Cardiac Voltage-gated Sodium Channel Research Articles

H558R: a common polymorphism may modify the efficacy and toxicity of flecainide in maintaining sinus rhythm; a cohort study

Abstract Background Certain mutations in the SCN5A gene alter the function of the type V voltage-gated cardiac sodium channel and the therapeutic effect of flecainide.(1) One such mutation, H558R, represents a polymorphism with a high prevalence associated with the development of atrial fibrillation (AF).(2–5) Purpose We aim to determine its relationship with the efficacy and adverse effects of flecainide, as well as describe for the first time its prevalence in a cohort of European patients with AF. Methods We conducted a cohort study collecting data through indirect methods, with the study being blinded to all parties involved except the statistician. Patients prescribed flecainide (dose of 100 mg/12 h) and followed up in our health area (n=450,000 people) between 2017 and 2021 were identified. A primary composite endpoint consisting of initial inefficacy and early toxicity and a secondary combined endpoint of toxicity or inefficacy during follow-up were predefined. Results Out of 104 patients who met the criteria and agreed to participate, 59 (56.4%) did not have the mutation (H558R-/-), 38 (36.5%) were heterozygous (H558R+/-), and 7 (6.7%) were homozygous (H558R+/+). This high prevalence in European patients with AF is significantly higher than that previously reported in healthy individuals (20.4% in Caucasians, 9.2% in Asians).(6) When compared with patients without the mutation, H558R+/- patients had a lower incidence of the primary endpoint (p=0.02, picture 1), with an absolute risk reduction (ARR) of -21.5% (95% confidence interval (CI): -38.4% to -4.6%) primarily due to a lower rate of initial inefficacy (ARR -18.35%, 95% CI: -1.37% to -35.3%), and a trend towards lower early toxicity (ARR -7.36; 95% CI: -19.9 to +5.2%). This difference remained in the multivariate analysis (p=0.03, table 1). Moreover, the H558R+/- genotype was associated with lower toxicity or inefficacy over an average follow-up of 492 days (p=0,04, picture 1), with a hazard ratio of 0.53 (95% CI: 0.27 to 0.999). Conclusions The H558R+/- mutation shows a marked prevalence in a selected sample of patients with AF. Its presence is clinically relevant both in terms of early efficacy or toxicity and during follow-up. If these results are confirmed, it may signify a step towards personalized treatment for patients with AF, not only from the standpoint of efficacy but also safety.Table 1.Picture 1.

Defining Cardiomyocyte Repolarization Response to Pharmacotherapy in Long-QT Syndrome Type 3.

Long-QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient-specific, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes model of long-QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. HiPSCswere generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient-derived LQT3 (SCN5A+/-) and genetically engineered LQT3 (SCN5A+/-) hiPSC-derived cardiomyocytesshowed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage-gated sodium channel (NaV1.5) blocker, shortened repolarization in both patient-derived LQT3 and geneticallyengineered LQT3 hiPSC-derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose-dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect invitro, but the LQT3 clones were not more disproportionately affected compared with the control. We demonstrated that patient-derived and genetically engineered LQT3 hiPSC-derived cardiomyocytesfaithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an invitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient-specific approach in high-risk patients with LQT3.

Abstract Mo066: A Bifunctional Actuator Reverses Na V 1.5 Dysfunction Linked To Cardiac Arrhythmias

Cardiac voltage-gated sodium channels (Na V 1.5) are critical for the initiation of action potentials within the heart. Alterations in Na V 1.5 function significantly contribute to diverse inherited and acquired arrhythmias, such as Long QT syndrome (LQT), Brugada syndrome (BrS), mixed-syndrome, and heart failure. These pathologies are closely associated with two distinct modifications in Na V 1.5 function: (1) a gain-of-function phenotype, characterized by incomplete inactivation of Na V 1.5 leading to a sustained Na + current ( I NaL ), and (2) a loss-of-function phenotype resulting in diminished peak Na + current ( I NaP ). Despite the broad understanding of Na V 1.5 implication in cardiac pathophysiology, the development of effective therapeutic strategies to reverse these dysfunctions remains challenging. Here, we introduce a novel bifunctional actuator that inhibits I NaL and upregulates I NaP , as a common molecular strategy to reverse pathophysiological changes in Nav-linked cardiac arrhythmias. To do so, we use a genetically encoded approach by engineering a selective nanobody (nb82) targeting Na V 1.5, linked to (1) a peptide inhibitor targeting I NaL (FixR), and (2) the catalytic domain of a deubiquitinase (DUB) to enhance Na V 1.5 membrane expression. Changes in I NaP and I NaL were assessed using whole-cell and multichannel electrophysiology techniques in both wild-type and channelopathic mutant channels. Our results demonstrate that NbFixR-DUB effectively reduces I NaL while concurrently increasing I NaP for multiple channelopathic mutations. Overall, this approach holds considerable promise for rectifying both loss-of-function and gain-of-function phenotypes in Nav channelopathies.

Popeye domain containing proteins modulate the voltage-gated cardiac sodium channel Nav1.5

Popeye domain containing (POPDC) proteins are predominantly expressed in the heart and skeletal muscle, modulating the K2P potassium channel TREK-1 in a cAMP-dependent manner. POPDC1 and POPDC2 variants cause cardiac conduction disorders with or without muscular dystrophy. Searching for POPDC2-modulated ion channels using a functional co-expression screen in Xenopus oocytes, we found POPDC proteins to modulate the cardiac sodium channel Nav1.5. POPDC proteins downregulate Nav1.5 currents in a cAMP-dependent manner by reducing the surface expression of the channel. POPDC2 and Nav1.5 are both expressed in different regions of the murine heart and consistently POPDC2 co-immunoprecipitates with Nav1.5 from native cardiac tissue. Strikingly, the knock-down of popdc2 in embryonic zebrafish caused an increased upstroke velocity and overshoot of cardiac action potentials. The POPDC modulation of Nav1.5 provides a new mechanism to regulate cardiac sodium channel densities under sympathetic stimulation, which is likely to have a functional impact on cardiac physiology and inherited arrhythmias.

Biophysical mechanisms of myocardium sodium channelopathies.

Genetic variants of gene SCN5A encoding the alpha-subunit of cardiac voltage-gated sodium channel Nav1.5 are associated with various diseases, including long QT syndrome (LQT3), Brugada syndrome (BrS1), and progressive cardiac conduction disease (PCCD). In the last decades, the great progress in understanding molecular and biophysical mechanisms of these diseases has been achieved. The LQT3 syndrome is associated with gain-of-function of sodium channels Nav1.5 due to impaired inactivation, enhanced activation, accelerated recovery from inactivation or the late current appearance. In contrast, BrS1 and PCCD are associated with the Nav1.5 loss-of-function, which in electrophysiological experiments can be manifested as reduced current density, enhanced fast or slow inactivation, impaired activation, or decelerated recovery from inactivation. Genetic variants associated with congenital arrhythmias can also disturb interactions of the Nav1.5 channel with different proteins or drugs and cause unexpected reactions to drug administration. Furthermore, mutations can affect post-translational modifications of the channels and their sensitivity to pH and temperature. Here we briefly review the current knowledge on biophysical mechanisms of LQT3, BrS1 and PCCD. We focus on limitations of studies that use heterologous expression systems and induced pluripotent stem cells (iPSC) derived cardiac myocytes and summarize our understanding of genotype-phenotype relations of SCN5A mutations.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through invitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

Abstract 15609: ParSE-seq: A Calibrated Multiplexed Assay for Clinical Interpretation of Putative Splice-Altering Variants

Background: Up to 10% of disease-associated variants are predicted to affect RNA-splicing. Interpreting the clinical significance of putative splice-altering variants outside the 2-base pair canonical splice sites remains difficult without functional studies. Goal: To develop a calibrated, high-throughput assay to adjudicate variant effects on RNA splicing. Methods: We established Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We used the tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select curated benign and pathogenic controls in SCN5A , which encodes the major voltage-gated cardiac sodium channel. We transfected 271 barcoded minigene constructs into HEK cells or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and calibrated to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data correlated to SpliceAI predictions. We studied selected variants by automated patch clamp (APC) in HEK cells and by assessing splicing and channel function directly in CRISPR-edited iPSC-CMs. Results: ParSE-seq revealed the splicing effect of 245 SCN5A variants in iPSC-CMs. The assay had concordant scores for 43/45 benign and pathogenic control variants, achieving both PS3- and BS3-strong levels of evidence after calibration. 41/114 exonic variants and 30/66 intronic variants with determinate scores disrupted splicing. 9 of 42 Variants of Uncertain Significance were reclassified, and 30 of 34 variants with conflicting interpretations were reinterpreted using the functional data. Predictions from SpliceAI performed well on experimental data (AUC = 0.96). We identified 3 clinically relevant splice-altering missense variants that had normal function when tested with a cDNA-based APC assay. A splice-altering intronic variant, c.1891-5C>G also disrupted splicing and sodium channel electrophysiology when introduced into iPSC-CMs by CRISPR-editing. Conclusions: ParSE-seq is an efficient, calibrated, high-throughput assay to facilitate splice-altering variant interpretation.

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous NaV β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ(myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/β NaV complexes.

Multifocal ectopic purkinje-related premature contractions and related cardiomyopathy.

In the past 20 years, genetic variants in SCN5A encoding the cardiac voltage-gated sodium channel Nav1.5 have been linked to a range of inherited cardiac arrhythmias: variants resulting in loss-of-function of Nav1.5 have been linked to sick sinus syndrome, atrial stand still, atrial fibrillation (AF) impaired pulse generation, progressive and non-progressive conduction defects, the Brugada Syndrome (BrS), and sudden cardiac death. SCN5A variants causing increased sodium current during the plateau phase of the cardiac action potential is associated with Long QT Syndrome type 3 (LQTS3), Torsade de Pointes ventricular tachycardia and SCD. Recently, gain-of-function variants have been linked to complex electrical phenotypes, such as the Multifocal Ectopic Purkinje-related Premature Contractions (MEPPC) syndrome. MEPPC is a rare condition characterized by a high burden of premature atrial contractions (PACs) and/or premature ventricular contractions (PVCs) often accompanied by dilated cardiomyopathy (DCM). MEPPC is inherited in an autosomal dominant fashion with an almost complete penetrance. The onset is often in childhood. The link between SCN5A variants, MEPPC and DCM is currently not well understood, but amino acid substitutions resulting in gain-of-function of Nav1.5 or introduction of gating pore currents potentially play an important role. DCM patients with a MEPPC phenotype respond relatively poorly to standard heart failure medical therapy and catheter ablation as the PVCs originate from all parts of the fascicular Purkinje fiber network. Class 1c sodium channel inhibitors, notably flecainide, have a remarkable positive effect on the ectopic burden and the associated cardiomyopathy. This highlights the importance of genetic screening of DCM patients to identify patients with SCN5A variants associated with MEPPC. Here we review the MEPPC phenotype, MEPPC-SCN5A associated variants, and pathogenesis as well as treatment options.

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Nav 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Nav 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. KEY POINTS: Nav 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Nav 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

HiPSC-derived cardiomyocyte to model Brugada syndrome: both asymptomatic and symptomatic mutation carriers reveal increased arrhythmogenicity

Brugada syndrome is an inherited cardiac arrhythmia disorder that is mainly associated with mutations of the cardiac voltage-gated sodium channel alpha subunit 5 (SCN5A) gene. The clinical symptoms include ventricular fibrillation and an increased risk of sudden cardiac death. Human-induced pluripotent stem cell (hiPSC) lines were derived from symptomatic and asymptomatic individuals carrying the R1913C mutation in the SCN5A gene. The present work aimed to observe the phenotype-specific differences in hiPSC-derived cardiomyocytes (CMs) obtained from symptomatic and asymptomatic mutation carriers. In this study, CM electrophysiological properties, beating abilities and calcium parameters were measured. Mutant CMs exhibited higher average sodium current densities than healthy CMs, but the differences were not statistically significant. Action potential durations were significantly shorter in CMs from the symptomatic individual, and a spike-and-dome morphology of action potential was exclusively observed in CMs from the symptomatic individual. More arrhythmias occurred in mutant CMs at single cell and cell aggregate levels compared with those observed in wild-type CMs. Moreover, there were no major differences in ionic currents or intracellular calcium dynamics between the CMs of asymptomatic and symptomatic individuals after the administration of adrenaline and flecainide.In conclusion, mutant CMs were more prone to arrhythmia than healthy CMs but did not explain why only one of the mutation carriers was symptomatic.

Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5

Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.

Inflammation provokes gating changes in the neuronal voltage-gated sodium channel, Nav1.6.

We recently showed that inflammation causes arrhythmogenic properties in cardiac voltage gated sodium channels (Nav). Here, we hypothesize that inflammation, via Nav, is a common trigger for other pathologies including neurological disorders. Based on our previous work, we further postulated that cannabidiol (CBD), the primary non-psychoactive constituent of Cannabis sativa, may protect against inflammation-induced neuropathy through its direct effects on neurological Nav1.6. To test these ideas, we used whole-cell patch clamp of human embryonic kidney (HEK 293) cells transiently transfected with SCN8A cDNA encoding neurological Nav1.6 α-subunit and incubated the cells under control conditions or in a cocktail of inflammatory mediators for 24 hours. Incubation in inflammatory mediators right-shifted the voltage dependence of both activation and steady-state fast inactivation, and increased late sodium current. Perfusion of CBD (IC50; 5 µM) during recording rescued all the gating changes in Nav1.6 provoked by incubation with inflammatory mediators. These findings suggest that inflammation, by affecting Nav, is a potential pathogenic trigger for neurological pathologies. Moreover, CBD, through its direct effects on Nav, could potentially mitigate the dysfunction induced by inflammation.

ARumenamides: A novel class of potential anti-arrhythmic compounds.

Loss-of-function mutations (LoF) in cardiac voltage-gated sodium channels (Nav1.5) are arrhythmogenic and cause, for example, Brugada syndrome. However, few attempts have been made to target LoF. A major impediment for the advancement of LoF therapeutics is the tendency for compounds to occlude the Nav1.5 central pore. This caveat in anti-LoF drug development may be mitigated with compounds that stabilize the activated state in Nav1.5 by binding to other sites besides the pore. We hypothesized that a novel family of compounds, called ARumenamides (ARs), with high affinity for the fenestrations would potentially reduce pore block. Six ARs,AR-051, AR-189, AR-674, AR-802, AR-807, and AR-811, were docked against a homology model of Nav1.5 built on NavAb and rNav1.5. Based on the virtual docking results, these ARs have a high affinity for Domain III-IV and Domain VI-I fenestrations. Upon functional characterization, a trend was observed in the effects of the six ARs on INa. An inverse correlation was established between the aromaticity of the AR's functional moieties and compound block. Due to its aromaticity, AR-811 blocked INa the least compared with other aromatic ARs, which also decelerated fast inactivation onset. AR-674, with its aliphatic functional group, significantly suppresses INa and enhances use-dependence in Nav1.5. AR-802 and AR-811 decelerated fast inactivation kinetics in Brugada/Long-QT syndrome mutant, E1784K, without affecting peak or late INa. The ARs we discovered seem to target LoF in Nav1.5 by preferentially occluding the fenestrations. We predict that bulkier and aromatic side groups have higher affinity for the hydrophobic milieu of the fenestrations, remaining there rather than in the central pore. Future refinements of AR compound structures and additional validation by molecular dynamic simulations and screening against more Brugada variants will further support their potential benefits in treating certain LoF cardiac arrhythmias.

A de novo arrhythmogenic Nav1.5 variant, E171Q, causes multiple biophysical defects.

An infant presenting antenatally with cardiac conduction disease and junctional tachycardia reminiscent of the multi-focal ectopic Purkinje-related premature contractions (MEPPC) syndrome was found to harbour the de novo E171Q variant of the cardiac voltage-gated sodium channel, Nav1.5. Functional analysis was performed using whole cell patch clamp and current clamp to compare WT Nav1.5 with E171Q expressed in HEK293 cells and iPSC-CMs. We found E171Q significantly right-shifted the voltage-dependence of both activation and steady-state fast inactivation, and increased the amplitude of late sodium current. These changes were reversed upon perfusion with 100 μM ranolazine. Incorporating the shifts in voltage-dependence and late current into the Ohara-Rudy cardiac action potential model produced a delay in action potential repolarization. Further investigation revealed a significant outward gating pore (omega) current in E171Q that was mitigated upon perfusion of 3 μM flecainide or with K+-free internal solution. Current clamp experiments in iPSC-CMs revealed action potential variability with delayed afterdepolarizations in myocytes expressing E171Q. The increase in gating pore current is consistent with disrupting the interaction between E171 and a corresponding positive charge in the S4 voltage sensor. This disruption could result in the completion of an ion-permeable pore, consistent with that previously described for Nav1.5 R222Q - a variant also associated with MEPPC - and Nav1.4 R222W.

The Antidepressant Paroxetine Reduces the Cardiac Sodium Current.

A considerable amount of literature has been published on antidepressants and cardiac ion channel dysfunction. The antidepressant paroxetine has been associated with Brugada syndrome and long QT syndrome, albeit on the basis of conflicting findings. The cardiac voltage-gated sodium channel (NaV1.5) is related to both of these syndromes, suggesting that paroxetine may have an effect on this channel. In the present study, we therefore carried out patch clamp experiments to examine the effect of paroxetine on human NaV1.5 channels stably expressed in human embryonic kidney 293 (HEK-293) cells as well as on action potentials of isolated rabbit left ventricular cardiomyocytes. Additionally, computer simulations were conducted to test the functional effects of the experimentally observed paroxetine-induced changes in the NaV1.5 current. We found that paroxetine led to a decrease in peak NaV1.5 current in a concentration-dependent manner with an IC50 of 6.8 ± 1.1 µM. In addition, paroxetine caused a significant hyperpolarizing shift in the steady-state inactivation of the NaV1.5 current as well as a significant increase in its rate of inactivation. Paroxetine (3 µM) affected the action potential of the left ventricular cardiomyocytes, significantly decreasing its maximum upstroke velocity and amplitude, both of which are mainly regulated by the NaV1.5 current. Our computer simulations demonstrated that paroxetine substantially reduces the fast sodium current of human left ventricular cardiomyocytes, thereby slowing conduction and reducing excitability in strands of cells, in particular if conduction and excitability are already inhibited by a loss-of-function mutation in the NaV1.5 encoding SCN5A gene. In conclusion, paroxetine acts as an inhibitor of NaV1.5 channels, which may enhance the effects of loss-of-function mutations in SCN5A.

ARumenamides: A novel class of potential antiarrhythmic compounds.

Background: Most therapeutics targeting cardiac voltage-gated sodium channels (Nav1.5) attenuate the sodium current (INa) conducted through the pore of the protein. Whereas these drugs may be beneficial for disease states associated with gain-of-function (GoF) in Nav1.5, few attempts have been made to therapeutically treat loss-of-function (LoF) conditions. The primary impediment to designing efficacious therapies for LoF is a tendency for drugs to occlude the Nav1.5 central pore. We hypothesized that molecular candidates with a high affinity for the fenestrations would potentially reduce pore block. Methods and Results: Virtual docking was performed on 21 compounds, selected based on their affinity for the fenestrations in Nav1.5, which included a class of sulfonamides and carboxamides we identify as ARumenamide (AR). Six ARs, AR-051, AR-189, AR-674, AR-802, AR-807 and AR-811, were further docked against Nav1.5 built on NavAb and rNav1.5. Based on the virtual docking results, these particular ARs have a high affinity for Domain III-IV and Domain VI-I fenestrations. Upon functional characterization, a trend was observed in the effects of the six ARs on INa. An inverse correlation was established between the aromaticity of the AR’s functional moieties and compound block. Due to its aromaticity, AR-811 blocked INa the least compared with other aromatic ARs, which also decelerated fast inactivation onset. AR-674, with its aliphatic functional group, significantly suppresses INa and enhances use-dependence in Nav1.5. AR-802 and AR-811, in particular, decelerated fast inactivation kinetics in the most common Brugada Syndrome Type 1 and Long-QT Syndrome Type 3 mutant, E1784K, without affecting peak or persistent INa. Conclusion: Our hypothesis that LoF in Nav1.5 may be therapeutically treated was supported by the discovery of ARs, which appear to preferentially block the fenestrations. ARs with aromatic functional groups as opposed to aliphatic groups efficaciously maintained Nav1.5 availability. We predict that these bulkier side groups may have a higher affinity for the hydrophobic milieu of the fenestrations, remaining there rather than in the central pore of the channel. Future refinements of AR compound structures and additional validation by molecular dynamic simulations and screening against more Brugada variants will further support their potential benefits in treating certain LoF cardiac arrhythmias.

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na+ channels.

Heme (Fe2+-protoporphyrin IX) is a well-known protein prosthetic group; however, heme and hemin (Fe3+-protoporphyrin IX) are also increasingly viewed as signaling molecules. Among the signaling targets are numerous ion channels, with intracellular-facing heme-binding sites modulated by heme and hemin in the sub-µM range. Much less is known about extracellular hemin, which is expected to be more abundant, in particular after hemolytic insults. Here we show that the human cardiac voltage-gated sodium channel hNaV1.5 is potently inhibited by extracellular hemin (IC 50≈80nM), while heme, dimethylhemin, and protoporphyrin IX are ineffective. Hemin is selective for hNaV1.5 channels: hNaV1.2, hNaV1.4, hNaV1.7, and hNaV1.8 are insensitive to 1µM hemin. Using domain chimeras of hNaV1.5 and rat rNaV1.2, domain II was identified as the critical determinant. Mutation N803G in the domain II S3/S4 linker largely diminished the impact of hemin on the cardiac channel. This profile is reminiscent ofthe interaction of some peptide voltage-sensor toxins with NaV channels. In line with a mechanism of select gating modifiers, the impact of hemin on NaV1.5 channels is reversely use dependent, compatible with an interaction of hemin and the voltage sensor of domain II. Extracellular hemin thus has potential to modulate the cardiac function.