RESUMO
Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 (DLG1) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current (Ito) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease.NEW & NOTEWORTHY The gene encoding SAP97 (DLG1) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1-encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.
Assuntos
Arritmias Cardíacas/metabolismo , Proteína 1 Homóloga a Discs-Large/metabolismo , Coração/fisiopatologia , Miocárdio/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Proteína 1 Homóloga a Discs-Large/genética , Humanos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismoRESUMO
The inward rectifier potassium current, IK1, contributes to the terminal phase of repolarization of the action potential (AP), as well as the value and stability of the resting membrane potential. Regional variation in IK1 has been noted in the canine heart, but the biophysical properties have not been directly compared. We examined the properties and functional contribution of IK1 in isolated myocytes from ventricular, atrial and Purkinje tissue. APs were recorded from canine left ventricular midmyocardium, left atrial and Purkinje tissue. The terminal rate of repolarization of the AP in ventricle, but not in Purkinje, depended on changes in external K(+) ([K(+)]o). Isolated ventricular myocytes had the greatest density of IK1 while atrial myocytes had the lowest. Furthermore, the outward component of IK1 in ventricular cells exhibited a prominent outward component and steep negative slope conductance, which was also enhanced in 10 mM [K(+)]o. In contrast, both Purkinje and atrial cells exhibited little outward IK1, even in the presence of 10 mM [K(+)]o, and both cell types showed more persistent current at positive potentials. Expression of Kir2.1 in the ventricle was 76.9-fold higher than that of atria and 5.8-fold higher than that of Purkinje, whereas the expression of Kir2.2 and Kir2.3 subunits was more evenly distributed in Purkinje and atria. Finally, AP clamp data showed distinct contributions of IK1 for each cell type. IK1 and Kir2 subunit expression varies dramatically in regions of the canine heart and these regional differences in Kir2 expression likely underlie regional distinctions in IK1 characteristics, contributing to variations in repolarization in response to in [K(+)]o changes.
Assuntos
Potenciais de Ação/fisiologia , Coração/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Cães , Feminino , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Ativação do Canal Iônico , Cinética , Masculino , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Poliaminas/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Potenciais da Membrana/fisiologia , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Canal de Potássio Kv1.4/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/citologia , Canais de Potássio Shal/metabolismoRESUMO
BACKGROUND: Developmental changes in the electrical characteristics of the ventricular myocardium are not well defined. This study examines the contribution of inwardly rectifying K(+) current (IK1), transient outward K(+) current (Ito), delayed rectifier K(+) currents (IKr and IKs) and sodium channel current (INa) to repolarization in the canine neonate myocardium. METHODS: Single myocytes isolated from the left ventricle of 2-3week old canine neonate hearts were studied using patch-clamp techniques. RESULTS: Neonate cells were ~6-fold smaller than those of adults (28.8±8.8 vs. 176±6.7pF). IK1 was larger in neonate myocytes and displayed a substantial inward component and an outward component with negative slope conductance, peaking at -60mV (4.13 pA/pF). IKr tail currents (at -40mV), were small (<20pA). IKs could not be detected, even after exposure to isoproterenol (100nM). Ito was also absent in the neonate, consistent with the absence of a phase 1 in the action potential. Peak INa, late INa and ICa were smaller in the neonate compared with adults. KCND3, KCNIP2 and KCNQ1 mRNA expression was half, while KCNH2 was equal and KCNJ2 was greater in the neonate when compared with adults. CONCLUSIONS: Two major repolarizing K(+) currents (IKs and Ito) present in adult ventricular cells are absent in the 2week old neonate. Peak and late INa are significantly smaller in the neonate. Our results suggest that the absence of these two currents in the neonate heart may increase the susceptibility to arrhythmias under certain long QT conditions.
Assuntos
Canais Iônicos/genética , Canais Iônicos/metabolismo , Função Ventricular/fisiologia , Potenciais de Ação , Animais , Animais Recém-Nascidos , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Cães , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Potássio/metabolismo , Canais de Potássio/fisiologia , Piridinas/farmacologia , Sódio/metabolismo , Função Ventricular/efeitos dos fármacosRESUMO
Heritable arrhythmia syndromes, including Brugada syndrome (BrS) and idiopathic ventricular fibrillation (IVF), may serve as the pathogenic basis for autopsy-negative sudden unexplained death (SUD) and sudden infant death syndrome (SIDS). Emerging evidence has linked perturbations in the transient outward current (I(to) ) conducted by the KCND3-encoded Kv4.3 pore-forming α-subunit to BrS or IVF. However, the contribution of KCND3 mutations to autopsy-negative SUD/SIDS is unknown. To investigate the potential association between KCND3 and SUD/SIDS, mutational analysis of KCND3 was conducted in 123 SUDS and 292 SIDS victims using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct sequencing. Overall, one SIDS case (<1.0%) and two SUDS cases (1.6%) harbored potentially pathogenic mutations in KCND3. The novel p.Val392Ile, p.Ser530Pro, and p.Gly600Arg mutations involved highly conserved residues and were absent in 1,560 reference alleles. Although the SIDS-associated p.Ser530Pro mutation demonstrated a wild-type (WT) electrophysiological phenotype when heterologously expressed, the SUDS-associated p.Val392Ile and p.Gly600Arg mutations significantly increased peak current density at +40 mV in comparison with WT by 100.4% (P < 0.05) and 50.4% (P < 0.05), respectively. p.Val392Ile also slowed recovery from inactivation 3.6-fold, indicating a mixed electrophysiological phenotype. This is the first report indicating that KCND3 may serve as a rare genetic substrate in the pathogenesis of SUDS but not SIDS cases.
Assuntos
Morte Súbita Cardíaca/etiologia , Mutação , Canais de Potássio Shal/genética , Morte Súbita do Lactente/genética , Potenciais de Ação , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Autopsia , Linhagem Celular , Criança , Pré-Escolar , Estudos de Coortes , Morte Súbita Cardíaca/epidemiologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Prevalência , Canais de Potássio Shal/metabolismo , Morte Súbita do Lactente/diagnóstico , Morte Súbita do Lactente/epidemiologia , Adulto JovemRESUMO
Block of Na(+) channel conductance by ranolazine displays marked atrial selectivity that is an order of magnitude higher that of other class I antiarrhythmic drugs. Here, we present a Markovian model of the Na(+) channel gating, which includes activation-inactivation coupling, aimed at elucidating the mechanisms underlying this potent atrial selectivity of ranolazine. The model incorporates experimentally observed differences between atrial and ventricular Na(+) channel gating, including a more negative position of the steady-state inactivation curve in atrial versus ventricular cells. The model assumes that ranolazine requires a hydrophilic access pathway to the channel binding site, which is modulated by both activation and inactivation gates of the channel. Kinetic rate constants were obtained using guarded receptor analysis of the use-dependent block of the fast Na(+) current (I(Na)). The model successfully reproduces all experimentally observed phenomena, including the shift of channel availability, the sensitivity of block to holding or diastolic potential, and the preferential block of slow versus fast I(Na.) Using atrial and ventricular action potential-shaped voltage pulses, the model confirms significantly greater use-dependent block of peak I(Na) in atrial versus ventricular cells. The model highlights the importance of action potential prolongation and of a steeper voltage dependence of the time constant of unbinding of ranolazine from the atrial Na(+) channel in the development of use-dependent I(Na) block. Our model predictions indicate that differences in channel gating properties as well as action potential morphology between atrial and ventricular cells contribute equally to the atrial selectivity of ranolazine. The model indicates that the steep voltage dependence of ranolazine interaction with the Na(+) channel at negative potentials underlies the mechanism of the predominant block of I(Na) in atrial cells by ranolazine.
Assuntos
Acetanilidas/farmacologia , Coração/efeitos dos fármacos , Piperazinas/farmacologia , Bloqueadores dos Canais de Sódio , Animais , Simulação por Computador , Relação Dose-Resposta a Droga , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Cinética , Cadeias de Markov , Modelos Biológicos , Modelos Estatísticos , Miócitos Cardíacos/efeitos dos fármacos , Ranolazina , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismoRESUMO
Atrial-selective inhibition of cardiac Na(+) channel current (I(Na)) and I(Na)-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study examined the basis for the atrial-selective actions of ranolazine. Whole cell I(Na) was recorded at 15°C in canine atrial and ventricular myocytes and in human embryonic kidney (HEK)-293 cells expressing SCN5A. Tonic block was negligible at holding potentials from -140 to -100 mV, suggesting minimal drug interactions with the closed state. Trains of 40 pulses were elicited over a range of holding potentials to determine use-dependent block. Guarded receptor formalism was used to analyze the development of block during pulse trains. Use-dependent block by ranolazine increased at more depolarized holding potentials, consistent with an interaction of the drug with either preopen or inactivated states, but was unaffected by longer pulse durations between 5 and 200 ms, suggesting a weak interaction with the inactivated state. Block was significantly increased at shorter diastolic intervals between 20 and 200 ms. Responses in atrial and ventricular myocytes and in HEK-293 cells displayed a similar pattern. Ranolazine is an open state blocker that unbinds from closed Na(+) channels unusually fast but is trapped in the inactivated state. Kinetic rates of ranolazine interactions with different states of atrial and ventricular Na(+) channels were similar. Our data suggest that the atrial selectivity of ranolazine is due to a more negative steady-state inactivation curve, less negative resting membrane potential, and shorter diastolic intervals in atrial cells compared with ventricular cells at rapid rates.
Assuntos
Acetanilidas/farmacologia , Piperazinas/farmacologia , Bloqueadores dos Canais de Sódio , Acetanilidas/metabolismo , Algoritmos , Animais , Sítios de Ligação/efeitos dos fármacos , Interpretação Estatística de Dados , Cães , Feminino , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cinética , Modelos Lineares , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Piperazinas/metabolismo , Ranolazina , Canais de Sódio/metabolismoRESUMO
BACKGROUND: Inherited loss of function mutations in SCN5A have been linked to overlapping syndromes including cardiac conduction disease and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood. METHODS: Direct sequencing was performed in a family with cardiac conduction disease. Wild-type (WT) and mutant channels were expressed in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant genes were used to assess channel trafficking. RESULTS: A novel SCN5A mutation, P1008S, was identified in all family members displaying first-degree atrioventricular block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (P < 0.001). Confocal microscopy showed that WT channels tagged with GFP were localized on the cell surface, whereas GFP-tagged P1008S channels remained trapped in intracellular organelles. Trafficking could be rescued by incubation at room temperature, but not by incubation with mexiletine (300 muM) at 37 degrees C. We also identified a novel polymorphism (D601E) in CACNB2b that slowed inactivation of L-type calcium current (I(Ca,L)), significantly increased total charge. Using the Luo-Rudy action potential (AP) model, we show that the reduction in sodium current (I(Na)) can cause loss of the right ventricular epicardial AP dome in the absence but not in the presence of the slowed inactivation of I(Ca,L). Slowed conduction was present in both cases. CONCLUSIONS: Our results suggest genetic variations leading to a loss-of-function in I(Na) coupled with a gain of function in I(Ca,L) may underlie the development of cardiac conduction disease without BrS.
Assuntos
Bradicardia/genética , Canais de Cálcio Tipo L/genética , Bloqueio Cardíaco/genética , Sistema de Condução Cardíaco/fisiopatologia , Proteínas Musculares/genética , Mutação , Polimorfismo de Nucleotídeo Único , Canais de Sódio/genética , Adolescente , Alelos , Análise de Variância , Bradicardia/fisiopatologia , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Feminino , Bloqueio Cardíaco/fisiopatologia , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5 , Linhagem , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Canal de Potássio ERG1 , Eletrocardiografia , Feminino , Variação Genética , Humanos , Dados de Sequência Molecular , Mutação , Fenótipo , Polimorfismo GenéticoRESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Sódio/metabolismo , Adulto , Ventrículos do Coração/metabolismo , Humanos , Técnicas de Patch-Clamp/métodosRESUMO
BACKGROUND: Timothy syndrome (TS) is a rare multisystem genetic disorder characterized by a myriad of abnormalities, including QT prolongation, syndactyly, and neurologic symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel; however, some cases remain genetically elusive. OBJECTIVE: The purpose of this study was to identify the genetic cause of TS in a patient who did not harbor a CACNA1C mutation in exon 8/A, and was negative for all other plausible genetic substrates. METHODS: Diagnostic exome sequencing was used to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole-cell patch-clamp technique, and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. RESULTS: Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiologic analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential as well as the development of spontaneous early afterdepolarizations. CONCLUSION: Through expanded whole exome sequencing, we identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiologic experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted.
Assuntos
Canais de Cálcio Tipo L/genética , Bloqueio Cardíaco/congênito , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Mutação/genética , Sindactilia/diagnóstico , Sindactilia/genética , Transtorno Autístico , Bloqueio Cardíaco/diagnóstico , Bloqueio Cardíaco/genética , Humanos , Lactente , MasculinoRESUMO
BACKGROUND: Hypothermia has been reported to induce ventricular tachycardia and fibrillation (VT/VF) in patients with early repolarization (ER) pattern. This study examines the cellular mechanisms underlying VT/VF associated with hypothermia in an experimental model of ER syndrome and examines the effectiveness of quinidine, cilostazol, and milrinone to prevent hypothermia-induced arrhythmias. METHODS AND RESULTS: Transmembrane action potentials were simultaneously recorded from 2 epicardial and 1 endocardial site of coronary-perfused canine left ventricular wedge preparations, together with a pseudo-ECG. A combination of NS5806 (3-10 µmol/L) and verapamil (1 µmol/L) was used to pharmacologically model the genetic mutations responsible for ER syndrome. Acetylcholine (3 µmol/L) was used to simulate increased parasympathetic tone, which is known to promote ER. In controls, lowering the temperature of the coronary perfusate to induce mild hypothermia (32°C-34°C) resulted in increased J-wave area on the ECG and accentuated epicardial action potential notch but no arrhythmic activity. In the setting of ER, hypothermia caused further accentuation of the epicardial action potential notch, leading to loss of the action potential dome at some sites but not others, thus creating the substrate for development of phase 2 reentry and VT/VF. Addition of the transient outward current antagonist quinidine (5 µmol/L) or the phosphodiesterase III inhibitors cilostazol (10 µmol/L) or milrinone (5 µmol/L) diminished the ER manifestations and prevented the hypothermia-induced phase 2 reentry and VT/VF. CONCLUSIONS: Hypothermia leads to VT/VF in the setting of ER by exaggerating repolarization abnormalities, leading to development of phase 2 reentry. Quinidine, cilostazol, and milrinone suppress the hypothermia-induced VT/VF by reversing the repolarization abnormalities.
Assuntos
Antiarrítmicos/farmacologia , Sistema de Condução Cardíaco/efeitos dos fármacos , Hipotermia Induzida , Milrinona/farmacologia , Inibidores da Fosfodiesterase 3/farmacologia , Quinidina/farmacologia , Taquicardia Ventricular/prevenção & controle , Tetrazóis/farmacologia , Fibrilação Ventricular/prevenção & controle , Potenciais de Ação , Animais , Cilostazol , Modelos Animais de Doenças , Cães , Eletrocardiografia , Sistema de Condução Cardíaco/fisiopatologia , Perfusão , Transdução de Sinais/efeitos dos fármacos , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/fisiopatologiaRESUMO
BACKGROUND: Wenxin Keli (WK), a Chinese herb extract, is reported to be effective in the treatment of atrial and ventricular cardiac arrhythmias. Recent studies suggest that WK inhibits the transient potassium outward current (I(to)). OBJECTIVE: To examine the effectiveness of WK, alone and in combination with quinidine, to suppress arrhythmogenesis in an experimental model of Brugada syndrome (BrS). METHODS: Action potential and electrocardiographic recordings were obtained from epicardial and endocardial sites of coronary-perfused canine right ventricular wedge preparations. The Ito agonist NS5806 (10-15 µM) was used to pharmacologically mimic a genetic predisposition to BrS. RESULTS: The Ito agonist induced Phase 2 reentry (P2R) in 13/19 preparations and polymorphic ventricular tachycardia (pVT) in 11/19 wedge preparations. WK (10 g/L) suppressed P2R and pVT in 100% (3/3) of preparations. A lower concentration of WK (5 g/L) suppressed P2R in 60% (3/5) and pVT in 50% (2/4), but in combination with a low concentration of quinidine (5 µM), was 100% effective in suppressing P2R and pVT. Quinidine alone suppressed P2R and pVT in 60% (3/5) and 50% (2/4), respectively, and in combination with WK (5 g/L) suppressed P2R and pVT by 80% (4/5) and 75% (3/4), respectively. WK reduced Ito, the L-type calcium current, and contractility in single cardiomyocytes, but dose-dependently increased contractility in intact wedge preparations, an effect mimicked by tyramine. CONCLUSIONS: Our data provide support for the hypothesis that WK, particularly in combination with quinidine, effectively suppresses arrhythmogenesis in an experimental model of BrS via inhibition of Ito and indirect adrenergic sympathomimetic effects.
Assuntos
Síndrome de Brugada/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Quinidina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/farmacologia , Síndrome de Brugada/patologia , Síndrome de Brugada/fisiopatologia , Modelos Animais de Doenças , Cães , Miócitos Cardíacos/patologia , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Potenciais de Ação , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potenciais de Ação/efeitos dos fármacos , Compostos de Bário/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cloretos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Piperidinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Brugada syndrome (BrS) is a sudden death-predisposing genetic condition characterized electrocardiographically by ST segment elevation in the leads V(1)-V(3). Given the prominent role of the transient outward current (I(to)) in BrS pathogenesis, we hypothesized that rare gain-of-function mutations in KCND3 may serve as a pathogenic substrate for BrS. METHODS: Comprehensive mutational analysis of KCND3-encoded Kv4.3 (I(to)) was conducted using polymerase chain reaction, denaturing high performance liquid chromatography, and direct sequencing of DNA derived from 86 unrelated BrS1-8 genotype-negative BrS patients. DNA from 780 healthy individuals was examined to assess allelic frequency for nonsynonymous variants. Putative BrS-associated Kv4.3 mutations were engineered and coexpressed with wild-type KChIP2 in HEK293 cells. Wild-type and mutant I(to) ion currents were recorded using whole-cell patch clamp. RESULTS: Two BrS1-8 genotype-negative cases possessed novel Kv4.3 missense mutations. Both Kv4.3-L450F and Kv4.3-G600R were absent in 1,560 reference alleles and involved residues highly conserved across species. Both Kv4.3-L450F and Kv4.3-G600R demonstrated a gain-of-function phenotype, increasing peak I(to) current density by 146.2% (n = 15, P <.05) and 50.4% (n = 15, P <.05), respectively. Simulations using a Luo-Rudy II action potential (AP) model demonstrated the stable loss of the AP dome as a result of the increased I(to) maximal conductance associated with the heterozygous expression of either L450F or G600R. CONCLUSIONS: These findings provide the first molecular and functional evidence implicating novel KCND3 gain-of-function mutations in the pathogenesis and phenotypic expression of BrS, with the potential for a lethal arrhythmia being precipitated by a genetically enhanced I(to) current gradient within the right ventricle where KCND3 expression is the highest.