Late Sodium Current Research Articles

NCC-3902, a novel late sodium current blocker, exhibits potent antiarrhythmic effect in isolated guinea pig pulmonary vein and canine rapid atrial pacing model

NCC-3902, a novel late sodium current blocker, exhibits potent antiarrhythmic effect in isolated guinea pig pulmonary vein and canine rapid atrial pacing model

Why Is Only Type 1 Electrocardiogram Diagnostic of Brugada Syndrome? Mechanistic Insights From Computer Modeling.

Three types of characteristic ST-segment elevation are associated with Brugada syndrome but only type 1 is diagnostic. Why only type 1 ECG is diagnostic remains unanswered. Computer simulations were performed in single cells, 1-dimensional cables, and 2-dimensional tissues to investigate the effects of the peak and late components of the transient outward potassium current (Ito), sodium current, and L-type calcium current (ICa,L) as well as other potassium currents on the genesis of ECG morphologies and phase 2 reentry (P2R). Although a sufficiently large peak Ito was required to result in the type 1 ECG pattern and P2R, increasing the late component of Ito converted type 1 ECG to type 2 ECG and suppressed P2R. Increasing the peak Ito promoted spiral wave breakup, potentiating the transition from tachycardia to fibrillation, but increasing the late Ito prevented spiral wave breakup by flattening the action potential duration restitution and preventing P2R. A sufficiently large ICa,L conductance was needed for P2R to occur, but once above the critical conductance, blocking ICa,L promoted P2R. However, selectively blocking the window and late components of ICa,L suppressed P2R, countering the effect of the late Ito. Blocking either the peak or late components of sodium current promoted P2R, with the late sodium current blockade having the larger effect. As expected, increasing other potassium currents potentiated P2R, with ATP-sensitive potassium current exhibiting a larger effect than rapid and slow component of the delayed rectifier potassium current. The peak Ito promotes type 1 ECG and P2R, whereas the late Ito converts type 1 ECG to type 2 ECG and suppresses P2R. Blocking the peak ICa,L and either the peak or the late sodium current promotes P2R, whereas blocking the window and late ICa,L suppresses P2R. These results provide important insights into the mechanisms of arrhythmogenesis and potential therapeutic targets for treatment of Brugada syndrome. Graphic Abstract: A graphic abstract is available for this article.

Arrhythmic Phenotypes Are a Defining Feature of Dilated Cardiomyopathy-Associated SCN5A Variants: A Systematic Review.

Variants in the SCN5A gene, that encodes the cardiac sodium channel, Nav1.5, are associated with a highly arrhythmogenic form of dilated cardiomyopathy (DCM). Our aim was to review the phenotypes, natural history, functional effects, and treatment outcomes of DCM-associated rare SCN5A variants. A systematic review of reported DCM-associated rare SCN5A variants was undertaken using PubMed and Embase. Eighteen SCN5A rare variants in 29 families with DCM (173 affected individuals) were identified. Eleven variants had undergone experimental evaluation, with 7 of these resulting in increased sustained current flow during the action potential (eg, increased window current) and at resting membrane potentials (eg, creation of a new gating pore current). These variants were located in transmembrane voltage-sensing domains and had a consistent phenotype characterized by frequent multifocal narrow and broad complex ventricular premature beats (VPB; 72% of affected relatives), ventricular arrhythmias (33%), atrial arrhythmias (32%), sudden cardiac death (13%), and DCM (56%). This VPB-predominant phenotype was not seen with 1 variant that increased late sodium current, or with variants that reduced peak current density or had mixed effects. In the latter groups, affected individuals mainly showed sinus node dysfunction, conduction defects, and atrial arrhythmias, with infrequent VPB and ventricular arrhythmias. DCM did not occur in the absence of arrhythmias for any variant. Twelve studies (23 total patients) reported treatment success in the VPB-predominant cardiomyopathy using sodium channel-blocking drug therapy. SCN5A variants can present with a diverse spectrum of primary arrhythmic features. A majority of DCM-associated variants cause a multifocal VPB-predominant cardiomyopathy that is reversible with sodium channel blocking drug therapy. Early recognition of the distinctive phenotype and prompt genetic testing to identify variant carriers are needed. Our findings have implications for interpretation and management of SCN5A variants found in DCM patients with and without arrhythmias.

Abstract 10418: Complex Action Potential Regulation in Cardiomyocytes from Induced Pluripotent Stem Cells (iPSC-CMs) Reveals a Novel Arrhythmia Susceptibility Mechanism

Background: While the cardiac sodium channel ( SCN5A ) variants S1103Y and R1193Q have been associated with LQT3 and drug-induced arrhythmia, they are too common to be highly arrhythmogenic: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. We have previously reported that S1103Y had no effect on QT in a large biobank or on action potential duration (APD) in iPSC-CMs. Here, we identify a mechanism underlying control of normal baseline repolarization with these variants and study whether the mechanism is generalizable. Methods: Late sodium current (I Na-L ), I Kr , and sensitivity to the blockers dofetilide (I Kr ) and GS967 (I Na-L ) were measured in natural or genome-edited iPSC-CMs carrying S1103Y, R1193Q, or R518X, a common KCNQ1 nonsense variant in the Jervell Lange-Nielsen (JLN) syndrome and unaffected family members. Results: Although APD was unaffected by S1103Y, I Na-L was increased 2~16-fold in SY/YY compared to SS cells (p<0.01) and GS967 shortened APDs only in SY/YY but not in SS cells (p<0.001). Further, I Kr was increased in SY/YY ([SY] 1.2 pA/pF, n=17, p<0.001, [YY] 1.2 pA/pF, n=11, p<0.01) compared to SS cells (0.67 pA/pF, n=20) and challenge with dofetilide prolonged APD by 100 msec at much lower concentrations in SY/YY (median 4.1 nM [SY], 4.2 [YY], n=5-11) compared to SS cells (249 nM, n=14, p<0.001); afterdepolarizations were seen more often in SY/YY (8/16) than in SS cells (1/14). R1193Q also displayed no difference in baseline APD but increased I Kr and I Na-L and increased dofetilide sensitivity. In R518X JLN iPSC-CMs, I Ks was absent as expected but I Kr was increased (1.9 pA/pF, n=5), and editing R518X to wild-type increased I Ks (1.3 pA/pF, n=10) and dramatically decreased I Kr (0.2 pA/pF, n=4). Conclusions: S1103Y and R1193Q increase I Na-L but unexpectedly do not affect baseline repolarization. We hypothesize that increased I Kr through as yet undetermined mechanisms maintains normal repolarization but at the cost of increased sensitivity to I Kr block. The findings with R518X suggest this may be a general mechanism. Our data show that preposition to genetically- or drug-mediated arrhythmias is determined by a complex interaction among multiple repolarizing currents that is not fully captured by heterologous expression systems.

Abstract 10583: A Novel Functional Variant, P.Gln419Pro, Residing Outside the SCN5A -Encoded Na V 1.5 Voltage-Sensing Domain Causes Multifocal Ectopic Purkinje-Related Premature Contractions

Background: Multifocal ectopic Purkinje-related premature contractions (MEPPC), the most recent addition to the spectrum of SCN5A -mediated cardiac channelopathies, is characterized by frequent premature ventricular complexes (PVCs) originating from the Purkinje system, atrial arrhythmias, and a predilection for PVC-mediated dilated cardiomyopathy. MEPPC-causative variants tend to cluster within the voltage sensing domain (VSD) of Na v 1.5. Objective: To describe the clinical and cellular electrophysiological phenotype associated with a novel, putative MEPPC-causative SCN5A variant (p.Gln419Pro-SCN5A) that resides outside the VSD of Na v 1.5. Methods: p.Gln419Pro-SCN5A was identified in a 21-year-old male with a history of frequent, multifocal PVCs and atrial tachycardia. Subsequently, three p.Gln419Pro-SCN5A-positive relatives, each with early onset atrial arrhythmias and/or frequent, multifocal PVCs, were identified. All individuals had structurally normal hearts by echocardiography and/or cardiac magnetic resonance imaging. p.Gln419Pro-SCN5A was engineered by site directed mutagenesis and heterologously expressed in TSA201 cells. The standard whole-cell patch clamp technique was used to measure SCN5A wild-type (WT) and p.Gln419Pro-SCN5A sodium currents. Results: Analysis of the current-voltage relationship revealed that neither peak sodium current densities nor late inward sodium current were impacted by p.Gln419Pro-SCN5A. However, p.Gln419Pro-SCN5A significantly shifted V 1/2 in inactivation by -4.6 mV from -84.5±0.7 mV (WT, n=10) to -89.1±0.4 mV (Gln419Pro, n=12, p<0.05 vs. WT) and V 1/2 in activation by -11.6 mV from -30.9±1.3 mV (WT, n=10) to -42.5±1.0 mV (Gln419Pro, n=12, p<0.05 vs. WT) leading to a larger hyperpolarized window current. Flecainide dramatically reduced PVC burden in p.Gln419Pro-SCN5A-positive patients. Conclusions: p.Gln419Pro-SCN5A represents only the fifth MEPPC-causative variant described to date and the first associated with a classic MEPPC phenotype localizing outside Na v 1.5’s VSD. As class I antiarrhythmic drugs are highly efficacious in MEPPC, this clinical entity merits consideration in all young patients found to have a high burden of Purkinje-related PVCs.

Abstract 10687: Structural Remodeling of Na V 16 Channel Clusters Contributes to Arrhythmogenic Late Sodium Current in the Presence of D96V Mutant Calmodulin

Calmodulin facilitates normal sodium channel (Na V ) inactivation, necessary to prevent proarrhythmic late sodium current (I Na ). Previous studies have implicated Na V 1.6 dysfunction in late I Na -mediated arrhythmias. Therefore we investigated Na V dysregulation by arrhythmogenic CaM mutant D96V. Chinese hamster ovary cells expressing Na V 1.6 were transfected with either WT- or D96V-CaM. STED microscopy revealed enlarged Na V 1.6 clusters in cells receiving D96V-CaM relative to WT. Therefore we explored Na V 1.6 remodeling in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V). Confocal microscopy confirmed expression of Na V 1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RyR2 in cD96V hearts, consistent with T-tubular localization. STORM microscopy enabled the quantitative assessment of Na V 1.6 clustering. In both WT and cD96V hearts, ~50% of Na V 1.6 clusters localized <100 nm from RyR2. However, Na V 1.6 density within these regions increased 67% in cD96V relative to WT. We probed the functional impact of this surprising remodeling using SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings. The number of NaVs as well as frequency of late Na V 1.6 burst openings were significantly increased in cD96V myocytes relative to WT (Fig 1). Previous work implicates aberrant late Na V 1.6 activity in proarrhythmic Ca2+ mishandling. Indeed, STORM revealed significant enrichment of NCX near Na V 1.6 in both WT and cD96V. Consistent with these data cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. This effect was reversed by cardiac-specific Na V 1.6 knockout. To our knowledge, this is the first report of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, which may shed mechanistic insight into calmodulinopathy.

Mutant D96V calmodulin induces unexpected remodeling of cardiac nanostructure and physiology

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided "smart" patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

Late Na+ Current Is [Ca2+]i-Dependent in Canine Ventricular Myocytes.

Enhancement of the late sodium current (INaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.

Ion Channel Impairment and Myofilament Ca2+ Sensitization: Two Parallel Mechanisms Underlying Arrhythmogenesis in Hypertrophic Cardiomyopathy.

Life-threatening ventricular arrhythmias are the main clinical burden in patients with hypertrophic cardiomyopathy (HCM), and frequently occur in young patients with mild structural disease. While massive hypertrophy, fibrosis and microvascular ischemia are the main mechanisms underlying sustained reentry-based ventricular arrhythmias in advanced HCM, cardiomyocyte-based functional arrhythmogenic mechanisms are likely prevalent at earlier stages of the disease. In this review, we will describe studies conducted in human surgical samples from HCM patients, transgenic animal models and human cultured cell lines derived from induced pluripotent stem cells. Current pieces of evidence concur to attribute the increased risk of ventricular arrhythmias in early HCM to different cellular mechanisms. The increase of late sodium current and L-type calcium current is an early observation in HCM, which follows post-translation channel modifications and increases the occurrence of early and delayed afterdepolarizations. Increased myofilament Ca2+ sensitivity, commonly observed in HCM, may promote afterdepolarizations and reentry arrhythmias with direct mechanisms. Decrease of K+-currents due to transcriptional regulation occurs in the advanced disease and contributes to reducing the repolarization-reserve and increasing the early afterdepolarizations (EADs). The presented evidence supports the idea that patients with early-stage HCM should be considered and managed as subjects with an acquired channelopathy rather than with a structural cardiac disease.

Vascular endothelial growth factor modulates pulmonary vein arrhythmogenesis via vascular endothelial growth factor receptor 1/NOS pathway

Atrial fibrillation (AF) is a common form of arrhythmia with serious public health impacts, but its underlying mechanisms are not yet fully understood. Vascular endothelial growth factor (VEGF) is highly expressed in the atrium of patients with AF, but whether VEGF affects AF pathogenesis remains unclear. Pulmonary veins (PVs) are important sources for the genesis of atrial tachycardia or AF. Therefore, this study assessed the effects of VEGF on PV electrophysiological properties and evaluated its underlying mechanisms. Conventional microelectrodes and whole-cell patch clamps were performed using isolated rabbit PV preparations or single isolated PV cardiomyocytes before and after VEGF or VEGF receptor (VEGFR), Akt, NOS inhibitor administration. We found that VEGF (0.1, 1, and 10 ng/mL) reduced the PV beating rate in a dose-dependent manner. Furthermore, VEGF (10 ng/mL) reduced late diastolic depolarization and diastolic tension. Isoproterenol increased PV beating and burst firing, which was attenuated by VEGF (1 ng/mL). In the presence of VEGFR-1 inhibition (ZM306416 at 10 μM) and L-NAME (100 μM), VEGF (1 ng/mL) did not alter PV spontaneous activity. In isolated PV cardiomyocytes, VEGF (1 ng/mL) decreased L-type calcium, sodium/calcium exchanger, and late sodium currents. In conclusion, we found that VEGF reduces PV arrhythmogenesis by modulating sodium/calcium homeostasis through VEGFR-1/NOS signaling pathway.

Late sustained sodium current in the adult human heart

Late sustained sodium current in the adult human heart

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes.

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD90. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (Ito) and late sodium current (INa,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of Ito. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly INa,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

The neuronal potassium current IA is a potential target for pain during chronic inflammation.

Voltage‐gated ion channels play a key role in the action potential (AP) initiation and its propagation in sensory neurons. Modulation of their activity during chronic inflammation creates a persistent pain state. In this study, we sought to determine how peripheral inflammation caused by complete Freund's adjuvant (CFA) alters the fast sodium (INa), L‐type calcium (ICaL), and potassium (IK) currents in primary afferent fibers to increase nociception. In our model, intraplantar administration of CFA induced mechanical allodynia and thermal hyperalgesia at day 14 post‐injection. Using whole‐cell patch‐clamp recording in dissociated small (C), medium (Aδ), and large‐sized (Aβ) rat dorsal root ganglion (DRG) neurons, we found that CFA prolonged the AP duration and increased the amplitude of the tetrodotoxin‐resistant (TTX‐r) INa in Aβ fibers. In addition, CFA accelerated the recovery of INa from inactivation in C and Aδ nociceptive fibers but enhanced the late sodium current (INaL) only in Aδ and Aβ neurons. Inflammation similarly reduced the amplitude of ICaL in each neuronal cell type. Fourteen days after injection, CFA reduced both components of IK (IKdr and IA) in Aδ fibers. We also found that IA was significantly larger in C and Aδ neurons in normal conditions and during chronic inflammation. Our data, therefore, suggest that targeting the transient potassium current IA represents an efficient way to shift the balance toward antinociception during inflammation, since its activation will selectively decrease the AP duration in nociceptive fibers. Altogether, our data indicate that complex interactions between IK, INa, and ICaL reduce pain threshold by concomitantly enhancing the activity of nociceptive neurons and reducing the inhibitory action of Aβ fibers during chronic inflammation.

Eugenol interacts with cardiac sodium channel and reduces heart excitability and arrhythmias

AimsEugenol is a natural compound found in the essential oils of many aromatic plants. The compound is used as a local anesthetic because of its inhibitory effect on the voltage-gated Na+ channels (Nav), which are expressed in the nociceptive neurons. Eugenol has shown wide range of activities in the cardiovascular system; most of these activities are attributed to the modulation of voltage-sensitive Ca2+ channels. However, its action on Nav1.5, the main subtype of Nav expressed in the mammalian myocardium, is unknown. The interaction of eugenol with Nav1.5 could also contribute to its antiarrhythmic properties in vitro and ex vivo. We investigated the compound's effect on sodium current (INa) and its possible cardiac antiarrhythmic activity. MethodsThe effect of eugenol on cardiac contractility was investigated using isolated atrium from guinea pig (for isometric force measurements). The compound's effect on INa was evaluated using human embryonic cell transiently expressing human Nav1.5 and patch-clamp technique. Key findingsEugenol caused negative inotropic and chronotropic effects in the atria. In the ex vivo arrhythmia model, eugenol decreased atrial pacing disturbance induced by ouabain. Eugenol reduced the INa in a concentration-dependent manner. Furthermore, the compound left-shifted the stationary inactivation curve, delayed recovery from inactivation of the INa, and preferentially blocked the channel in the inactivated state. Importantly, eugenol was able to attenuate the late sodium current. All these aspects are considered to be antiarrhythmic. SignificanceOverall, our findings demonstrate that eugenol has antiarrhythmic activity due, at least in part, to its interaction with Nav1.5.

Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

Mexiletine rescues the mixed phenotype in SCN5A-1795insD hiPSC-CMs

Abstract Funding Acknowledgements Type of funding sources: None. Background The sodium channel blocker mexiletine can reduce late sodium current (INa) in patients with LQT3 syndrome, and additionally restore the decreased peak INa associated with SCN5A loss of function mutations. Purpose To investigate whether mexiletinecan rescue the mixed phenotype associated with the SCN5A-1795insD mutation in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Methods and Results HEK293 cells transfected with SCN5A-1795insD and SCN5A-WT and hiPSC-CMs from a patient carrying the SCN5A-1795insD mutation were incubated with a therapeutic dose of mexiletine (10 µM) or vehicle (H2O) for 48h. Peak INa, late INa and action potential (AP) properties were assessed by patch-clamp analysis. In HEK-293 cells transfected with SCN5A-1795insD or SCN5A-WT, exposure to mexiletine caused a significant increase in peak INa, in addition to a small increase in late INa in HEK-293 cells transfected with SCN5A-1795insD. In 1795insD hiPSC-CMs, peak INa was significantly increased whereas late INa was unchanged after mexiletine treatment. Accordingly, mexiletine increased AP upstroke velocity in SCN5A-1795insD hiPSC-CMs (indicating a rescue of INa availability), while AP amplitude, resting membrane potential and AP duration were unaffected. Conclusions Chronic treatment with a therapeutic concentration of mexiletine is capable of rescuing the mixed phenotype in SCN5A-1795insD hiPSC-CMs.

Post myocardial infarction ionic remodelling promotes repolarisation dispersions and abnormalities in acute and chronic stages

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Wellcome Trust Background Sudden cardiac death after a myocardial infarction (MI) was reported in both the acute stage and the chronic stage with the development of heart failure (HF). Around 50% of these deaths were due to ventricular tachyarrhythmia which is related to the temporal dispersion of repolarisation caused by the post-MI remodelling of ionic currents. While variable extent of ionic current changes was observed in multiple animal species in different post-MI phases, the proarrhythmic effects of such remodelling in post infarction human hearts is not fully explored. Purpose The goal of this study is to explore the proarrhythmic effects of post-MI ionic remodelling in human ventricular myocyte models of electrophysiology for both the acute and the chronic stages. Methods The ToR-ORd human ventricular cellular model was used as the baseline model, and a new formulation of the calcium activated potassium current was implemented. We constructed and calibrated a population of 253 human ventricular cell models (POM) to represent electrophysiological variability of normal zone (NZ) myocytes. For the acute phase, three types of border zone (BZ) remodelling (Acute BZ1-3) were introduced based on dog experimental data collected within one week after infarction. For the chronic phase with the development of HF, BZ (Chronic BZ) and one type of remote zone (RZ) remodelling (Chronic RZ1) were introduced based on minipig data collected 5 months post-MI. Another type of RZ remodelling (Chronic RZ2) was introduced based on experimental data from failing human hearts. The cellular models were paced at cycle lengths of 1000ms, 500ms, 400ms and 300ms for 500 beats before the analysis of repolarization abnormalities. Results For both the acute and the chronic stages post-MI, the extent of repolarisation dispersion between BZ and the corresponding NZ and RZ varied from 2ms up to 120ms, and largely depended on the level of potassium current remodelling. A weaker calcium transient was always observed in the BZ. Early after depolarisations (EADs) and repolarisation failure (RF) were observed in the chronic stages, caused by the reactivation of L-type calcium current due to the augmentation of late sodium current and the inhibition of hERG current. Beat-to-beat repolarization alternans was observed in both the acute and the chronic stages, with the key ionic mechanisms being the inhibition of SERCA activity and the slower calcium release due to enhanced CaMKII. Conclusions Post-MI ionic remodelling of the acute and chronic stages can cause a substantial dispersion of repolarisation in human hearts by inducing the prolongation of action potential durations, EADs, RFs, and alternans, providing substrates for the generation and maintenance of lethal reentrant waves. Abstract Figure. BZ and RZ ionic current remodelling

Human induced pluripotent stem cell-derived atrial cardiomyocytes carrying an SCN5A mutation identify nitric oxide signaling as a mediator of atrial fibrillation.

SummaryMutations in SCN5A, encoding the cardiac sodium channel, are linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain unclear. To characterize the pathogenesis of AF-linked SCN5A mutations, we generated patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) from two kindreds carrying SCN5A mutations (E428K and N470K) and isogenic controls using CRISPR-Cas9 gene editing. We showed that mutant AF iPSC-aCMs exhibited spontaneous arrhythmogenic activity with beat-to-beat irregularity, prolonged action potential duration, and triggered-like beats. Single-cell recording revealed enhanced late sodium currents (INa,L) in AF iPSC-aCMs that were absent in a heterologous expression model. Gene expression profiling of AF iPSC-aCMs showed differential expression of the nitric oxide (NO)-mediated signaling pathway underlying enhanced INa,L. We showed that patient-specific AF iPSC-aCMs exhibited striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses supported that the NO signaling pathway modulated the INa,L and triggered AF.

A novel outpatient regimen in management of fluoropyrimidine-induced cardiotoxicity.

e15613 Background: Fluoropyrimidines, such as 5-fluorouracil (5-FU) and capecitabine, are commonly used chemotherapies for solid tumors, and essential for curative intent treatment of colorectal cancer. But sometimes, their use may be limited by cardiac toxicity limiting the possibility of cure in some patients. Cardiotoxicity could be asymptomatic (EKG changes) or manifested as chest pain, arrhythmias, acute coronary syndrome or death. Rechallenging may be daunting and it may result in interruption or even discontinuation of planned chemo. Traditionally, nitrates and/or IV/oral calcium channel blockers (CCB) have been used for management of fluoropyrimidine-induced cardiotoxicity but without much benefit. Ranolazine, an oral antianginal drug approved for chronic angina, diminishes myocardial ischemia by reducing calcium overload caused by inhibition of late sodium current and it does not affect heart rate or blood pressure. Our objective was to evaluate the efficacy of our novel approach using ranolazine with other traditional drugs. Methods: 8 patients (median age 49.5 yrs) with fluoropyrimidine induced cardiotoxicity were retrospectively analyzed. They were rechallenged with the planned fluoropyrimidine regimen with our 3 drug cardioprotective regimen (KU protocol). This included oral Ranolazine 1000mg BID and Amlodipine 2.5 mg daily to start the day before starting 5FU infusion/oral capecitabine and to continue it until the day after completion of infusion/treatment, and Nitroglycerin paste 1 inch every 6 hours starting before infusion and continue until it was completed. These meds were discontinued upon completion of chemo. Results: 8 patients were rechallenged with fluoropyrimidine utilizing KU protocol, 6 patients (75%) were able to complete previously planned fluoropyrimidine regimen. One pt (*) discontinued capecitabine due to recurrent chest pain and treatment was switched to 5FU based regimen with KU protocol, which pt was able to complete without chest pain. Another pt (**), 5FU was stopped due to severe diarrhea, not due to cardiotoxicity. All pts tolerated KU protocol well. Conclusions: In our small, single center experience, we were able to safely and effectively rechallenge pts with fluoropyrimidines and complete curative intent treatment with our KU protocol. This protocol uses FDA approved oral and transcutaneous drugs without requiring a healthcare personnel to administer an IV CCB that can cause precipitous bradycardia and/or hypotension. Our results need validation in a larger cohort. [Table: see text]

Effects of phosphodiesterase-1 inhibitor on pulmonary vein electrophysiology and arrhythmogenesis.

Phosphodiesterase (PDE) isoform inhibitors have mechanical and electrical effects on the heart. Inhibition of PDE-1 enzymes is a novel strategy for treating heart failure. However, the electrophysiological effects of PDE-1 inhibition on the heart remain unclear. This study explored the effects of PDE-1 inhibition using ITI-214 on electrical activity in the pulmonary vein (PV), the most common trigger of atrial fibrillation, and investigated the underlying ionic mechanisms. Conventional microelectrodes or whole-cell patch clamps were employed to study the effects of ITI-214 (0.1-10μM) on PV electrical activity, mechanical responses and ionic currents in isolated rabbit PV tissue specimens and isolated single PV cardiomyocytes. ITI-214 at 1μM and 10μM (but not 0.1μM) significantly reduced PV spontaneous beating rate (10±2% and 10±3%, respectively) and PV diastolic tension (11±3% and 17±3%, respectively). ITI-24 (1μM) significantly reduced late sodium current (INa-Late ), L-type calcium current (ICa-L ) and the reverse mode of the sodium-calcium exchanger (NCX), but it did not affect peak sodium currents. ITI-214 reduces PV spontaneous activity and PV diastolic tension by reducing INa-Late , ICa-L and NCX current. Considering its therapeutic potential in heart failure, targeting PDE-1 inhibition may provide a novel strategy for managing atrial arrhythmogenesis.