Delayed Rectifier Potassium Channel Research Articles

Blockade of the Human Ether A-Go-Go-Related Gene (hERG) Potassium Channel by Fentanyl.

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). Drug-mediated or medical condition-mediated disruption of hERG function is the primary cause of acquired long-QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. Fentanyl abuse poses a serious health concern, with abuse and death rates rising over recent years. As fentanyl has a propensity to cause sudden death, we investigated its effects on the hERG channel. The effects of norfentanyl, the main metabolite, and naloxone, an antidote used in fentanyl overdose, were also examined. Currents of hERG channels stably expressed in HEK293 cells were recorded using the whole-cell voltage-clamp method. When hERG tail currents were analyzed upon -50 mV repolarization after a 50 mV depolarization, fentanyl and naloxone blocked hERG current (IhERG) with IC50 values of 0.9 and 74.3 μM, respectively, whereas norfentanyl did not block. However, fentanyl-mediated block of IhERG was voltage dependent. When a voltage protocol that mimics a human ventricular action potential (AP) was used, fentanyl blocked IhERG with an IC50 of 0.3 μM. Furthermore, fentanyl (0.5 μM) prolonged AP duration and blocked IKr in ventricular myocytes isolated from neonatal rats. The concentrations of fentanyl used in this study were higher than seen with clinical use but overlap with postmortem overdose concentrations. Although mechanisms of fentanyl-related sudden death need further investigation, blockade of hERG channels may contribute to the death of individuals with high-concentration overdose or compromised cardiac repolarization.

Human ether‑à‑go‑go‑related gene mutation L539fs/47‑hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy that often results in fatal arrhythmias. LQTS mutations not only lead to abnormal myocardial electrical activities but are associated with heart contraction abnormalities, cardiomyopathy and congenital heart defects. In vivo and in vitro studies have found that LQTS mutations are associated with cardiomyocyte apoptosis, cardiac developmental disorders and even embryonic mortality. Cardiac delayed rectifier potassium channel dysfunction due to the human ether-à-go-go-related gene (hERG) mutation causes congenital LQTS type 2. The majority of LQTS 2 mutations are characterized by mutant protein accumulation in the endoplasmic reticulum (ER). Unfolded or misfolded protein retention in the ER causes an unfolded protein reaction, which is characteristic of ER stress (ERS). Therefore, the present study hypothesized that LQTS mutations can cause cardiac structural abnormalities via ERS-mediated cardiomyocyte apoptosis. To test this hypothesis, 293 cells were transiently transfected with an L539fs/47-hERG plasmid to generate an LQTS 2 model. L539fs/47-hERG is an LQTS 2 mutation, which consists of a 19-bp deletion at 1619-1637 and a point mutation at 1692. Using confocal laser scanning microscopy analysis, it was verified that the L539fs/47-hERG protein was retained in the ER. Hoechst 33342 apoptosis staining indicated that apoptosis was increased in the L539fs/47-hERG-transfected cells, and this be reversed by treatment with 4-phenyl butyric acid. Western blot analysis revealed increased expression levels of the ERS chaperone glucose regulated protein 78 and pro-apoptotic ERS-induced factors, including protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation-initiation factor-2α and C/EBP homologous protein, in the L539fs/47-hERG-transfected cells. The B-cell lymphoma (Bcl-2)-associated X protein/Bcl-2 ratio and caspase-12 were also increased in the mutated cells. These results demonstrate that L539fs/47-hERG induces cell apoptosis and the potential molecular mechanism involves the activation of ERS and ERS-mediated cell apoptosis.

Drug-Induced QT Interval Prolongation: Mechanisms, Risk Factors, Genetics and Clinical Management

Long QT syndrome LQTS characterized by prolongation of the QT interval, may occur as congenital or drug-induced forms. Drug-induced QT interval prolongation DI-QTP is closely associated with severe ventricular arrhythmias [especially torsade de pointes TdP ] and sudden cardiac death. In particular, development of DI-QTP is generally associated with multiple risk factors. Cardiac and non-cardiac drugs may cause QT interval prolongation QTP and TdP. Most of the QT-prolonging drugs act by blocking the rapid component of the delayed rectifier potassium channel whereas a smaller number of drugs act by modifying Ca2+ and Na+ currents. In addition, pharmacokinetic drug interactions are among the reasons of DI-QTP. The corrected QT interval QTc according to heart rate by Bazett’s formula is the most commonly used. Genetic susceptibility is another important issue in predicting DI-QTP and TdP risk. Silent mutations and/or polymorphisms associated with cardiac ion channels may cause a risk for DI-QTP. Firstly, for treatment, drugs that cause QTP should be stopped rapidly, electrolyte abnormalities and other pathologies should be rapidly corrected. Intravenous magnesium sulphate, overdrive pacing, isoproterenol and plasma alkalinisation via sodium bicarbonate are the main useful treatments for DI-QTP and related TdP therapy. Class 1B antiarrhythmic drugs and intravenous potassium are thought to may be effective in TdP.The purpose of this article is to review the underlying mechanisms of QTP, risk factors and genetics of DI-QTP, how to measurement of QT interval and treatment of acquired LQTS.Key-words: Drugs, QT interval, QT prolongation and torsade de pointes

Anti-Ro52 antibody acts on the S5-pore linker of hERG to chronically reduce channel expression.

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr). Malfunction of hERG/IKr is the primary cause of acquired long QT syndrome (LQTS), an electrical disorder of the heart that can cause arrhythmias and sudden death. Patients with autoimmune diseases display a high incidence of LQTS. While dysfunction of hERG channels induced by autoantibodies such as anti-Ro52 may play a role in this pathology, the underlying mechanisms are not well understood. Here, we investigated the acute and chronic effects of anti-Ro52 antibody on hERG channels stably expressed in human embryonic kidney (hERG-HEK) 293 cells as well as IKr in neonatal rat ventricular myocytes. Using whole-cell patch clamp, western blot analyses, and immunocytochemistry, we found that a 12-h treatment of hERG-HEK cells with patients' sera containing anti-Ro52 autoantibody decreased the hERG current (IhERG) by 32% compared to cells treated with autoantibody-negative patients' sera. Commercial anti-Ro52 antibody at 100 µg/mL did not acutely block IhERG. Instead, a 12-h treatment with anti-Ro52 antibody at a concentration of 4 µg/mL significantly reduced mature hERG protein expression and IhERG. Specifically, anti-Ro52 antibody did not acutely block hERG current but chronically facilitated hERG endocytic degradation. The extracellular S5-pore linker of hERG, which forms the turret of the channel on the outside of the cell, is the target region for anti-Ro52-mediated hERG reduction since its replacement with the analogous region of EAG abolished the anti-Ro52 effect. In neonatal rat ventricular myocytes, 100 µg/mL anti-Ro52 antibody did not acutely block IKr, but a 12-h treatment of cells with 4 µg/mL anti-Ro52 antibody selectively reduced IKr and prolonged the action potential duration. Our results indicate that anti-Ro52 antibody acts on the hERG S5-pore linker to chronically decrease hERG expression and current. These findings provide novel insights into hERG regulation and anti-Ro52 antibody-associated LQTS.

Modulation of Ion Channels in the Superior Cervical Ganglion Neurons by Myocardial Ischemia and Fluvastatin Treatment.

Background: The superior cervical ganglion (SCG) of the autonomic nervous system plays an important role in different cardiovascular diseases. In this study, we investigated the effects of ischemia and fluvastatin treatment on the ion channel characteristics of SCG neurons in a rabbit myocardial ischemia (MI) model.Methods: MI was induced by abdominal subcutaneous injections of isoproterenol (ISO). The properties of the delayed rectifier potassium channel current (IK), sodium channel current (INa), and action potential (APs) on isolated SCG neurons in the control, MI-7d, MI-14d, fluvastatin-7d (fluvastatin pretreated 14 days and treated 7 days after ISO-induced MI), and fluvastatin-14d (fluvastatin pretreated 14 days and treated 14 days after ISO-induced MI) groups were studied. In addition, the RNA expressions of KCNQ3 and SCN9A in the SCG tissue were determined by performing real-time PCR. Intracellular calcium concentration was monitored using laser scanning confocal microscopy.Results: Compared with the control group, the current amplitude of IK and INa were increased in the MI-7d and MI-14d groups. KCNQ3 RNA (corresponding to channel proteins of IK) expression and SCN9A RNA (corresponding to channel proteins of INa) expression were also increased in MI groups. Activation and inactivation curves for INa in the two MI groups shifted negatively compared with the control group. These changes were reversed by fluvastatin treatment. Intracellular calcium concentration in SCG neurons was not altered significantly by MI or fluvastatin treatment. By contrast, increased AP amplitude and shortened APD90 were observed in the MI-7d and MI-14d groups. These changes were reversed in the fluvastatin-treated MI group.Conclusion: Fluvastatin treatment partly reversed the characteristics of SCG neurons in MI. The ion channel of SCG neurons could be one of the potential targets of fluvastatin in treating coronary heart diseases.

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

SummaryHuman induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, “Optopatch,” pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Effect of down-regulation of IKs repolarization-reserve on ventricular arrhythmogenesis in a guinea pig model of cardiac hypertrophy

To observe the changes of rapidly activated delayed rectifier potassium channel (IKr) and slowly activated delayed rectifier potassium channel (IKs) in cardiac hypertrophy and to evaluate the effects of IKr and IKs blocker on the incidence of ventricular arrhythmias in guinea pigs with left ventricular hypertrophy (LVH). Methods: Guinea pigs were divided into a sham operation group and a left ventricular hypertrophy (LVH) group. LVH model was prepared. Whole cell patch-clamp technique was used to record IKr and IKs tail currents in a guinea pig model with LVH. The changes of QTc and the incidence rate of ventricular arrhythmias in LVH guinea pigs were observed by using the IKr and IKs blockers. Results: Compared with cardiac cells in the control group, the interventricular septal thickness at end systole (IVSs), left ventricular posterior wall thickness at end systole (LVPWs), QTc interval and cell capacitance in guinea pigs with LVH were significantly increased (P<0.05); while IKs densities were significantly reduced [+60 mV: (0.36±0.03) pA/pF vs (0.58±0.05) pA/pF, P<0.01]. However, LVH exerted no significant effect on IKr densities. IKr blocker markedly prolonged the QTc interval (P<0.01) and increased the incidence of ventricular arrhythmias in guinea pigs with LVH compared with the control guinea pigs. In contrast, IKs blocker produced modest increase in QTc interval in guinea pigs of control group with no increase in LVH animals. IKs blocker did not induce ventricular arrhythmias incidence in either control or LVH animals. Conclusion: The cardiac hypertrophy-induced arrhythmogenesis is due to the down-regulation of IKs.

Modeling Short QT Syndrome Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BackgroundShort QT syndrome (SQTS), a disorder associated with characteristic ECG QT‐segment abbreviation, predisposes affected patients to sudden cardiac death. Despite some progress in assessing the organ‐level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged because of a lack of appropriate human cellular models of the disorder. The objective of this study was to establish a cellular model of SQTS using human‐induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs).Methods and ResultsThis study recruited 1 patient with short QT syndrome type 1 carrying a mutation (N588K) in KCNH2 as well as 2 healthy control subjects. We generated hiPSCs from their skin fibroblasts, and differentiated hiPSCs into cardiomyocytes (hiPSC‐CMs) for physiological and pharmacological studies. The hiPSC‐CMs from the patient showed increased rapidly activating delayed rectifier potassium channel current (IK r) density and shortened action potential duration compared with healthy control hiPSC‐CMs. Furthermore, they demonstrated abnormal calcium transients and rhythmic activities. Carbachol increased the arrhythmic events in SQTS but not in control cells. Gene and protein expression profiling showed increased KCNH2 expression in SQTS cells. Quinidine but not sotalol or metoprolol prolonged the action potential duration and abolished arrhythmic activity induced by carbachol.ConclusionsPatient‐specific hiPSC‐CMs are able to recapitulate single‐cell phenotype features of SQTS and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects.

Restoration of mutant hERG stability by inhibition of HDAC6

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.

Testosterone-mediated upregulation of delayed rectifier potassium channel in cardiomyocytes causes abbreviation of QT intervals in rats.

Men have shorter rate-corrected QT intervals (QTc) than women, especially at the period of adolescence or later. The aim of this study was to elucidate the long-term effects of testosterone on cardiac excitability parameters including electrocardiogram (ECG) and potassium channel current. Testosterone shortened QT intervals in ECG in castrated male rats, not immediately after, but on day 2 or later. Expression of Kv7.1 (KCNQ1) mRNA was significantly upregulated by testosterone in cardiomyocytes of male and female rats. Short-term application of testosterone was without effect on delayed rectifier potassium channel current (IKs), whereas IKs was significantly increased in cardiomyocytes treated with dihydrotestosterone for 24h, which was mimicked by isoproterenol (24h). Gene-selective inhibitors of a transcription factor SP1, mithramycin, abolished the effects of testosterone on Kv7.1. Testosterone increases Kv7.1-IKs possibly through a pathway related to a transcription factor SP1, suggesting a genomic effect of testosterone as an active factor for cardiac excitability.

Sevoflurane causes greater QTc interval prolongation in chronically hyperglycemic patients than in normoglycemic patients.

QTc interval prolongation is a serious diabetic complication and increases mortality rate. Hyperglycemia inhibits the rapid component of delayed rectifier potassium channel currents (Ikr) and prolongs the QTc interval on electrocardiograms. Sevoflurane also inhibits the Ikr and causes QTc interval prolongation. In fact, torsade de pointes occurred in a patient with poorly controlled diabetes mellitus during sevoflurane anesthesia. We enrolled 74 patients, including 37 normoglycemic patients (glycated hemoglobin [HbA1c]: <6.5%) (NG group) and 37 chronically hyperglycemic patients (HbA1c: ≥6.5%) (HG group). Anesthesia was induced with 2 mg/kg propofol and 0.3 μg/kg/min remifentanil, and maintained with 2% sevoflurane in 40% O2 and 0.2–0.3 μg/kg/min remifentanil. The QT interval and Tp-e interval (from the peak to the end of the T wave) were measured before and at 5, 10, 30, 60, 90, and 120 min after the administration of sevoflurane and adjusted for the patient’s heart rate (QTc and Tp-ec, respectively). P-values of <0.05 were considered statistically significant. The QTc and the Tp-ec intervals of the two groups did not differ significantly before the administration of sevoflurane. The QTc interval gradually increased with time in both groups and was significantly longer than the baseline value at 10 min after the administration of sevoflurane in both groups. The QTc interval of the HG group was significantly longer than that of the NG group at 90 min and 120 min after the administration of sevoflurane. The Tp-ec interval was not affected by sevoflurane in either group.We have demonstrated that sevoflurane significantly prolongs the QTc interval, and that the extent of the prolongation is significantly greater in chronically hyperglycemic patients than in normoglycemic patients. Although Tp-ec is not affected by sevoflurane, it should be noted that the simultaneous blockade of potassium channels would increase the risk of arrhythmias.

Therapeutic effects of a taurine-magnesium coordination compound on experimental models of type 2 short QT syndrome.

Short QT syndrome (SQTS) is a genetic arrhythmogenic disease that can cause malignant arrhythmia and sudden cardiac death. The current therapies for SQTS have application restrictions. We previously found that Mg· (NH2CH2CH2SO3)2· H2O, a taurine-magnesium coordination compound (TMCC) exerted anti-arrhythmic effects with low toxicity. In this study we established 3 different models to assess the potential anti-arrhythmic effects of TMCC on type 2 short QT syndrome (SQT2). In Langendorff guinea pig-perfused hearts, perfusion of pinacidil (20 μmol/L) significantly shortened the QT interval and QTpeak and increased rTp-Te (P<0.05 vs control). Subsequently, perfusion of TMCC (1-4 mmol/L) dose-dependently increased the QT interval and QTpeak (P<0.01 vs pinacidil). TMCC perfusion also reversed the rTp-Te value to the normal range. In guinea pig ventricular myocytes, perfusion of trapidil (1 mmol/L) significantly shortened the action potential duration at 50% (APD50) and 90% repolarization (APD90), which was significantly reversed by TMCC (0.01-1 mmol/L, P<0.05 vs trapidil). In HEK293 cells that stably expressed the outward delayed rectifier potassium channels (IKs), perfusion of TMCC (0.01-1 mmol/L) dose-dependently inhibited the IKs current with an IC50 value of 201.1 μmol/L. The present study provides evidence that TMCC can extend the repolarization period and inhibit the repolarizing current, IKs, thereby representing a therapeutic candidate for ventricular arrhythmia in SQT2.

Attenuation of ischemia/reperfusion-induced inhibition of the rapid component of delayed rectifier potassium current by Isosteviol through scavenging reactive oxygen species

Isosteviol has been demonstrated to play a protective role during ischemia reperfusion (I/R) myocardial infarction. However, the underlying electrophysiological mechanisms of isosteviol are still unknown. Our previous study showed that the rapid component of the delayed rectifier potassium channel (IKr) plays an important role in the prolongation of I/R-induced QT interval-related arrhythmia. This study aimed to investigate whether isosteviol could attenuate I/R-induced prolongation of the action potential duration (APD) along with inhibition of IKr, and we aimed to clarify the electrophysiological mechanism of isosteviol to determine its cardioprotective effects in guinea pigs. We observed that the APD90 were 298.5±41.6ms in control, 528.6±56.7ms during I/R, and reduced to 327.8±40.5ms after 10μmol/L of isosteviol treatment. The IKr currents were 1.44±0.06 pA·pF−1in the control group, 0.50±0.07pA·pF−1during I/R, and recovered to 1.20±0.12pA·pF−1after 10μmol/L of isoteviol treatment. Moreover, isosteviol reduced the over-production of reactive oxygen species (ROS) during I/R. Importantly, isosteviol does not affect the IKr and human ether-a-go-go-related gene currents of normal cardiomyocytes. It attenuated the I/R-induced inhibition of IKr due to reduced over-production of ROS. Furthermore, isosteviol is safe and has no cardiotoxicity, and it might be beneficial for coronary reperfusion therapy.

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 μM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 μM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and Ikr blocker.

Voltage-sensitive potassium channels expressed after 20-Hydroxyecdysone treatment of a mosquito cell line

The goal of this research was to express receptors and ion channels in hormone-treated insect cell lines. Treatment of Anopheles gambiae Sua1B cells with 20-hydroxyecdysone showed an inhibition of cell growth over a time course of three days, with no change in cellular morphology. The effect of 20-hydroxyecdysone was enhanced in the presence of the potassium channel blocker 4-aminopyridine, but not tetraethylammonium. Concentration-response curves of 4-aminopyridine in the presence of 42 μM (1 mg/ml) 20-hydroxyecdysone showed similar IC50 values (6–10 μM) across 3 day exposures. Whole cell patch clamp confirmed the expression of delayed-rectifier (Kv2) potassium channels in hormone-supplemented Sua1B cells, whereas untreated Sua1B cells showed no evidence of Kv2 expression. The hormone-induced expression of Kv2 channels occurred in as little as 4 h after treatment, but were not observed after 24 h of exposure to 20-hydroxyecdysone, suggesting they played a role in cell death. The expressed channels had current-voltage relationships diagnostic for the Kv2 subtype, and were inhibited with an IC50 = 13 mM of tetraethylammonium. Overall, these parameters were similar to Anopheles gambiae Kv2 potassium channels expressed in HEK-293 cells. The induced presence of ion channels (and possibly receptors) in these cells has potential utility for high throughput screening and basic neuroscience research.

In silico assessment of the effects of quinidine, disopyramide and E-4031 on short QT syndrome variant 1 in the human ventricles.

AimsShort QT syndrome (SQTS) is an inherited disorder associated with abnormally abbreviated QT intervals and an increased incidence of atrial and ventricular arrhythmias. SQT1 variant (linked to the rapid delayed rectifier potassium channel current, IKr) of SQTS, results from an inactivation-attenuated, gain-of-function mutation (N588K) in the KCNH2-encoded potassium channels. Pro-arrhythmogenic effects of SQT1 have been well characterized, but less is known about the possible pharmacological antiarrhythmic treatment of SQT1. Therefore, this study aimed to assess the potential effects of E-4031, disopyramide and quinidine on SQT1 using a mathematical model of human ventricular electrophysiology.MethodsThe ten Tusscher et al. biophysically detailed model of the human ventricular action potential (AP) was modified to incorporate IKr Markov chain (MC) formulations based on experimental data of the kinetics of the N588K mutation of the KCNH2-encoded subunit of the IKr channels. The modified ventricular cell model was then integrated into one-dimensional (1D) strand, 2D regular and realistic tissues with transmural heterogeneities. The channel-blocking effect of the drugs on ion currents in healthy and SQT1 cells was modeled using half-maximal inhibitory concentration (IC50) and Hill coefficient (nH) values from literatures. Effects of drugs on cell AP duration (APD), effective refractory period (ERP) and pseudo-ECG traces were calculated. Effects of drugs on the ventricular temporal and spatial vulnerability to re-entrant excitation waves were measured. Re-entry was simulated in both 2D regular and realistic ventricular tissue.ResultsAt the single cell level, the drugs E-4031 and disopyramide had hardly noticeable effects on the ventricular cell APD at 90% repolarization (APD90), whereas quinidine caused a significant prolongation of APD90. Quinidine prolonged and decreased the maximal transmural AP heterogeneity (δV); this led to the decreased transmural heterogeneity of APD across the 1D strand. Quinidine caused QT prolongation and a decrease in the T-wave amplitude, and increased ERP and decreased temporal susceptibility of the tissue to the initiation of re-entry and increased the minimum substrate size necessary to prevent re-entry in the 2D regular model, and further terminated re-entrant waves in the 2D realistic model. Quinidine exhibited significantly better therapeutic effects on SQT1 than E-4031 and disopyramide.ConclusionsThe simulated pharmacological actions of quinidine exhibited antiarrhythmic effects on SQT1. This study substantiates a causal link between quinidine and QT interval prolongation in SQT1 and suggests that quinidine may be a potential pharmacological agent for treating SQT1 patients.

Regulation of Histone Acetylation on Expression Profiles of Potassium Channels During Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells.

The cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) is a dynamic and complex process that involved in the precision regulation of histone acetylation. The formation of action potential (AP) in mature cardiomyocytes is based on the expression pattern of Na+ , Ca2+ , and K+ ion channels, in which the slow delayed rectifier potassium current (IKs ), the rapid delayed rectifier potassium current (IKr ) and the inwardly rectifying Kir current (IK1 ) mainly contribute to repolarization for AP in different species. However, the expression status of potassium channels conducted IKs , IKr , and IK1 in cardiomyocyte differentiation are not fully defined. Here, we investigated the expression pattern of the slow delayed rectifier potassium channel and the rapid delayed rectifier potassium channel using a model of mouse cardiomyocyte differentiation under different conditions of histone acetylation. We found that expression levels of both the delayed rectifier potassium channel and the inwardly rectifying potassium channel were more sensitive to histone hyperacetylation during differentiation from mESCs into cardiomyocytes. Especially, histone H4 hyperacetylation induced by Class I HDACs inhibitors promoted the expression profiles of potassium channels (Kcnj2, Kcnj3, Kcnj5, Kcnj11, and Kcnh2) in the process. Our results provide a clue for expression status of potassium channels which may be essential to forming functional cardiomyocyte in the cardiac lineage commitment of mESC. J. Cell. Biochem. 118: 4460-4467, 2017. © 2017 Wiley Periodicals, Inc.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals.

SummaryCardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min), cyclic strain (5%), and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

078 - hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state

Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels.

Computational models of O-LM cells are recruited by low or high theta frequency inputs depending on h-channel distributions.

Although biophysical details of inhibitory neurons are becoming known, it is challenging to map these details onto function. Oriens-lacunosum/moleculare (O-LM) cells are inhibitory cells in the hippocampus that gate information flow, firing while phase-locked to theta rhythms. We build on our existing computational model database of O-LM cells to link model with function. We place our models in high-conductance states and modulate inhibitory inputs at a wide range of frequencies. We find preferred spiking recruitment of models at high (4-9 Hz) or low (2-5 Hz) theta depending on, respectively, the presence or absence of h-channels on their dendrites. This also depends on slow delayed-rectifier potassium channels, and preferred theta ranges shift when h-channels are potentiated by cyclic AMP. Our results suggest that O-LM cells can be differentially recruited by frequency-modulated inputs depending on specific channel types and distributions. This work exposes a strategy for understanding how biophysical characteristics contribute to function.