We identified two different inherited mutations in KCNH2 gene, or human ether-a-go-go related gene (hERG), which are linked to Long QT Syndrome. The first mutation was in a 1-day-old infant, whereas the second was in a 14-year-old girl. The two KCNH2 mutations were transiently transfected into either human embryonic kidney (HEK) cells or human induced pluripotent stem-cell derived cardiomyocytes. We performed associated multiscale computer simulations to elucidate the arrhythmogenic potentials of the KCNH2 mutations. Genetic screening of the first and second index patients revealed a heterozygous missense mutation in KCNH2, resulting in an amino acid change (P632L) in the outer loop of the channel and substitution at position 428 from serine to proline (S428P), respectively. Heterologous expression of P632L and S428P into HEK cells produced no hERG current compared to the wild type (WT). Moreover, the co-transfection of WT and P632L yielded no hERG current; however, the co-transfection of WT and S428P yielded partial hERG current. Action potentials were prolonged in a complete or partial blockade of hERG current from computer simulations which was more severe in Purkinje than ventricular myocytes. Three dimensional simulations revealed a higher susceptibility to reentry in the presence of hERG current blockade. Our experimental findings suggest that both P632L and S428P mutations may impair the KCNH2 gene. The Purkinje cells exhibit a more severe phenotype than ventricular myocytes, and the hERG current blockade renders the ventricles an arrhythmogenic substrate from computer modeling.

Cardiac arrhythmias are responsible for 200-300 thousand deaths/year. Despite considerable research effort, much remains to be elucidated concerning the underlying mechanisms of arrhythmogenesis. Mutations in transient receptor potential melastatin 4 (TRPM4), a widely expressed Ca 2+ -activated nonselective cation channel, have been associated with causing cardiac arrhythmias. However, direct genotype-phenotype correlation of arrhythmogenic TRPM4 mutant variants are often complicated, as this channel is not primary to the cardiac action potential. Here, we assessed the electrophysiological and post-transcriptional molecular properties of a single mutation (R892C)-TRPM4 associated with short QT syndrome (SQTS) and a triple mutation (R250C|A432T|G582S)-TRPM4 associated with long QT syndrome (LQTS), using stably transfected HEK293 cells. Overall, protein expression of the triple mutant was found to be significantly reduced, with increased proteasomal degradation, but enhanced SUMOylation, as compared to either WT or the single R892 mutant TRPM4 channel. Consequently, expression of R250C|A432T|G582S-TRPM4 was significantly lower at the cellular membrane than either R892C- and WT-TRPM4. In contrast, while total expression of TRPM4 was not significantly different between WT and the R892C single mutant, although the R892C exhibited increase aggregation. Patch-clamp cellular electrophysiology experiments indicated that both single and triple TRPM4 mutant channels could be activated by lower Ca 2+ concentrations compared to WT. However, R892C-TRPM4 channels inactivated faster, while R250C|A432T|G582S-TRPM4 channels inactivated much slower compared to WT. These data as obtained in our homologous recombinant overexpression system reveal that while the R892C-TRPM4 mutant variant exhibited normal-to-higher levels of expression and increased Ca 2+ -activation sensitivity, its tendency to aggregate combined with faster inactivation can result in overall loss-of-function compared to WT, correlative with SQTS. Conversely, while the R250C|A432T|G582S-TRPM4 mutant variant exhibited reduced expression and perturbed trafficking, its increased Ca 2+ -activation sensitivity and slow inactivation can result in overall gain-of-function compared to WT, correlative with LQTS.

Cardiac arrhythmias are responsible for 200-300 thousand deaths/year. Despite considerable research effort, much remains to be elucidated concerning the underlying mechanisms of arrhythmogenesis. Mutations in transient receptor potential melastatin 4 (TRPM4), a widely expressed Ca 2+ -activated nonselective cation channel, have been associated with causing cardiac arrhythmias. However, direct genotype-phenotype correlation of arrhythmogenic TRPM4 mutant variants are often complicated, as this channel is not primary to the cardiac action potential. Here, we assessed the electrophysiological and post-transcriptional molecular properties of a single mutation (R892C)-TRPM4 associated with short QT syndrome (SQTS) and a triple mutation (R250C|A432T|G582S)-TRPM4 associated with long QT syndrome (LQTS), using stably transfected HEK293 cells. Overall, protein expression of the triple mutant was found to be significantly reduced, with increased proteasomal degradation, but enhanced SUMOylation, as compared to either WT or the single R892 mutant TRPM4 channel. Consequently, expression of R250C|A432T|G582S-TRPM4 was significantly lower at the cellular membrane than either R892C- and WT-TRPM4. In contrast, while total expression of TRPM4 was not significantly different between WT and the R892C single mutant, although the R892C exhibited increase aggregation. Patch-clamp cellular electrophysiology experiments indicated that both single and triple TRPM4 mutant channels could be activated by lower Ca 2+ concentrations compared to WT. However, R892C-TRPM4 channels inactivated faster, while R250C|A432T|G582S-TRPM4 channels inactivated much slower compared to WT. These data as obtained in our homologous recombinant overexpression system reveal that while the R892C-TRPM4 mutant variant exhibited normal-to-higher levels of expression and increased Ca 2+ -activation sensitivity, its tendency to aggregate combined with faster inactivation can result in overall loss-of-function compared to WT, correlative with SQTS. Conversely, while the R250C|A432T|G582S-TRPM4 mutant variant exhibited reduced expression and perturbed trafficking, its increased Ca 2+ -activation sensitivity and slow inactivation can result in overall gain-of-function compared to WT, correlative with LQTS.

Background: Mutations in the gene KCNH2 have been associated with both Short and Long QT syndrome. In this study, we identified a 1-day old infant that exhibited T-wave alternans coupled with Long QT Syndrome leading to aborted sudden infant death. Methods: Genetic analysis of the patient revealed a mutation in KCNH2 resulting in a proline to leucine substitution at position 632 (P632L). Functional electrophysiological studies were performed with the hERG mutation transiently transfected into either Human Embryonic Kidney (HEK) cells or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The functional effects of P632L were incorporated into biophysical computational models of hiPSC-CM as well as adult human cardiomyocytes to further assess its arrhythmogenic potential. Results: The male infant displayed multiple repolarization disorders including T-wave alternans and a QTc =510 ms that appeared 1 day after birth. Genetic screening revealed a heterozygous missense mutation (P632L) in KCNH2 in the infant. Patch clamp analysis of HEK cells transiently transfected with P632L mutation showed a complete loss of function of HERG current compared to WT HERG. Transient transfection of P632L into WT hiPSC myocytes resulted in prolongation of the hiPSC action potential compared to untransfected hiPSC myocytes. Co-transfection of WT and non-functional P632L channels into HEK cells resulted in a dramatic loss of current suggesting a dominant negative effect. Implementing the effects of a complete block of HERG current in the hiPSC-CM computer model prolonged the action potential by 45% and depolarized the resting membrane potentials. In adult human myocyte models, the effects of HERG blockade were more severe in Purkinje cells than that in ventricular myocytes (71% vs. 16% APD prolongation, respectively). Conclusions: The mutation P632L causes a complete loss of HERG current and results in QT prolongation. Numerical simulations suggest a more severe phenotype in Purkinje cells than in ventricular myocytes which could be proarrhythmic.

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.

Background: We have identified a novel form of abnormal Ca 2+ wave activity in normal and failing dog atrial myocytes which occurs during the action potential (AP) and is absent during diastole. The goal of this study was to determine if triggered Ca 2+ waves affect cellular electrophysiological properties. Methods: Simultaneous recordings of intracellular Ca 2+ and APs allowed measurements of maximum diastolic potential and AP duration during triggered calcium waves (TCWs) in isolated dog atrial myocytes. Computer simulations then explored electrophysiological behavior arising from TCWs at the tissue scale. Results: At 3.3 to 5 Hz, TCWs occurred during the AP and often outlasted several AP cycles. Maximum diastolic potential was reduced, and AP duration was significantly prolonged during TCWs. All electrophysiological responses to TCWs were abolished by SEA0400 and ORM10103, indicating that Na-Ca exchange current caused depolarization. The time constant of recovery from inactivation of Ca 2+ current was 40 to 70 ms in atrial myocytes (depending on holding potential) so this current could be responsible for AP activation during depolarization induced by TCWs. Modeling studies demonstrated that the characteristic properties of TCWs are potentially arrhythmogenic by promoting both conduction block and reentry arising from the depolarization induced by TCWs. Conclusions: Triggered Ca 2+ waves activate inward NCX and dramatically reduce atrial maximum diastolic potential and prolong AP duration, establishing the substrate for reentry which could contribute to the initiation and maintenance of atrial arrhythmias.

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 INa but reduced ICa in the proband compared with control. Coexpression of PXDNL-R391Q with SCN5A-WT displayed lower INa density compared to PXDNL-WT. In addition, coexpression of PXDNL-R391Q with KCND3-WT displayed significantly higher Ito 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 INa and ICa and gain of function of Ito.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. We have previously shown that the maximum diastolic potential (MDP) in hiPSC-CMs depends on the rapid delayed rectifier K+ current (IKr). We examined the effect of a dual Ito and IKr agonist (NS3623) on hiPSC-CMs and determined its contribution to action potentials (APs), extracellular field potentials (EFPs) and ionic currents. hiPSC-CM monolayers were used to record EFPs using CardioExcyte 96, and AP recordings were made using high resistance electrodes. Whole cell patch clamp was used to record IKr in single hiPSC-CMs. All measurements were made at 36° C. EFP signals from hiPSC-CMs monolayers showed a pseudo-QRS complex and T-wave similar to that observed in native ventricular tissue. Application of NS3623 (5 μM) resulted in a small shortening of the QT interval. Similarly, AP recordings in the presence of NS3623 resulted in hyperpolarization of the MDP (from −70.4±1.9 to −73.9±1.9 mV) and shortening of APD (from 196.6±28.0 to 176.6±25.7 ms). Voltage clamp analysis of IKr tail currents revealed a 47±9% increase in IKr following application of NS3623. A minor inhibition of Ito was observed following NS3623. AP clamp experiments revealed the IKr transient was largest during repolarization with some contribution during phase 4 depolarization. A robust IKr is present in hiPSC-CMs which can be augmented by the agonist NS3623. Application of the agonist resulted a slowing of the spontaneous rate and shortening of the APD but minimal effect on MDP. Although we have previously shown that the MDP in hiPSC-CMs critically depends on IKr, increasing the magnitude of IKr resulted only in a small hyperpolarization of the MDP.

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.

Downregulation of ion currents as well as a loss of T-tubules is documented during heart failure. A similar reduction of both has been observed in cultured ventricular myocytes. We further assessed the functional impact of cell-culture on excitation-contraction (EC) coupling in dog ventricular, atrial and Purkinje myocytes. Ventricular, atrial and Purkinje myocytes were isolated and cultured for up to 48 hours. Myocytes were stained with di-8-ANEPPS to visualize ultrastructure and Ca2+ transients (CaTs) were recorded by confocal microscopy. Ion channel currents were assessed via patch electrode voltage clamp. Membrane staining with di-8-ANEPPS indicated dog ventricular myocytes having an extensive T-tubular network; atrial myocytes having a rudimentary T-tubule system at best, and Purkinje myocytes having no T-tubules. Transverse X-t lines scans of electrically stimulated ventricular myocytes showed CaTs rising synchronously across the cell, whereas in Purkinje myocytes the CaT rise showed a U-shaped profile. CaT rise in atrial myocytes typically showed a U-shaped profile, but less pronounced than that in Purkinje myocytes. Ventricular myocytes cultured for 48 hours indicated a dramatic loss of T-tubules and a switch from a synchronous CaT rise to a U-shaped profile. In cultured atrial cells, no loss of T-tubules nor alterations in CaTs was noted. ICa in ventricular myocytes cultured for 48 hours was reduced by 21%. Comparative assessment of culture-induced changes in ion channel currents in atrial myocytes is ongoing. While cultured ventricular myocytes show a loss of T-tubules and phenotypic switch from a homogeneous CaT rise to a ‘U’-shaped profile, atrial myocytes cultured identically show little change in ultrastructure and CaT profile. These results suggest that character and degree of change in EC coupling induced by similar pathological insults markedly depends on cardiac myocyte type.

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. β2 and β4 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/β subunit expression. These results provide a potential mechanism for the right ventricular manifestation of BrS.

Background: The proposed Comprehensive in Vitro Pro-Arrhythmia Assay (CiPA) initiative aims to examine proarrhythmic drug effects on multiple cardiac ion channels in adult cardiac tissue as well as in human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes. In this study, we examined the biophysical properties of INa in hiPSC myocytes and compared them to adult canine atrial and ventricular myocytes. Methods: Left atrial and ventricular myocytes were isolated from dog hearts, whereas hiPSC myocytes were obtained from commercially available sources. Action potential recordings from all tissue types were made using high resistance electrodes. Patch clamp techniques were used to record INa from single cells. Results: Analysis of the action potential characteristics showed a higher upstroke velocity in atrial and ventricular tissue (185.4±18.4 and 183.3±14.2 V/s, respectively) compared to hiPSC (33.1±3.3 V/s) beating clusters. Voltage clamp recordings of fast INa showed that current density was lower in hiPSC myocytes compared with atrial and ventricular myocytes (−25.8±4.28 pA/pF vs. −119.6±6.7 pA/pF and −70.0±9.28 pA/pF, respectively, p<0.05). Steady state-inactivation was also significantly different between the 3 cardiac tissue types with atrial cells showing the most negative V½ and ventricular cells showing the least negative. Application of the INa blocker lidocaine (100 µM) resulted in a differential effect on Vmax and INa blockade with atrial tissue and hiPSC showing the greatest reduction in both parameters whereas ventricular tissue showing the smallest reduction. Conclusions: These data show that the density of INa in hiPSC myocytes was much lower compared to native myocytes. In addition, the response of hiPSC cells to a prototypical sodium channel blocker indicates caution should be exercised when comparing the results obtained in these cells to native ventricular cells.

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 Navβ1 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.

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 I-Na 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 I-Na. Results: Voltage clamp recordings showed a large I-Na 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 I-Na 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 I-Na while application of a sinoatrial node waveform elicited no I-Na. 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 I-Na in adult atrial, ventricular, and hiPSC-CMs. The depolarized maximum diastolic potential coupled with the presence of phase 4 depolarization limits the contribution of I-Na 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.

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.

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.

Background: The inward rectifier potassium current, IK1, regulates the terminal phase of repolarization of the action potential, as well as 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. Methods and Results: Action potentials (AP) were recorded from canine left ventricular midmyocardium, left atrial and Purkinje tissue. The terminal rate of repolarization of the AP (as assessed by the minimum dV/dt) 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 showed that 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. Conclusions: IK1 and Kir2 subunit expression vary dramatically in regions of the canine heart, these variations in IK1 properties could potentially explain the differences in the AP rate of repolarization between heart regions in response to [K+]o changes.

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.

BackgroundHypertrophic 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.MethodsCardiomyocytes 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 (I-Na) characteristics.ResultsAction 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 M). Whole cell patch-clamp recording yielded robust peak I-Na and large late I-Na (1.1% of peak I-Na vs 0.1-0.2% in healthy controls). A large window current was observed as well. Ranolazine (10 M) shifted steady-state V-0.5 of inactivation by -8 mV, reduced late I-Na by 82%, and significantly diminished the window current.ConclusionOur 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 I-Na and thus to reduce the prolonged APD in the setting of HCM. These results suggest a potential therapeutic role for ranolazine in HCM.

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 (I-K1), transient outward K+ current (I-to), delayed rectifier K+ currents (I-Kr and I-Ks) and sodium channel current (I-Na) to repolarization in the canine neonate myocardium. Methods: Single myocytes isolated from the left ventricle of 2-3 week 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 +/- 6.7 pF). I-K1 was larger in neonate myocytes and displayed a substantial inward component and an outward component with negative slope conductance, peaking at -60 mV (4.13 pA/pF). I-Kr tail currents (at -40 mV), were small (<20 pA). I-Ks could not be detected, even after exposure to isoproterenol (100 nM). I-to was also absent in the neonate, consistent with the absence of a phase 1 in the action potential. Peak I-Na, late I-Na and I-Ca 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 (I-Ks and I-to) present in adult ventricular cells are absent in the 2 week old neonate. Peak and late I-Na 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. (C) 2013 Elsevier Ltd. All rights reserved.