , Flecainida/uso terapéutico , Genes Dominantes , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Linaje , Canales de Potasio de Rectificación Interna/química , Taquicardia Ventricular/complicaciones , Taquicardia Ventricular/tratamiento farmacológico , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatología , UltrasonografíaRESUMEN
BACKGROUND: L-type calcium channel (LTCC) mutations have been associated with Brugada syndrome (BrS), short QT (SQT) syndrome, and Timothy syndrome (LQT8). Little is known about the extent to which LTCC mutations contribute to the J-wave syndromes associated with sudden cardiac death. OBJECTIVE: The purpose of this study was to identify mutations in the α1, ß2, and α2δ subunits of LTCC (Ca(v)1.2) among 205 probands diagnosed with BrS, idiopathic ventricular fibrillation (IVF), and early repolarization syndrome (ERS). CACNA1C, CACNB2b, and CACNA2D1 genes of 162 probands with BrS and BrS+SQT, 19 with IVF, and 24 with ERS were screened by direct sequencing. METHODS/RESULTS: Overall, 23 distinct mutations were identified. A total of 12.3%, 5.2%, and 16% of BrS/BrS+SQT, IVF, and ERS probands displayed mutations in α1, ß2, and α2δ subunits of LTCC, respectively. When rare polymorphisms were included, the yield increased to 17.9%, 21%, and 29.1% for BrS/BrS+SQT, IVF, and ERS probands, respectively. Functional expression of two CACNA1C mutations associated with BrS and BrS+SQT led to loss of function in calcium channel current. BrS probands displaying a normal QTc had additional variations known to prolong the QT interval. CONCLUSION: The study results indicate that mutations in the LTCCs are detected in a high percentage of probands with J-wave syndromes associated with inherited cardiac arrhythmias, suggesting that genetic screening of Ca(v) genes may be a valuable diagnostic tool in identifying individuals at risk. These results are the first to identify CACNA2D1 as a novel BrS susceptibility gene and CACNA1C, CACNB2, and CACNA2D1 as possible novel ERS susceptibility genes.