Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a "pseudo" QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

Susceptibility to Ventricular Arrhythmias Resulting from Mutations in FKBP1B, PXDNL, and SCN9A Evaluated in hiPSC Cardiomyocytes.

BACKGROUND: We report an inherited cardiac arrhythmia syndrome consisting of Brugada and Early Repolarization Syndrome associated with variants in SCN9A, PXDNL, and FKBP1B. The proband inherited the 3 mutations and exhibited palpitations and arrhythmia-mediated syncope, whereas the parents and sister, who carried one or two of the mutations, were asymptomatic. METHODS AND RESULTS: We assessed the functional impact of these mutations in induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) derived from the proband and an unaffected family member. Current and voltage clamp recordings, as well as confocal microscopy analysis of Ca2+ transients, were evaluated in hiPSC-CMs from the proband and compared these results with hiPSC-CMs from undiseased controls. Genetic analysis using next-generation DNA sequencing revealed heterozygous mutations in SCN9A, PXDNL, and FKBP1B in the proband. The proband displayed right bundle branch block and exhibited episodes of syncope. The father carried a mutation in FKBP1B, whereas the mother and sister carried the SCN9A mutation. None of the 3 family members screened developed cardiac events. Action potential recordings from control hiPSC-CM showed spontaneous activity and a low upstroke velocity. In contrast, the hiPSC-CM from the proband showed irregular spontaneous activity. Confocal microscopy of the hiPSC-CM of the proband revealed low fluorescence intensity Ca2+ transients that were episodic in nature. Patch-clamp measurements in hiPSC-CM showed no difference in I Na but reduced I Ca in the proband compared with control. Coexpression of PXDNL-R391Q with SCN5A-WT displayed lower I Na density compared to PXDNL-WT. In addition, coexpression of PXDNL-R391Q with KCND3-WT displayed significantly higher I to density compared to PXDNL-WT. CONCLUSION: SCN9A, PXDNL, and FKBP1B variants appeared to alter spontaneous activity in hiPSC-CM. Only the proband carrying all 3 mutations displayed the ERS/BrS phenotype, whereas one nor two mutations alone did not produce the clinical phenotype. Our results suggest a polygenic cause of the BrS/ERS arrhythmic phenotype due to mutations in these three gene variants caused a very significant loss of function of I Na and I Ca and gain of function of I to.

Pharmacological enhancement of repolarization reserve in human induced pluripotent stem cells derived cardiomyocytes.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for many applications including safety pharmacology. However, a deficiency or complete absence of several K+ currents suggests repolarization reserve is low in hiPSC-CMs. We determined whether a dual Ito and IKr activator can improve repolarization reserve in hiPSC-CMs resulting in a more electrophysiologically mature phenotype. METHODS AND RESULTS: Human iPSC were maintained on growth factor and differentiated into the cardiac phenotype by addition of selective Wnt molecules. Current and voltage clamp recordings in single cells were made using patch electrodes. Extracellular field potentials were made using a microelectrode array on hiPSC monolayers. Action potential recordings from hiPSC-CMs following application of an IKr inhibitor resulted in depolarization of the membrane potential and prolongation of the APD. A flattening of the T-wave was noted on the pseudo-ECG. In contrast, application of the IKr and Ito agonist, NS3623, resulted in hyperpolarization of the membrane, slowing of the spontaneous rate and shortening of the APD. Voltage clamp recording showed a significant increase in IKr; no enhancement of Ito in hiPSC-CMs was noted. AP clamp experiments revealed that IKr plays a role in both phase 3 repolarization and phase 4 depolarization. mRNA analysis revealed that KCNH2 is abundantly expressed in hiPSC-CM, consistent with electrophysiological recordings. CONCLUSIONS: Although NS3623 is a dual Ito and IKr activator in ventricular myocytes, application of this compound to hiPSC-CMs enhanced only IKr and no effect on Ito was noted. Our results suggest IKr enhancement can improve repolarization reserve in this cell type. The disconnect between a dramatic increase in Ito in adult myocytes versus the lack of effect in hiPSC-CMs suggest that the translation of pharmacological effects in hiPSC-CM to adult myocytes should be viewed with caution.

Interventricular differences in sodium current and its potential role in Brugada syndrome.

Brugada syndrome (BrS) is an inherited disease associated with ST elevation in the right precordial leads, polymorphic ventricular tachycardia (PVT), and sudden cardiac death in adults. Mutations in the cardiac sodium channel account for a large fraction of BrS cases. BrS manifests in the right ventricle (RV), which led us to examine the biophysical and molecular properties of sodium channel in myocytes isolated from the left (LV) and right ventricle. Patch clamp was used to record sodium current (INa ) in single canine RV and LV epicardial (epi) and endocardial (endo) myocytes. Action potentials were recorded from multicellular preparations and single cells. mRNA and proteins were determined using quantitative RT-PCR and Western blot. Although LV wedge preparations were thicker than RV wedges, transmural ECG recordings showed no difference in the width of the QRS complex or transmural conduction time. Action potential characteristics showed RV epi and endo had a lower Vmax compared with LV epi and endo cells. Peak INa density was significantly lower in epi and endo RV cells compared with epi and endo LV cells. Recovery from inactivation of INa in RV cells was slightly faster and half maximal steady-state inactivation was more positive. ß2 and ß4 mRNA was detected at very low levels in both ventricles, which was confirmed at the protein level. Our observations demonstrate that Vmax and Na+ current are smaller in RV, presumably due to differential Nav 1.5/ß subunit expression. These results provide a potential mechanism for the right ventricular manifestation of BrS.

Biophysical comparison of sodium currents in native cardiac myocytes and human induced pluripotent stem cell-derived cardiomyocytes.

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.

Atrial electrophysiological and molecular remodelling induced by obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) affects 9-24% of the adult population. OSA is associated with atrial disease, including atrial enlargement, fibrosis and arrhythmias. Despite the link between OSA and cardiac disease, the molecular changes in the heart which occur with OSA remain elusive. To study OSA-induced cardiac changes, we utilized a recently developed rat model which closely recapitulates the characteristics of OSA. Male Sprague Dawley rats, aged 50-70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Rats were given 60 apnoeas per hour of either 13 sec. (moderate apnoea) or 23 sec. (severe apnoea), 8 hrs per day for 2 weeks. Controls received implants, but no inflations were made. Pulse oximetry measurements were taken at regular intervals, and post-apnoea ECGs were recorded. Rats had longer P wave durations and increased T wave amplitudes following chronic OSA. Proteomic analysis of the atrial tissue homogenates revealed that three of the nine enzymes in glycolysis, and two proteins related to oxidative phosphorylation, were down regulated in the severe apnoea group. Several sarcomeric and pro-hypertrophic proteins were also up regulated with OSA. Chronic OSA causes proteins changes in the atria which suggest impairment of energy metabolism and enhancement of hypertrophy.

Biophysical and molecular comparison of sodium current in cells isolated from canine atria and pulmonary vein.

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.

Atria are More Sensitive Than Ventricles to GS-458967-Induced Inhibition of Late Sodium Current.

INTRODUCTION: The differential response of atrial and ventricular cells to late sodium channel current (late INa) inhibition has not been thoroughly investigated. The aim of the present study was to compare the atrioventricular differences in electrophysiological actions of GS-458967, a potent late INa blocker. METHODS AND MATERIALS: Canine coronary-perfused atrial and ventricular preparations and isolated ventricular myocytes were used. Transmembrane action potentials were recorded using standard microelectrode recording techniques. RESULTS: In coronary-perfused preparations paced at a cycle length (CL) of 500 ms, GS-458967 (100-300 nmol/L) significantly abbreviated action potential duration at 50% to 90% (APD50-90) in atria but not in the ventricles. GS-458967 (≥100 nmol/L) prolonged the effective refractory period (ERP) in atria due to the development of postrepolarization refractoriness (PRR) but did not alter ERP in the ventricles. The maximum rate of rise in the action potential upstroke (Vmax) was significantly reduced at concentrations ≥100 nmol/L in atria but not in the ventricles (CL = 300 ms). At slower pacing rates (CL = 2000 ms) and higher concentrations, GS-458967 (100-1000 nmol/L) still failed to abbreviate ventricular APD. However, when APD was prolonged by the rapidly activating delayed rectifier potassium channel blocker E-4031 (1 µmol/L), addition of 1 µmol/L GS-458967 abbreviated APD in the ventricles at slow rates. In contrast, GS-458967 (300 nmol/L) consistently abbreviated APD in untreated isolated ventricular myocytes. CONCLUSION: In canine coronary-perfused preparations, GS-458967 abbreviates APD, induces PRR, and reduces Vmax in atria but has no significant effect on these parameters in the ventricles, indicating an atrial-selective effect of GS-458967 on both peak and late INa-mediated parameters. In multicellular preparations, GS-458967 abbreviated ventricular APD only under long QT conditions, suggesting a pathology-specific action of GS-458967 in canine ventricular myocardium.

Electrophysiologic characteristics and pharmacologic response of human cardiomyocytes isolated from a patient with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiac disorder encountered in the clinic. Data relative to the electrophysiologic characteristics and pharmacologic responsiveness of human tissues and cells isolated from patients with HCM are rare. As a consequence, cellular mechanisms underlying arrhythmogenicity are poorly understood. METHODS: Cardiomyocytes were enzymatically dissociated from a septal myectomy surgically removed from a patient with obstructive HCM. Sharp microelectrodes and patch-clamp techniques were used to evaluate action potential and sodium channel current (INa ) characteristics. RESULTS: Action potential morphology recorded was typical of an M cell, but with a longer than normal duration (APD) and a relatively steep APD-rate relationship. APD at all rates was significantly reduced following exposure to ranolazine (10 µM). Whole cell patch-clamp recording yielded robust peak INa and large late INa (1.1% of peak INa vs 0.1-0.2% in healthy controls). A large window current was observed as well. Ranolazine (10 µM) shifted steady-state V0.5 of inactivation by -8 mV, reduced late INa by 82%, and significantly diminished the window current. CONCLUSION: Our results indicate the presence of cells with M-cell characteristics in the septum of the human heart, as has previously been described in the canine heart. They also point to an ameliorative effect of ranolazine to reduce augmented late INa and thus to reduce the prolonged APD in the setting of HCM. These results suggest a potential therapeutic role for ranolazine in HCM.

Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes.

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.

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr).

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 (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) 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.

Cellular basis for the electrocardiographic and arrhythmic manifestations of Timothy syndrome: effects of ranolazine.

BACKGROUND: Timothy syndrome is a multisystem disorder associated with QT interval prolongation and ventricular cardiac arrhythmias. The syndrome has been linked to mutations in Ca(V)1.2 resulting in gain of function of the L-type calcium current (I(Ca,L)). Ranolazine is an antianginal agent shown to exert an antiarrhythmic effect in experimental models of long QT syndrome. OBJECTIVE: The purpose of this study was to develop and characterize an experimental model of Timothy syndrome by using BayK8644 to mimic the gain of function of I(Ca,L) and to examine the effects of ranolazine. METHODS: Action potentials from epicardial and M regions and a pseudo-electrocardiogram (ECG) were simultaneously recorded from coronary-perfused left ventricular wedge preparations, before and after addition of BayK8644 (1 microM). RESULTS: BayK8644 preferentially prolonged action potential duration of the M cell, leading to prolongation of the QT interval and an increase in transmural dispersion of repolarization (from 44.3 +/- 7 ms to 86.5 +/- 25 ms). Stimulation at cycle lengths of 250-500 ms led to ST-T wave alternans due to alternation of the plateau voltage of the M cell action potential as well as development of delayed afterdepolarizations in epicardial and M cell action potentials. Ventricular extrasystoles and tachycardia (monomorphic, bidirectional, or torsades de pointes) developed spontaneously or after rapid pacing. Peak and late I(Na) were unaffected by BayK8644. Clinically relevant concentrations of ranolazine (10 microM) suppressed all actions of BayK8644. CONCLUSION: A left ventricular wedge model of long QT syndrome created by augmentation of I(Ca,L) recapitulates the ECG and arrhythmic manifestations of Timothy syndrome, which can be suppressed by ranolazine.

Electrophysiologic properties and antiarrhythmic actions of a novel antianginal agent.

Ranolazine is a novel antianginal agent capable of producing anti-ischemic effects at plasma concentrations of 2 to 6 microM without a significant reduction of heart rate or blood pressure. This review summarizes the electrophysiologic properties of ranolazine. Ranolazine significantly blocks I(Kr) (IC(50) = 12 microM), late I(Na), late I(Ca), peak I(Ca), I(Na-Ca) (IC(50) = 5.9, 50, 296, and 91 microM, respectively) and I(Ks) (17% at 30 microM), but causes little or no inhibition of I(to) or I(K1). In left ventricular tissue and wedge preparations, ranolazine produces a concentration-dependent prolongation of action potential duration (APD) in epicardium, but abbreviation of APD of M cells, leading to either no change or a reduction in transmural dispersion of repolarization (TDR). The result is a modest prolongation of the QT interval. Prolongation of APD and QT by ranolazine is fundamentally different from that of other drugs that block I(Kr) and induce torsade de pointes in that APD prolongation is rate-independent (ie, does not display reverse rate-dependent prolongation of APD) and is not associated with early after depolarizations, triggered activity, increased spatial dispersion of repolarization, or polymorphic ventricular tachycardia. Torsade de pointes arrhythmias were not observed spontaneously nor could they be induced with programmed electrical stimulation in the presence of ranolazine at concentrations as high as 100 microM. Indeed, ranolazine was found to possess significant antiarrhythmic activity, acting to suppress the arrhythmogenic effects of other QT-prolonging drugs. Ranolazine produces ion channel effects similar to those observed after chronic exposure to amiodarone (reduced late I(Na), I(Kr), I(Ks), and I(Ca)). Ranolazine's actions to reduce TDR and suppress early after depolarization suggest that in addition to its anti-anginal actions, the drug possesses antiarrhythmic activity.

Ionic and cellular basis for the predominance of the Brugada syndrome phenotype in males.

BACKGROUND: The Brugada syndrome displays an autosomal dominant mode of transmission with low penetrance. Despite equal genetic transmission of the disease, the clinical phenotype is 8 to 10 times more prevalent in males than in females. The basis for this intriguing sex-related distinction is unknown. The present study tests the hypothesis that the disparity in expression of the Brugada phenotype is a result of a more prominent I(to)-mediated action potential notch in the right ventricular (RV) epicardium of males versus females. METHODS AND RESULTS: We studied epicardial tissue slices, arterially perfused wedge preparations, and dissociated epicardial myocytes isolated from male and female canine hearts. RV epicardium action potential phase 1 amplitude was 64.8+/-2.0% of that of phase 2 in males compared with 73.8+/-4.4% in females (P<0.05) at a cycle length of 2000 ms. I(to) density was 26% smaller and time constant for inactivation 17% smaller at +40 mV in female versus male RV epicardial cells (P<0.05). The other functional characteristics of I(to), including the voltage dependence of inactivation and time course of reactivation, were no different between the sexes. Pinacidil caused loss of action potential dome in male, but not female, RV epicardial tissue slices. Terfenadine (5 micromol/L) induced phase 2 reentry in 6 of 7 male but only 2 of 7 female arterially perfused wedge preparations. Two of 6 male and 1 of 2 female preparations developed polymorphic ventricular tachycardia/ventricular fibrillation. CONCLUSIONS: Our results suggest that the predominance of the Brugada phenotype in males is a result of the presence of a more prominent I(to) in males versus females.