Increase In Late Sodium Current Research Articles

The fungicide tebuconazole modulates the sodium current of human NaV1.5 channels expressed in HEK293cells.

The fungicide Tebuconazole is a widely used pesticide in agriculture and may cause cardiotoxicity. In our present investigation the effect of Tebuconazole on the sodium current (INa) of human cardiac sodium channels (NaV1.5) was studied using a heterologous expression system and whole-cell patch-clamp techniques. Tebuconazole reduced the amplitude of the peak INa in a concentration- and voltage-dependent manner. At the holding potential of -120 mV the IC50 was estimated at 204.1±34.3μM, while at -80 mV the IC50 was 0.3±0.1μM. The effect of the fungicide is more pronounced at more depolarized potentials, indicating a state-dependent interaction. Tebuconazole caused a negative shift in the half-maximal inactivation voltage and delayed recovery from fast inactivation of INa. Also, it enhanced closed-state inactivation, exhibited use-dependent block in a voltage-dependent manner. Furthermore, Tebuconazole reduced the increase in late sodium current induced by the pyrethroid insecticide β-Cyfluthrin. These results suggest that Tebuconazole can interact with NaV1.5 channels and modulate INa. The observed effects may lead to decreased cardiac excitability through reduced INa availability, which could be a new mechanism of cardiotoxicity to be attributed to the fungicide.

Late sodium current: incomplete inactivation triggers seizures, myotonias, arrhythmias, and pain syndromes.

Acquired and inherited dysfunction in voltage-gated sodium channels underlies a wide range of diseases. In addition to defects in trafficking and expression, sodium channelopathies are caused by dysfunction in one or several gating properties, for instance activation or inactivation. Disruption of channel inactivation leads to increased late sodium current, which is a common defect in seizure disorders, cardiac arrhythmias skeletal muscle myotonia and pain. An increase in late sodium current leads to repetitive action potentials in neurons and skeletal muscles, and prolonged action potential duration in the heart. In this Topical Review, we compare the effects of late sodium current in brain, heart, skeletal muscle and peripheral nerves.

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.

High-throughput screening of animal venoms for identification of compounds active on the cardiac Nav1.5 channel

Dysfunctions of the cardiac sodium channels (Nav1.5) have been associated with many cardiac diseases such as long QT syndrome or Brugada syndrome. In order to study the role of this channel in diseases or to restore function, molecules that specifically target this sodium channel are needed. Highly specific molecules for a given isoform of sodium channel are hard to discover with usual chemical libraries. Animal venoms, and especially spider venoms, contain each tens of peptides acting on ion channels and represent therefore interesting starting compound libraries for drug discovery. By screening spider venoms on Nav, we aimed to identify new toxins targeting specifically Nav1.5 channel which can be used as: – tools to study role of Nav1.5; – as pharmacological drug to prevent Nav1.5-related arrhythmias. For that purpose, 16 venoms isolated from spiders were first fractionated to generate libraries. Each fraction has been tested on several stable Nav cell lines using Syncropatch384PE system (Nanion) to ensure selectivity. False-positive fractions were excluded based on detection of material in HPLC and mass spectrometry analyses. We identified 10 native fractions active on Nav1.5. Among them, 5 inhibit sodium currents and 5 slow-down inactivation with increase in late sodium current. Theses fractions have been re-fractionated with a complementary purification approach until identification of isolated peptides. Validation of effects of these peptides on properties of Nav1.5 currents is ongoing. Sequences of positive hits are currently under study and chemical syntheses will be performed to get synthetic versions of these peptides. Complete pharmacological characterization of synthetic peptides is in progress. We developed a complete pipeline using automated patch-clamp that allows us to isolate and validate new toxins specific for human Nav1.5 sodium channel from large libraries of spider venoms.

Effects of amiodarone on rodent ventricular cardiomyocytes: Novel perspectives from a cellular model of Long QT Syndrome Type 3

AimsAmiodarone (AMIO) is currently used in medical practice to reverse ventricular tachycardia. Here we determine the effects of AMIO in the electromechanical properties of isolated left ventricle myocyte (LVM) from mice and guinea pig and in a cellular model of Long QT Syndrome Type 3 (LQTS-3) using anemone neurotoxin 2 (ATX II), which induces increase of late sodium current in LVM. Main methods and key findingsUsing patch-clamp technique, fluorescence imaging to detect cellular Ca2+ transient and sarcomere detection systems we evaluate the effect of AMIO in healthy LVM. AMIO produced a significant reduction in the percentage of sarcomere shortening (0.1, 1 and 10 μM) in a range of pacing frequencies, however, without significant attenuation of Ca2+ transient. Also, 10 μM of AMIO caused the opposite effect on action potential repolarization of mouse and guinea pig LVM. When LVM from mouse and guinea pig were paced in a range of pacing frequencies and exposed to ATX (10 nM), AMIO (10 μM) was only able to abrogate electromechanical arrhythmias in LVM from guinea pig at lower pacing frequency. SignificanceAMIO has negative inotropic effect with opposite effect on action potential waveform in mouse and guinea pig LVM. Furthermore, the antiarrhythmic action of AMIO in LQTS-3 is species and frequency-dependent, which indicates that AMIO may be beneficial for some types of arrhythmias related to late sodium current.

Prolonged QT Syndrome in Newborn Diagnostic Dilemma

Introduction: Long QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the SCN5A gene, which encodes the cardiac sodium channel Nav1.51. Mutations result in a pathological increase in late sodium current leading to delayed ventricular repolarization, torsade de pointes and sudden death(1). To the best of our knowledge, LQT3 has not been associated with manifestations including metabolic abnormalities during the newborn period. Case Report Patient presented on day seven of life with lethargy, hypotonia, hypothermia, and respiratory distress to his primary pediatrician. He was …

Increase of late sodium current contributes to enhanced susceptibility to atrial fibrillation in diabetic mice

Studies demonstrated that the incidence of atrial fibrillation is significantly increased in patients with diabetes mellitus. Increase of late sodium current (INaL) has been associated with atrial arrhythmias. However, the role of INaL in the setting of atrial fibrillation in diabetes mellitus remained unknown. In this study, we investigated the alteration of INaL in the atria of diabetic mice and the therapeutic effect of its inhibitor (GS967) on the susceptibility of atrial fibrillation. The whole-cell patch-clamp technique was used to detect single cell electrical activities. The results showed that the density of INaL in diabetic cardiomyocytes was larger than that of the control cells at the holding potential of −100 mV. The action potential duration at both 50% and 90% repolarization, APD50 and APD90, respectively, was markedly increased in diabetic mice than in controls. GS967 application inhibited INaL and shortened APD of diabetic mice. High-frequency electrical stimuli were used to induce atrial arrhythmias. We found that the occurrence rate of atrial fibrillation was significantly increased in diabetic mice, which was alleviated by the administration of GS967. In GS967-treated diabetic mice, the INaL current density was reduced and APD was shortened. In conclusion, the susceptibility to atrial fibrillation was increased in diabetic mice, which is associated with the increased late sodium current and the consequent prolongation of action potential. Inhibition of INaL by GS967 is beneficial against the occurrence of atrial fibrillation in diabetic mice.

Sudden Infant Death and Modulation of Late Sodium Current by Hypoxia, Investigated in Induced Pluripotent Stem Cells

Sudden infant death (SIDS) occurs in about 0.4 per 1000 life births in the United States and other western countries. Recognized risk factors for SIDS are prone sleeping position, maternal smoking, and bed sharing. These factors point to the potential involvement of respiratory stress in the syndrome.Furthermore, in about 20% of SIDS cases, mutations in cardiac ion channels have been found. Of those, about 50% affect the cardiac sodium channel, which is also affected in individuals suffering from long-QT syndrome type 3 (LQT3). In these mutated ion channels, the late component of the sodium current during the plateau phase of the cardiac action potential is markedly increased, due to incomplete inactivation of the channels.We hypothesise that a combination of hypoxia, induced by respiratory stress, and cardiac sodium channel mutations that increase the late sodium current, cause an synergistic increase in late sodium current, which predisposes vulnerable infants to sudden arrhythmic death.Preliminary data using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) supports this hypothesis. Using microelectrode arrays, we were able to show that in a pharmacological model of LQT3, hypoxic conditions dramatically increase the field potential upstroke velocity, which is an indirect marker of the sodium channel availability.Cells were treated with Anthopleurin-A, which destabilises the inactive state of cardiac sodium channels. Under hypoxic conditions, the field potential upstroke velocity was increased by about 4-fold, compared to normoxic conditions. This effect was not present in untreated control cells. The late sodium current blocker Ranolazine was able to reduce this effect in a dose dependent manner, suggesting that indeed the late sodium current was responsible for this effect.

Somatic Mosaicism of Novel SCN5A Mutation in Purkinje System (PS) may Underlie 2:1 Block in an Infant with Long QT Syndrome (LQTS)

Somatic mosaicism is a condition that arises when DNA mutations lead to genetic variations within an individual. Recently, an infant with LQTS characterized by episodes of 2:1 block (i.e., 2 atrial/His-bundle activations for every one ventricular activation) was found to have a novel SCN5a mutation (V1762L) in ∼20% of cardiomyocytes, causing a 10-fold increase in late sodium current within affected cells. However, the mechanism by which V1762L mosaicism could give rise to this clinical phenotype remained unknown. We hypothesized that 2:1 block could arise from 20% V1762L mosaicism in the PS, regardless of its spatial distribution.To test this hypothesis, we used a biophysically-detailed 3D computational model of the ventricles and PS calibrated to approximate the size/geometry of the human neonatal heart. Versions of the model incorporating 20% V1762L mosaicism with diffuse or clustered distributions in the ventricles and PS were generated using a stochastic approach. To examine the arrhythmogenic propensity of the heart models, simulated normal sinus rhythm (coupling interval, CI=400ms) was interrupted by two consecutive abnormally timed stimuli (variable CIs: 300-700ms in 50ms intervals).Ventricular mosaicism alone did not lead to 2:1 block. However, once mosaicism was incorporated into the PS, a majority of all pacing sequences in each model (diffuse: 79%, clustered: 80%) resulted in 2:1 block. Even pacing sequences with CIs within the physiological range for infant heart rates (CI=400-600ms) resulted in 2:1 block (diffuse: 48%, clustered: 52%). Left bundle branch block, a highly arrhythmogenic activation pattern, was also observed (diffuse: 4%, clustered: 2%).We demonstrated a possible link between cell-scale phenotypic variability and organ-scale arrhythmogenesis by showing that 20% V1762L mosaicism in the PS, regardless of spatial distribution, can result in 2:1 block.

Mutation Specific Drug Response and Cardiac Risk in Long QT Type 3

Long QT type 3 (LQT3) is caused by mutations that cause an increase in the cardiac sodium current during late phases of the cardiac action potential. For a number of LQT3 mutants this is caused by a failure to inactivate fully, and a consequent increase in late sodium current. For several mutations, shifts in voltage dependence of activation and inactivation cause increased sodium channel contribution to more depolarized voltages, an effect referred as increase in window current. The goal of this study was to perform a study in a large number of LQT3 associated mutations and compare the biophysical effect of the mutation to cardiac risk in LQT3 patients and response to treatment. We measured the function of eight common mutations associated with LQT3 with different mechanism underlying channel dysfunction. We compared the functional effects of the channel with the clinical course of these patients and observed that for the two mutations tested with increased sustained current but without increase in current availability (window current), D1784K and D1790G, the patients had significant lower risk of cardiac events, suggesting an increase in current availability may be associated with increased risk for these patients. In addition, we measured mutation specific effects of ranolazine for these mutants. For all mutants tested, ranolazine preferentially blocked late sodium currents. Ranolazine shifts steady-state availability of the inactivation was dependent on the specific mutant tested. Our results suggest that ranolazine may have a mutation dependent effect. Mutations dysfunction may affect drug binding and drug actions in the channel and may alter treatment effectiveness in patients.

GW24-e0582 The role and mechanism of late sodium current in the development of electromechanical alternans and arrhythmia in heart failure

ObjectivesHeart failure is associated with electromechanical alternans that often heralds the development of malignant arrhythmias and sudden death, but the exact mechanism is unclear. Our previous studies have shown that...

Sophocarpine Attenuates the Na+-dependent Ca2+ Overload Induced by Anemonia Sulcata Toxin—Increased Late Sodium Current in Rabbit Ventricular Myocytes

Many studies indicate that an increase in late sodium current (I(Na.L)) of cardiomyocytes causes intracellular Na overload and subsequently raises the reverse Na/Ca exchanger current (INCX), ultimately resulting in intracellular Ca overload. Therefore, using drugs to inhibit the increased INa.L under various pathological conditions can lower intracellular Ca overload. This study was intended to explore the effect of sophocarpine (SOP) on the increase in INa.L, INCX, calcium transient and contraction in rabbit ventricular myocytes induced by Anemonia sulcata toxin II (ATX II), an opener of sodium channel, with the application of whole-cell patch-clamp techniques, the video-based motion edge detection system, and the intracellular calcium concentration determination system. The results indicate that tetrodotoxin (TTX, 4 μM ) obviously decreased INa.L and INCX enlarged by ATX II (30 nM), and SOP (20, 40, and 80 μM) also inhibited both the parameters concentration dependently in rabbit ventricular myocytes. However, transient sodium current remained unaffected by the above-mentioned concentrations of ATX II, TTX, and SOP. In addition, SOP also reversed diastolic calcium concentration, calcium transient amplitude, and ventricular muscle contractility augmented by ATX II. Its effects were similar to those of TTX, a specific inhibitor of the sodium channel. In conclusion, SOP inhibits INa.L, INCX, diastolic Ca concentration, and contractility in rabbit ventricular myocytes, which suggests that relief of intracellular Ca overload through inhibiting INa.L is likely to become a new therapeutic mechanism of SOP against arrhythmia and myocyte damage associated with intracellular Ca overload.

Unique mixed phenotype and unexpected functional effect revealed by novel compound heterozygosity mutations involving SCN5A

Functional characterization of mutations involving the SCN5A-encoded cardiac sodium channel has established the pathogenic mechanisms for type 3 long QT syndrome and type 1 Brugada syndrome and has provided key insights into the physiological importance of essential structure-function domains. This study sought to present the clinical and biophysical phenotypes discerned from compound heterozygosity mutations in SCN5A on different alleles in a toddler diagnosed with QT prolongation and fever-induced ventricular arrhythmias. A 22-month-old boy presented emergently with fever and refractory ventricular tachycardia. Despite restoration of sinus rhythm, the infant sustained profound neurological injury and died. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open-reading frame/splice mutational analysis of the 12 known long QT syndrome susceptibility genes was performed. The infant had 2 SCN5A mutations: a maternally inherited N-terminal frame shift/deletion (R34fs/60) and a paternally inherited missense mutation, R1195H. The mutations were engineered by site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. As expected, the frame-shifted and prematurely truncated peptide, SCN5A-R34fs/60, showed no current. SCN5A-R1195H had normal peak and late current but abnormal voltage-dependent gating parameters. Surprisingly, co-expression of SCN5A-R34fs/60 with SCN5A-R1195H elicited a significant increase in late sodium current, whereas co-expression of SCN5A-WT with SCN5A-R34fs/60 did not. A severe clinical phenotype characterized by fever-induced monomorphic ventricular tachycardia and QT interval prolongation emerged in a toddler with compound heterozygosity involving SCN5A: R34fs/60, and R1195H. Unexpectedly, the 94-amino-acid fusion peptide derived from the R34fs/60 mutation accentuated the late sodium current of R1195H-containing Na(V)1.5 channels in vitro.

Ranolazine Shortens Repolarization in Patients with Sustained Inward Sodium Current Due to Type‐3 Long‐QT Syndrome

One form of the hereditary long-QT syndrome, LQT3-Delta KPQ, is associated with sustained inward sodium current during membrane depolarization. Ranolazine reduces late sodium channel current, and we hypothesized that ranolazine would have beneficial effects on electrical and mechanical cardiac function in LQT3 patients with the SCN5A-DeltaKPQ mutation. We assessed the effects of 8-hour intravenous ranolazine infusions (45 mg/h for 3 hours followed by 90 mg/h for 5 hours) on ventricular repolarization and myocardial relaxation in 5 LQT3 patients with the SCN5A-Delta KPQ mutation. Changes in electrocardiographic repolarization parameters from before to during ranolazine infusion were evaluated by time-matched, paired t-test analyses. Cardiac ultrasound recordings were obtained before ranolazine infusion and just before completion of the 8-hour ranolazine infusion. Ranolazine shortened QTc by 26 +/- 3 ms (P < 0.0001) in a concentration-dependent manner. At peak ranolazine infusion, there was a significant 13% shortening in left ventricular isovolumic relaxation time, a significant 25% increase in mitral E-wave velocity, and a meaningful 22% decrease in mitral E-wave deceleration time compared with the baseline. No adverse effects of ranolazine were observed in the study patients. Ranolazine at therapeutic concentrations shortened a prolonged QTc interval and improved diastolic relaxation in patients with the LQT3-Delta KPQ mutation, a genetic disorder that is known to cause an increase in late sodium current.

An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes

This study determined the role of a slowly inactivating component of sodium current (I(Na)), late I(Na), to induce delayed afterdepolarizations (DADs) and triggered activity. We hypothesized that an increase of late I(Na) may induce not only early afterdepolarizations (EADs), but also intracellular calcium overload and DADs. Guinea pig atrial myocytes were studied using the whole cell patch-clamp technique. Anemone toxin II (ATX-II) (5-10 nmol/l) was used to enhance late I(Na). Ranolazine (10 micromol/l) and TTX (2 micromol/l) were applied to block ATX-II-induced late I(Na). ATX-II prolonged action potential duration and induced EADs. In the continuous presence of ATX-II, following the appearance of EADs, both DADs and sustained triggered activity occurred. Triggered activity was abolished and DADs were reduced by either ranolazine or TTX. Consistent with induction of DADs, ATX-II induced the transient inward current (I(TI)). The amplitude of I(TI) was significantly reduced by ranolazine. ATX-II induced only EADs, but no DADs, in the presence of the sodium-calcium exchange inhibitor KB-R7943 or the sarcoplasmic reticulum calcium release channel inhibitor ryanodine, or when the calcium chelator EGTA or BAPTA was included in the pipette solution. In conclusion, an increase of late I(Na), in addition to inducing EADs, can cause cellular calcium overload and induce DADs and sustained triggered activity in atrial myocytes. The data reveal that an increase of late I(Na) is a novel mechanism for initiation of atrial arrhythmic activity.

Novel mechanism for sudden infant death syndrome: Persistent late sodium current secondary to mutations in caveolin-3

Sudden infant death syndrome (SIDS) is one of the leading causes of death during the first year of life. Long QT syndrome (LQTS)-associated mutations may be responsible for 5% to 10% of SIDS cases. We recently established CAV3-encoded caveolin-3 as a novel LQTS-associated gene with mutations producing a gain-of-function, LQT3-like molecular/cellular phenotype. The purpose of this study was to determine the prevalence and functional properties of CAV3 mutations in SIDS. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, postmortem genetic testing of CAV3 was performed on genomic DNA isolated from frozen necropsy tissue on a population-based cohort of unrelated cases of SIDS (N = 134, 57 females, average age = 2.7 months). CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel. Overall, three distinct CAV3 mutations (V14L, T78M, and L79R) were identified in three of 50 black infants (6-month-old male, 2-month-old female, and 8 month-old female), whereas no mutations were detected in 83 white infants (P <.05). CAV3 mutations were more likely in decedents 6 months or older (2/12) than in infants who died before 6 months (1/124, P = .02). Voltage clamp studies showed that all three CAV3 mutations caused a significant fivefold increase in late sodium current compared with controls. This study provides the first molecular and functional evidence implicating CAV3 as a pathogenic basis of SIDS. The LQT3-like phenotype of increased late sodium current supports an arrhythmogenic mechanism for some cases of SIDS.

Mutant Caveolin-3 Induces Persistent Late Sodium Current and Is Associated With Long-QT Syndrome

Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects approximately 1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a "final common pathway" hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A-encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa(v)1.5. We identified 4 novel mutations in CAV3-encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa(v)1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.