Cardiac Sodium Current Research Articles

Phospho-ablation of cardiac sodium channel Nav1.5 mitigates susceptibility to atrial fibrillation and improves glucose homeostasis under conditions of diet-induced obesity

BackgroundAtrial fibrillation (AF) is the most common sustained arrhythmia, with growing evidence identifying obesity as an important risk factor for the development of AF. Although defective atrial myocyte excitability due to stress-induced remodeling of ion channels is commonly observed in the setting of AF, little is known about the mechanistic link between obesity and AF. Recent studies have identified increased cardiac late sodium current (INa,L) downstream of calmodulin-dependent kinase II (CaMKII) activation as an important driver of AF susceptibility.MethodsHere, we investigated a possible role for CaMKII-dependent INa,L in obesity-induced AF using wild-type (WT) and whole-body knock-in mice that ablates phosphorylation of the Nav1.5 sodium channel and prevents augmentation of the late sodium current (S571A; SA mice).ResultsA high-fat diet (HFD) increased susceptibility to arrhythmias in WT mice, while SA mice were protected from this effect. Unexpectedly, SA mice had improved glucose homeostasis and decreased body weight compared to WT mice. However, SA mice also had reduced food consumption compared to WT mice. Controlling for food consumption through pair feeding of WT and SA mice abrogated differences in weight gain and AF inducibility, but not atrial fibrosis, premature atrial contractions or metabolic capacity.ConclusionsThese data demonstrate a novel role for CaMKII-dependent regulation of Nav1.5 in mediating susceptibility to arrhythmias and whole-body metabolism under conditions of diet-induced obesity.

Small G-protein RhoA is a potential inhibitor of cardiac fast sodium current.

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and epithelial Na+ channels. The present study was aimed to check the RhoA potential regulatory effects on Na+ current (INa) transferred by Na+ channel cardiac isoform NaV1.5 in heterologous expression system and in native rat cardiomyocytes. Whole-cell patch-clamp experiments showed that coexpression of NaV1.5 with the wild-type RhoA in CHO-K1 cell line caused 2.7-fold decrease of INa density with minimal influence on steady-state activation and inactivation. This effect was reproduced by the coexpression with a constitutively active RhoA, but not with a dominant negative RhoA. In isolated ventricular rat cardiomyocytes, a 5-h incubation with the RhoA activator narciclasine (5 × 10-6M) reduced the maximal INa density by 38.8%. The RhoA-selective inhibitor rhosin (10-5M) increased the maximal INa density by 25.3%. Experiments with sharp microelectrode recordings in isolated right ventricular wall preparations showed that 5 × 10-6M narciclasine induced a significant reduction of action potential upstroke velocity after 2h of incubation. Thus, RhoA might be considered as a potential negative regulator of sodium channels cardiac isoform NaV1.5.

The selective late sodium current inhibitor eleclazine reduces atrial fibrillation dominant frequency and facilitates the suppression of arrhythmia in HL-1 cells

Abstract Background The cardiac late sodium current (INaL) has been increasingly implicated in the initiation of atrial fibrillation (AF). In fact, it has been reported that the augmentation of INaL in pathophysiological conditions prolongs repolarization and facilitates the appearance of afterdepolarizations, which can act as triggers of arrhythmic activity. Eleclazine is a novel selective inhibitor of INaL and is undergoing clinical testing for the treatment of cardiac arrhythmias. Purpose The aim of this study was to investigate the effects of eleclazine on spectral characteristics of atrial fibrillation in cultured atrial myocyte monolayer in order to assess whether this inhibitor could protect against cardiac arrhythmias. Methods Confluent HL-1 murine atrial myocyte monolayer with spontaneous fibrillatory activity was cultured in 1.5 cm diameter petri dishes (n=10). A high-resolution optical mapping system was used to record fibrillatory activity under basal conditions (without drug), and under eleclazine at increasing concentrations (1, 3 and 5 μM). Power spectra of optical signals were estimated by using Welch periodogram and dominant frequency (frequency with the largest peak in the spectrum between 0.05 and 30 Hz) was determined. The incidence of spontaneous defibrillation was analyzed under control and drug conditions. An ANOVA and a chi-squared test were used. Significance was reached when p<0.05. Results Eleclazine at 1, 3 and 5 μM significantly decreased dominant frequency with respect to basal conditions (basal: 4.74±1.31 Hz; 1μM: 3.59±1.17 Hz, p<0.001; 3μM: 3.19±0.64 Hz, p<0.01; 5μM: 2.58±0.40 Hz, p<0.01). The magnitude of drug-induced decrease in AF activation frequency was enhanced by increasing concentrations (1μM: 27%, 3μM: 42%; 5μM: 46%; p<0.05). After the analysis of optical signals, we observed that the incidence of spontaneous defibrillation in atrial monolayers was significantly greater under eleclazine 5μM action than under control conditions (chi-square=5.00, p=0.025). In fact, under the action of eleclazine 5μM, fibrillatory activity was suppressed in 4 of the 10 monolayers, phenomenon that did not occur in any case of control situation. Conclusions Selective late INa inhibition with eleclazine reduced dominant frequency of atrial fibrillation and facilitates the termination of arrhythmia in cultured atrial myocyte monolayer. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III, Ministerio de Innovaciόn y Ciencia, Spain; Generalitat Valenciana, Spain

Chloroquine or Hydroxychloroquine for COVID-19: Is Cardiotoxicity a Concern?

Chloroquine or Hydroxychloroquine for COVID-19: Is Cardiotoxicity a Concern?

A study of the outward background current conductance gK1, the pacemaker current conductance gf, and the gap junction conductance gj as determinants of biological pacing in single cells and in a two-cell syncytium using the dynamic clamp.

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A.

Electrocardiological effects of ranolazine and lidocaine on normal and diabetic rat atrium.

Cellular changes occurring in diabetic cardiomyopathy include disturbances of calcium and sodium homeostasis. Voltage-gated sodium channels are responsible for the initiation of cardiac action potentials, and the excitability would create relevance. The effect of ranolazine as a sodium channel blocker on atrium electromechanical parameters is investigated and compared with lidocaine in streptozocin-treated diabetic rats. After an 8-week induction of diabetes type I, the effect of cumulative concentrations of ranolazine and lidocaine on the electrophysiology of isolated atrium was studied. Ranolazine's effects were evaluated on cardiac sodium current in normal- and high-glucose medium, with whole-cell patch-clamp technique. Ranolazine at therapeutic concentrations had no significant statistical effect on refractory period in normal and diabetic isolated heart. Ranolazine (10μM) caused a hyperpolarizing shift of V1/2 for steady-state inactivation in normal media, while it significantly elicited a depolarizing shift in high-glucose media (p < 0.05). It is concluded that in the isolated rat atrium preparation, ranolazine and lidocaine have no beneficial on diabetic cardiomyopathy. Although refractoriness and contractility were not much different in normal and diabetic atria, there was a definite effect of ranolazine and lidocaine on sodium current in varying concentrations. This may have significance in future therapeutics.

Abstract TP216: Identifying Common Genetic Variants Associated With Fatal Stroke in Incident Ischemic Stroke Patients

Introduction: There is an unmet need to understand the genetic factors associated with stroke outcome. The purpose of this study is to identify the common genetic variants, if any, associated with incident of ischemic stroke in a community-based population and to determine if any genetic risk can be enriched in fatal stroke patients. Methods: Cases and controls of ischemic stroke were identified by leveraging the comprehensive Electronic Health Record, and endophenotypes, e.g. the fatal or nonfatal stroke was defined by those dying within one month (fatal incidence) or surviving to 12 months (nonfatal incidence) of their first-ever stroke incident. The GWAS was carried out in a nested case-control design by considering all patients with age &gt;79 and without any stroke-related diagnostic codes as low-risk control. A linear mixed regression model (SAIGE) with saddlepoint approximation, adjusted for covariates was conducted to account for the relatedness and case-control imbalance. Results: No genome-wide significant association was identified for ischemic stroke (n=1,179) versus control (n=7,489). When stratifying stroke patients by the fatality (n=61/1,118), rs80094021 (MAF = 0.038; OR=3.332±0.635; p=9.04E-11) and SNPs in LD with, located at an intronic region of TIAM1, were the top associated signal. All these SNPs served as eQTL for TIAM1(β=0.144, p=5.8E-13, eQTLGen). Rs79694970, the second top SNP (MAF = 0.020; OR=4.179±0.931; p=9.27E-09), is located near GPD1L and functions as eQTL for GPD1L(β=-0.279, p=6.1E-28). GPD1L plays a role in the DPD1L-dependent SCN5A phosphorylation pathway, thereby regulating cardiac sodium current. Dysfunction of sodium current can cause fatal ventricular arrhythmia. According to PheWAS data from UK Biobank, rs80094021 showed a moderate association with high cholesterol (p=0.0045), coronary atherosclerosis (p=0.0075), and angina (p=0.012), with the same direction for the minor allele which increased the risk for fatal stroke. rs79694970 were associated with ischemic stroke (large artery atherosclerosis) with p=0.0016. Conclusion: Through stratification of ischemic stroke by fatality some genetic risk factors and genes with biological relevant to the pathogenesis of stroke could be identified.

Ginsenoside Rb1 exerts antiarrhythmic effects by inhibiting INa and ICaL in rabbit ventricular myocytes

Ginsenoside Rb1 exerts its pharmacological action by regulating sodium, potassium and calcium ion channels in the membranes of nerve cells. These ion channels are also present in cardiomyocytes, but no studies have been reported to date regarding the effects of Rb1 on cardiac sodium currents (INa), L-type calcium currents (ICaL) and action potentials (APs). Additionally, the antiarrhythmic potential of Rb1 has not been assessed. In this study, we used a whole-cell patch clamp technique to assess the effect of Rb1 on these ion channels. The results showed that Rb1 inhibited INa and ICaL, reduced the action potential amplitude (APA) and maximum upstroke velocity (Vmax), and shortened the action potential duration (APD) in a concentration-dependent manner but had no effect on the inward rectifier potassium current (IK1), delayed rectifier potassium current (IK) or resting membrane potential (RMP). We also designed a pathological model at the cellular and organ level to verify the role of Rb1. The results showed that Rb1 abolished high calcium-induced delayed afterdepolarizations (DADs), depressed the increase in intracellular calcium ([Ca2+]i), relieved calcium overload and protected cardiomyocytes. Rb1 can also reduce the occurrence of ventricular premature beats (VPBs) and ventricular tachycardia (VT) in ischemia-reperfusion (I-R) injury.

P4775Efficacy and safety of vernakalant for cardioversion of recent-onset atrial fibrillation in real-world clinical practice: the SPECTRUM post-approval safety study

Abstract Background Vernakalant is an antiarrhythmic agent designed for pharmacological conversion of recent onset of atrial fibrillation (AF) with combined action on cardiac potassium and sodium currents mainly concentrated in the atria. Purpose SPECTRUM was a post-authorisation safety study of vernakalant, conducted to collect information about real-life conditions of use and appropriate dosing, and to quantify possible medically significant risks associated with the use of vernakalant in real-world clinical practice. Methods This prospective and retrospective registry was conducted from Sep 2011 to Apr 2018 in 53 hospitals in EU countries including Austria, Denmark, Finland, Germany, Spain and Sweden. A total of 1,778 patients with 2,009 episodes of recent-onset AF received vernakalant and were followed up for 24 hours after the last infusion or until hospital discharge/end of medical encounter to obtain information on medically significant health outcomes of interest (HOIs, defined as significant hypotension, significant ventricular arrhythmia, significant atrial flutter, significant bradycardia), and serious adverse events (SAEs). Results In more than 99% of treatments, vernakalant was used in accordance to the labelled indication for conversion of AF for non-surgery (94.7%) or post-cardiac surgery patients (5.2%). Vernakalant was administered in the emergency department in 64.2% of cases, with a median stay of 7.5 hours and successfully converted 70.2% (95% CI: 68.1–72.2) of patients in the effectiveness analysis population with a median time to conversion of 11 minutes (95% CI: 8.0–27.0). A total of 19 HOIs were reported in 17 patients (0.8%, 95% CI: 0.5– .4%) with individual HOIs ranging from &lt;0.1% to 0.7% suggesting these HOIs are uncommon. Significant bradycardia was the most common HOI observed in 15 patients (0.8%, 95% CI: 0.4–1.2%), with all events occurring within the first two hours (0.8%, 95% CI: 0.4–1.2%). The incidence of significant hypotension was 0.1% (2/2,009), significant atrial flutter (with 1:1 conduction) was 0.1% (2/2,009), and significant ventricular arrhythmia (sustained ventricular tachycardia) was &lt;0.1% (1/2,009). A total of 28 SAEs, including all HOIs, were observed (1.3%, 95% CI: 0.8–1.9%); all patients fully recovered, except one who recovered with sequelae after an SAE of pericardial effusion definitely not related to vernakalant. There were no cases of torsades de pointes, ventricular fibrillation, or deaths reported in the SPECTRUM study. Conclusion(s) SPECTRUM is, to our knowledge, the largest drug registry conducted on the cardioversion of recent onset AF. The cumulative data from 2,009 vernakalant treatment episodes demonstrate an incidence of HOIs and SAEs similar or lower to what has been reported in earlier vernakalant IV clinical trials. The observed conversion rate was higher than reported in pivotal trials supporting vernakalant's efficacy and allowing early discharge. Acknowledgement/Funding Study funded by Correvio International Sarl, Geneva, Switzerland

Abstract 811: The Role of Mitochondrial Stress and the Late Sodium Current in Ibrutinib-Mediated Atrial Fibrillation

Background: Ibrutinib (a Bruton’s tyrosine kinase inhibitor, BTKI) is rapidly becoming the treatment of choice for many B-cell malignancies, but its proarrhythmic effects may limit its widespread use. GWAS have consistently identified an AF risk locus on chromosome (chr) 4q25, and the nearest gene Pitx2c has been implicated in AF development as evidenced by increased AF in Pitx2c +/- mice which serve as an experimental model for clinical AF. Objective: To assess the role of the late cardiac Na current (I Na,L ) and oxidative stress in mediating increased susceptibility to AF in Pitx2c +/- mice which are genetically prone to developing AF as well as in atrial human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to ibrutinib. Method: Pitx2c +/- mice were administered ibrutinib (30mg/kg/day IP) for 14 days while atrial hiPSC-CMs were exposed to ibrutinib for 48 hrs. Results: There was a graded increase in AF episodes and burden with the number of days the Pitx2c +/- mice were exposed to ibrutinib versus controls ( Fig. 1A ). The action potential duration at 90% repolarization and the I Na-L were markedly prolonged ( Fig. 1B-C ). Furthermore, we observed a significant increase in mitochondrial fragmentation ( Fig. 1D-E ) and superoxide production ( Fig. 1F-G ) in ibrutinib treated atrial hiPSC-CMs versus controls. Conclusion: We showed in Pitx2c +/- mice and atrial hiPSC-CMs that ibrutinib-mediated AF may in part be related to enhanced I Na-L and mitochondrial dysfunction leading to increased oxidative stress. Patients undergoing ibrutinib treatment may benefit from novel therapies which target mitochondrial dysfunction or the late cardiac sodium current.

SCN5A variant R222Q generated abnormal changes in cardiac sodium current and action potentials in murine myocytes and Purkinje cells

The cardiac sodium channel (SCN5A) mutation R222Q neutralizes a positive charge in the domain I voltage sensor. Mutation carriers display very frequent ectopy and dilated cardiomyopathy. To describe the effect of SCN5A R222Q on murine myocyte and Purkinje fiber electrophysiology, and identify underlying mechanisms. We generated mice carrying humanized wild-type (H) and mutant (RQ) SCN5A channels. We characterized whole-heart and isolated ventricular and Purkinje myocyte properties. RQ/RQ mice were not viable. INa from RQ/H ventricular myocytes displayed increased "window current" and hyperpolarizing shifts in both inactivation and activation compared to H/H, as previously reported in heterologous expression systems. Surprisingly, action potentials were markedly abbreviated in RQ/H myocytes (action potential durations at 90% repolarization: 12.6± 1.3 ms vs 29.1 ± 1.0 ms in H/H, P < .01, n = 10 each). We identified a large [K+]o-dependent outward gating pore current in RQ/H but not H/H myocytes, and decreasing [K+]o elicited early afterdepolarizations (EADs) and triggered activity in isolated myocytes and ectopic beats in whole hearts. Further, RQ/H Purkinje cells displayed striking, consistent low-voltage EADs. Invivo, however, RQ/H mice displayed little ectopy and contractile function was normal. While SCN5A R222Q increases plateau inward sodium current, action potentials were unexpectedly shortened, likely reflecting an outward gating-pore current. Low extracellular potassium increased this pore current, and was arrhythmogenic invitro and exvivo.

In utero exposure to nicotine abolishes the postnatal response of the cardiac sodium current to isoproterenol in newborn rabbit atrium

In utero exposure to tobacco smoke is associated with sudden infant death syndrome (SIDS) and cardiac arrhythmias in newborns. The arrhythmogenic mechanisms seem linked to alterations of the cardiac sodium current (INa). We previously reported that in utero exposure to nicotine delays the postnatal development of the heart sinoatrial node in rabbits and altered expression of the sodium channels NaV1.5 and NaV1.1 in the atrium surrounding it. These channels react differently to sympathetic stimulation. The purpose of this study was to test whether nicotine altered the response of INa to stimulation by the β-adrenoreceptor agonist isoproterenol in atrial myocytes. Our hypothesis is that changes in the sympathetic response of sinoatrial node peripheral cells may create a substrate for arrhythmia. Using the patch-clamp technique we measured the effect of nicotine on the response of INa to adrenergic stimulation in isolated cardiomyocytes. Isoproterenol increased INa by 50% in newborn sham rabbits but had no effect in newborn rabbits exposed to nicotine in utero. Our data also show that nicotine increases the late sodium current, an effect that may promote QT prolongation. We provide the first evidence linking fetal exposure to nicotine to long-term alterations of INa response to isoproterenol. These changes may impair INa adaptation to sympathetic tone and prevent awakening from sleep apnea, thus leading to arrhythmias that could potentially be involved in SIDS. Our data also raise concerns about the use of nicotine replacement therapies for pregnant women.

Multiple mechanisms underlie increased cardiac late sodium current

We recently reported a quantitative relationship between the degree of functional perturbation reported in the literature for 356 variants in the cardiac sodium channel gene SCN5A and the penetrance of resulting arrhythmia phenotypes. In the course of that work, we identified multiple SCN5A variants, including R1193Q, that are common in populations but are reported in human embryonic kidney (HEK) cells to generate large late sodium current (INa-L). The purpose of this study was to compare the functional properties of R1193Q with those of the well-studied type 3 long QT syndrome mutation ΔKPQ. We compared functional properties of SCN5A R1193Q with those of ΔKPQ in Chinese hamster ovary (CHO) cells at baseline and after exposure to intracellular phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which inhibits INa-L generated by decreased Phosphoinositide 3-kinase (PI3K) activity. We also used CRISPR/Cas9 editing to generate R1193Q in human-induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs). Both R1193Q and ΔKPQ generated robust INa-L in CHO cells. PIP3 abrogated the late current phenotype in R1193Q cells but had no effect on ΔKPQ. Homozygous R1193Q hiPSC-CMs displayed increased INa-L and long action potentials with frequent triggered beats, which were reversed with the addition of PIP3. The consistency between the late current produced in HEK cells, CHO cells, and hiPSC-CMs suggests that the late current is a feature of the SCN5A R1193Q variant in human cardiomyocytes but that the mechanism by which the late current is produced is distinct and indirect, as compared with the more highly penetrant ΔKPQ. These data suggest that observing a late current in an invitro setting does not necessarily translate to highly pathogenic type 3 long QT syndrome phenotype but depends on the underlying mechanism.

Lamin A mutation impairs interaction with nucleoporin NUP155 and disrupts nucleocytoplasmic transport in atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Here, we show the identification and functional characterization of one AF-associated mutation p.Arg399Cys in lamin A/C. Co-immunoprecipitation and GST pull-down assays demonstrate that lamin A/C interacts with NUP155, which is a nucleoporin and causes AF when mutated. Lamin A/C mutation p.Arg399Cys impairs the interaction between lamin A/C and NUP155, and increases extractability of NUP155 from the nuclear envelope (NE). Mutation p.Arg399Cys leads to aggregation of lamin A/C in the nucleus, although it does not impair the integrity of NE upon cellular stress. Mutation p.Arg399Cys inhibits the export of HSP70 mRNA and the nuclear import of HSP70 protein. Electrophysiological studies show that mutation p.Arg399Cys decreases the peak cardiac sodium current by decreasing the cell surface expression level of cardiac sodium channel Nav 1.5, but does not affect IKr potassium current. In conclusion, our results indicate that lamin A/C mutation p.Arg399Cys weakens the interaction between nuclear lamina (lamin A/C) and the nuclear pore complex (NUP155), leading to the development of AF. The findings provide a novel molecular mechanism for the pathogenesis of AF.

Concomitant SK current activation and sodium current inhibition cause J wave syndrome.

The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.

Abstract 17231: Sodium Channel Blockers Shorten Prolonged QTC in Patients With Rett Syndrome

Introduction: Rett syndrome (RTT) is an X-linked disorder caused by mutations in the MECP2 gene resulting in various neurological, respiratory, and gastrointestinal symptoms. Up to 25% of deaths in RTT are sudden. Prolonged corrected QT (QTc) has also been reported and thought to be a cause of sudden death in RTT. Previous studies in mice with RTT showed that MECP2 mutations lead to persistent increased cardiac sodium current causing prolonged QTc and increasing risks of ventricular tachycardia. The aim of this study was to determine whether sodium channel blockers lidocaine (acutely) and mexiletine (chronically) shorten QTc in RTT patients. Hypothesis: Sodium channel blockers such as lidocaine (acutely) and mexiletine (chronically) significantly shorten QTc (≥ 30ms from baseline) in RTT patients with prolonged QTc. Methods: RTT patients with prolonged QTc were prospectively enrolled to receive a lidocaine attenuation test. The test consisted of a loading dose of 1 mg/kg intravenous lidocaine followed by a continuous infusion at 50 μg / (kg·min). Patients were deemed responders should their QTc shorten by 30 ms or more. These patients were then prescribed mexiletine for chronic therapy. Results: A total of 6 patients (all females) were given lidocaine and mexiletine. The median age of the cohort at the time of testing was 12 years (8-19 years), and the median QTc before therapy was 480.5 ms (476-506 ms). All 6 patients responded positively to lidocaine with median QTc shortening of 44.5 ms (33-54 ms, P = 0.018). The median daily dose of mexiletine was 8.75 mg/kg. The median duration of therapy was 5 months (4-12 months). Mexiletine shortened QTc by an average of 47 ms at latest follow-up ( P = 0.016). One patient developed whole-body tremors as a side effect of mexiletine, which was stopped. Conclusions: Sodium channel blockers that target late sodium channels were effective in shortening QTc thereby confirming the presence of late sodium current causing prolonged QTc in RTT. Therefore mexiletine could be used clinically in RTT patients with long QTc. Larger and longer studies are needed to confirm these drugs’ positive effects in shortening the QTc and reducing arrhythmic events in RTT patients.

Predicting changes to INa from missense mutations in human SCN5A

Mutations in SCN5A can alter the cardiac sodium current INa and increase the risk of potentially lethal conditions such as Brugada and long-QT syndromes. The relation between mutations and their clinical phenotypes is complex, and systems to predict clinical severity of unclassified SCN5A variants perform poorly. We investigated if instead we could predict changes to INa, leaving the link from INa to clinical phenotype for mechanistic simulation studies. An exhaustive list of nonsynonymous missense mutations and resulting changes to INa was compiled. We then applied machine-learning methods to this dataset, and found that changes to INa could be predicted with higher sensitivity and specificity than most existing predictors of clinical significance. The substituted residues’ location on the protein correlated with channel function and strongly contributed to predictions, while conservedness and physico-chemical properties did not. However, predictions were not sufficiently accurate to form a basis for mechanistic studies. These results show that changes to INa, the mechanism through which SCN5A mutations create cardiac risk, are already difficult to predict using purely in-silico methods. This partly explains the limited success of systems to predict clinical significance of SCN5A variants, and underscores the need for functional studies of INa in risk assessment.

Differences in Functional Expression of Connexin43 and NaV1.5 by Pan- and Class-Selective Histone Deacetylase Inhibition in Heart.

Class-selective histone deacetylase (HDAC) inhibitors were designed to improve safety profiles and therapeutic effectiveness in the treatment of multiple cancers relative to pan-HDAC inhibitors. However, the underlying mechanisms for their therapeutic and cardiotoxic potentials remain poorly understood. Cardiac sodium currents (INa) and gap junction conductance (gj) were measured by whole cell patch clamp techniques on primary cultures of neonatal cardiomyocytes. Cardiac NaV1.5 sodium channel and connexin43 (Cx43) gap junction protein levels were assessed by Western blot analyses. Panobinostat produced concentration-dependent reductions in ventricular gj, peak INa density, and NaV1.5 protein expression levels. Membrane voltage (Vm)-dependent activation of INa was shifted by +3 to 6 mV with no effect on inactivation. Entinostat (1 μM) did not affect ventricular gj, peak INa density, or INa activation. However, the INa half-inactivation voltage (V½) was shifted by −3.5 mV. Ricolinostat had only minor effects on ventricular gj and INa properties, though INa activation was shifted by −4 mV. Cx43 and NaV1.5 protein expression levels were not altered by class-selective HDAC inhibitors. The lack of effects of class-selective HDAC inhibitors on ventricular gj and INa may help explain the improved cardiac safety profile of entinostat and ricolinostat.

Inhibition of the α-Subunit of Phosphoinositide 3-Kinase in Heart Increases Late Sodium Current and Is Arrhythmogenic.

Although inhibition of phosphoinositide 3-kinase (PI3K) is an emerging strategy in cancer therapy, we and others have reported that this action can also contribute to drug-induced QT prolongation and arrhythmias by increasing cardiac late sodium current (INaL). Previous studies in mice implicate the PI3K-α isoform in arrhythmia susceptibility. Here, we have determined the effects of new anticancer drugs targeting specific PI3K isoforms on INaL and action potentials (APs) in mouse cardiomyocytes and Chinese hamster ovary cells (CHO). Chronic exposure (10-100 nM; 5-48 hours) to PI3K-α-specific subunit inhibitors BYL710 (alpelisib) and A66 and a pan-PI3K inhibitor (BKM120) increased INaL in SCN5A-transfected CHO cells and mouse cardiomyocytes. The specific inhibitors (10-100 nM for 5 hours) markedly prolonged APs and generated triggered activity in mouse cardiomyocytes (9/12) but not in controls (0/6), and BKM120 caused similar effects (3/6). The inclusion of water-soluble PIP3, a downstream effector of the PI3K signaling pathway, in the pipette solution reversed these arrhythmogenic effects. By contrast, inhibition of PI3K-β, -γ, and -δ isoforms did not alter INaL or APs. We conclude that inhibition of cardiac PI3K-α is arrhythmogenic by increasing INaL and this effect is not seen with inhibition of other PI3K isoforms. These results highlight a mechanism underlying potential cardiotoxicity of PI3K-α inhibitors.

Sinus Bradycardia in Carriers of the SCN5A-1795insD Mutation: Unraveling the Mechanism through Computer Simulations.

The SCN5A gene encodes the pore-forming α-subunit of the ion channel that carries the cardiac fast sodium current (INa). The 1795insD mutation in SCN5A causes sinus bradycardia, with a mean heart rate of 70 beats/min in mutation carriers vs. 77 beats/min in non-carriers from the same family (lowest heart rate 41 vs. 47 beats/min). To unravel the underlying mechanism, we incorporated the mutation-induced changes in INa into a recently developed comprehensive computational model of a single human sinoatrial node cell (Fabbri–Severi model). The 1795insD mutation reduced the beating rate of the model cell from 74 to 69 beats/min (from 49 to 43 beats/min in the simulated presence of 20 nmol/L acetylcholine). The mutation-induced persistent INa per se resulted in a substantial increase in beating rate. This gain-of-function effect was almost completely counteracted by the loss-of-function effect of the reduction in INa conductance. The further loss-of-function effect of the shifts in steady-state activation and inactivation resulted in an overall loss-of-function effect of the 1795insD mutation. We conclude that the experimentally identified mutation-induced changes in INa can explain the clinically observed sinus bradycardia. Furthermore, we conclude that the Fabbri–Severi model may prove a useful tool in understanding cardiac pacemaker activity in humans.