Cardiac Voltage-gated Sodium Channel Research Articles

MIR448 antagomir reduces arrhythmic risk after myocardial infarction by upregulating the cardiac sodium channel.

Cardiac ischemia is associated with arrhythmias; however, effective therapies are currently limited. The cardiac voltage-gated sodium channel α subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a miR, miR-448. miR-448 expression is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3′-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF-1α and NF-κB were major transcriptional regulators for MIR448. Moreover, hypoxia relieved MIR448 transcriptional suppression by RE1 silencing transcription factor. Therefore, miR-448 inhibition reduced arrhythmic risk after myocardial infarction. Here, we show that ischemia drove miR-448 expression, reduced Nav1.5 current, and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy.

Propranolol Attenuates Late Sodium Current in a Long QT Syndrome Type 3-Human Induced Pluripotent Stem Cell Model.

BackgroundLong QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the SCN5A gene, which encodes the α subunit of the cardiac voltage-gated sodium channel. LQT3 patients present bradycardia and lethal arrhythmias during rest or sleep. Further, the efficacy of β-blockers, the drug used for their treatment, is uncertain. Recently, a large multicenter LQT3 cohort study demonstrated that β-blocker therapy reduced the risk of life-threatening cardiac events in female patients; however, the detailed mechanism of action remains unclear.ObjectivesThis study aimed to establish LQT3-human induced pluripotent stem cells (hiPSCs) and to investigate the effect of propranolol in this model.MethodAn hiPSCs cell line was established from peripheral blood mononuclear cells of a boy with LQT3 carrying the SCN5A-N1774D mutation. He had suffered from repetitive torsades de pointes (TdPs) with QT prolongation since birth (QTc 680 ms), which were effectively treated with propranolol, as it suppressed lethal arrhythmias. Furthermore, hiPSCs were differentiated into cardiomyocytes (CMs), on which electrophysiological functional assays were performed using the patch-clamp method.ResultsN1774D-hiPSC-CMs exhibited significantly prolonged action potential durations (APDs) in comparison to those of the control cells (N1774D: 440 ± 37 ms vs. control: 272 ± 22 ms; at 1 Hz pacing; p < 0.01). Furthermore, N1774D-hiPSC-CMs presented gain-of-function features: a hyperpolarized shift of steady-state activation and increased late sodium current compared to those of the control cells. 5 μM propranolol shortened APDs and inhibited late sodium current in N1774D-hiPSC-CMs, but did not significantly affect in the control cells. In addition, even in the presence of intrapipette guanosine diphosphate βs (GDPβs), an inhibitor of G proteins, propranolol reduced late sodium current in N1774D cells. Therefore, these results suggested a unique inhibitory effect of propranolol on late sodium current unrelated to β-adrenergic receptor block in N1774D-hiPSC-CMs.ConclusionWe successfully recapitulated the clinical phenotype of LQT3 using patient-derived hiPSC-CMs and determined that the mechanism, by which propranolol inhibited the late sodium current, was independent of β-adrenergic receptor signaling pathway.

E1784K, the most common Brugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channel inactivation through two separate mechanisms.

Inheritable and de novo variants in the cardiac voltage-gated sodium channel, Nav1.5, are responsible for both long-QT syndrome type 3 (LQT3) and Brugada syndrome type 1 (BrS1). Interestingly, a subset of Nav1.5 variants can cause both LQT3 and BrS1. Many of these variants are found in channel structures that form the channel fast inactivation machinery, altering the rate, voltage dependence, and completeness of the fast inactivation process. We used a series of mutants at position 1784 to show that the most common inheritable Nav1.5 variant, E1784K, alters fast inactivation through two separable mechanisms: (1) a charge-dependent interaction that increases the noninactivating current characteristic of E1784K; and (2) a hyperpolarized voltage dependence and accelerated rate of fast inactivation that decreases the peak sodium current. Using a homology model built on the NavPaS structure, we find that the charge-dependent interaction is between E1784 and K1493 in the DIII-DIV linker of the channel, five residues downstream of the putative inactivation gate. This interaction can be disrupted by a positive charge at position 1784 and rescued with the K1493E/E1784K double mutant that abolishes the noninactivating current. However, the double mutant does not restore either the voltage dependence or rates of fast inactivation. Conversely, a mutant at the bottom of DIVS4, K1641D, causes a hyperpolarizing shift in the voltage dependence of fast inactivation and accelerates the rate of fast inactivation without causing an increase in noninactivating current. These findings provide novel mechanistic insights into how the most common inheritable arrhythmogenic mixed syndrome variant, E1784K, simultaneously decreases transient sodium currents and increases noninactivating currents, leading to both BrS1 and LQT3.

Single-Molecule Localization of the Cardiac Voltage-Gated Sodium Channel Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains.

Mutations in the gene encoding the cardiac voltage-gated sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy and computational modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, the lateral membrane groove, and T-tubules in cardiomyocytes from wild-type (N=3), dystrophin-deficient (mdx; N=3) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV; N=3). We moreover assessed T-tubules sodium current by recording whole-cell sodium currents in control (N=5) and detubulated (N=5) wild-type cardiomyocytes. We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a nonuniform redistribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A T-tubules sodium current could, however, not be demonstrated. Nav1.5 mutations may site-specifically affect Nav1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.

Dataset of electrophysiological patch-clamp recordings of the effect of the compounds deltamethrin, ATx-II and β4-peptide on human cardiac Nav1.5 sodium channel gating properties.

This article describes the effect of the pyrethroid insecticide deltamethrin on the cardiac voltage-gated sodium channel Nav1.5. Two concentrations of deltamethrin were used and the effects were compared with those of the sea anemone toxin ATx-II and β4-peptide, which is the C-terminus of the Nav channel β-subunit. Activation, fast inactivation, deactivation, persistent currents and resurgent currents of Nav1.5 channels were assessed in the presence of these compounds. The data display not only the effect of separately applied compounds on Nav1.5 channels but also investigates how combinations of these substances affect Nav1.5 channel gating properties.The dataset presented in this article is related to the research article “Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5″ (Sarah Thull, Cristian Neacsu, Andrias O. O'Reilly, Stefanie Bothe, Ralf Hausmann, Tobias Huth, Jannis Meents, Angelika Lampert, doi: 10.1016/j.taap.2020.11501), that investigates the effect of the pyrethroid insecticide deltamethrin on Nav channel gating properties and explains the mechanism underlying hooked, resurgent-like tail currents induced by deltamethrin in Nav1.5 channels.

Protective Effect of Cannabidiol Against Oxidative Stress and Cytotoxicity Evoked by High Glucose in Cardiac Voltage-Gated Sodium Channels

Cardiovascular complications are the major cause of mortality in diabetic patients. However, the molecular mechanisms underlying diabetes-associated arrhythmias are unclear. We hypothesized that high glucose could adversely affect Nav1.5, the major cardiac sodium channel isoform, at least partially via oxidative stress. We further hypothesized that cannabidiol (CBD), one of the main constituents of Cannabis sativa, could guard against diabetes induced cardiomyopathy. We postulated that CBD, through its effects on Nav1.5, could protect against high glucose elicited oxidative stress and cytotoxicity. To test these ideas, we used Chinese hamster ovarian (CHO) cells transiently transfected with cDNA encoding Nav1.5 α-subunit and incubated the cells in control (10 mM) and high glucose (50 or 100 mM) conditions for 24 hours. High glucose evoked cell death associated with elevation in reactive oxygen species (ROS), right shifted the voltage dependence of conductance and steady state fast inactivation, and increased persistent current leading to prolongation of action potential (hyperexcitability) which could result in long QT-3 arrhythmia. CBD mitigated the deleterious effects provoked by high glucose. Perfusion with lidocaine (a sodium channels inhibitor with anti-oxidant effects), or co-incubation of Tempol (an anti-oxidant) elicited protection, comparable to CBD, against the deleterious effects of high glucose. These findings suggest that, through its favorable anti-oxidant and sodium channel inhibitory effects, CBD may protect against high-glucose induced arrhythmia and cytotoxicity.

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

ABSTRACT The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 NTD: Y87C–here newly identified–, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80–105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.

β1 and β3 subunits amplify mechanosensitivity of the cardiac voltage-gated sodium channel Nav1.5

In cardiomyocytes, electrical activity is coupled to cellular contraction, thus exposing all proteins expressed in the sarcolemma to mechanical stress. The voltage-gated sodium channel Nav1.5 is the main contributor to the rising phase of the action potential in the heart. There is growing evidence that gating and kinetics of Nav1.5 are modulated by mechanical forces and pathogenic variants that affect mechanosensitivity have been linked to arrhythmias. Recently, the sodium channel β1 subunit has been described to stabilise gating against mechanical stress of Nav1.7 expressed in neurons. Here, we tested the effect of β1 and β3 subunits on mechanosensitivity of the cardiac Nav1.5. β1 amplifies stress-induced shifts of V1/2 of steady-state fast inactivation to hyperpolarised potentials (ΔV1/2: 6.2 mV without and 10.7 mV with β1 co-expression). β3, on the other hand, almost doubles stress-induced speeding of time to sodium current transient peak (Δtime to peak at - 30 mV: 0.19 ms without and 0.37 ms with β3 co-expression). Our findings may indicate that in cardiomyocytes, the interdependence of electrical activity and contraction is used as a means of fine tuning cardiac sodium channel function, allowing quicker but more strongly inactivating sodium currents under conditions of increased mechanical stress. This regulation may help to shorten action potential duration during tachycardia, to prevent re-entry phenomena and thus arrhythmias.

Abstract 893: MicroRNA-448 Regulates the Cardiac Sodium Channel During Ischemia

Background: The cardiac voltage-gated sodium channel ( SCN5A ; encoding Na v 1.5) plays a key role in cardiac conduction. The sodium channel is downregulated in cardiomyopathy, contributing to arrhythmic risk. In part, this downregulation is because of abnormal mRNA splicing and reduced mRNA stability. The reason for decreased mRNA stability is unclear, however. Objective: Here, we examined whether microRNA-448 (miR-448)can contribute to SCN5A mRNA instability. Methods: Datasets from the Gene Expression Omnibus database were utilized to screen miR-448 in human and mouse heart tissues. The relationship between SCN5A and miR-448 was predicted by three independent prediction tools. The expressions of SCN5A and miR-448 were determined by quantitative real time polymerase chain reaction. Protein levels of Na v 1.5 were determined by Western blot. The effect of miR-448 mimic on sodium current was determined in induced pluripotent stem cells-derived cardiomyocytes. Mouse myocardial infarction was created by left anterior descending coronary artery ligation. Results: The expression of miR-448 is increased in stressed heart after myocardial infarction. The binding sequence for miR-448 is well-conserved in 3’-UTR of SCN5A and miR-448 directly bound to a consensus cis element, suppressing SCN5A expression and sodium currents. miR-448 expression wass increased by hypoxia. Activated by hypoxia, HIF1α and NF-κB activated were major transcriptional regulator for MIR448 . Conclusion: These results indicated that miR-448 contributed to post-transcriptional modification of SCN5A . miR-448 is likely one cause of sodium channel downregulation during ischemia and may represent a target for antagomir therapy to reduce arrhythmic risk associated with cardiomyopathy.. Keywords: SCN5A, miR-448, Cardiomyocyte, Hypoxia, NF-κB, HIF1α

Characterization of a novel SCN5A genetic variant A1294G associated with mixed clinical phenotype

Mutations in gene SCN5A, which encodes cardiac voltage-gated sodium channel Nav1.5, are associated with multiple clinical phenotypes. Here we describe a novel A1294G genetic variant detected in a male patient with combined clinical phenotype including atrioventricular II block, Brugada-like ECG, septal fibrosis, right ventricular dilatation and decreased left ventricular contractility. Residue A1294 is located in the IIIS3–S4 extracellular loop, in proximity to several residues whose mutations are associated with sodium channelopathies. The wild-type channel Nav1.5 and mutant Nav1.5-A1294G were expressed in the CHO–K1 and HEK293T cells and whole-cell sodium currents were recorded using the patch-clamp method. The A1294G channels demonstrated a negative shift of steady-state inactivation, accelerated fast and slow inactivation and decelerated recovery from intermediate inactivation. Our study reveals biophysical mechanism of the Nav1.5-A1294G dysfunction, which may underlie the combined phenotypic manifestation observed in the patient.

Mutations in NaV1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel.

Mutations in the SCN5A gene, encoding the cardiac voltage-gated sodium channel NaV1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca2+-dependent mechanisms and the involvement of β-subunit (NaVβ) in NaV1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the SCN5A gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of NaV1.5, both are putative CaM-binding domains. These mutants were functionally studied in human embryonic kidney (HEK) cells and HL-1 cells using whole-cell patch clamp technique. Calmodulin (CaM) interaction and cell-surface expression of heterologously expressed NaV1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered NaV1.5 non-conductive. NaV1.5K1493del altered the gating properties of co-expressed functional NaV1.5, in a Ca2+ and NaVβ1-dependent manner. NaV1.5A1924T impaired NaVβ1-dependent gating regulation. Ca2+-dependent CaM-interaction with NaV1.5 was blunted in NaV1.5K1493del. Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca2+-dependent gating regulation of NaV1.5 channels. Our results highlight the role of NaV1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca2+-sensing machinery of NaV1.5 involves NaVβ1.

Identification of rare variants in cardiac sodium channel β4-subunit gene SCN4B associated with ventricular tachycardia.

Ventricular tachycardia (VT) causes sudden cardiac death, however, the majority of risk genes for VT remain unknown. SCN4B encodes a β-subunit, Navβ4, for the voltage-gated cardiac sodium channel complex involved in generation and conduction of the cardiac action potential. We hypothesized that genomic variants in SCN4B increase the risk of VT. We used high-resolution melt analysis followed by Sanger sequencing to screen 199 VT patients to identify nonsynonymous variants in SCN4B. Two nonsynonymous heterozygous variants in SCN4B were identified in VT patients, including p.Gly8Ser in four VT patients and p.Ala145Ser in one VT patient. Case-control association studies were used to assess the association between variant p.Gly8Ser and VT in two independent populations for VT (299 VT cases vs. 981 controls in population 1 and 270 VT patients vs. 639 controls in population 2). Significant association was identified between p.Gly8Ser and VT in population 1 (P = 1.21 × 10-4, odds ratio or OR = 11.04), and the finding was confirmed in population 2 (P = 0.03, OR = 3.62). The association remained highly significant in the combined population (P = 3.09 × 10-5, OR = 6.17). Significant association was also identified between p.Gly8Ser and idiopathic VT (P = 1.89 × 10-5, OR = 7.27). Functional analysis with Western blotting showed that both p.Gly8Ser and p.Ala145Ser variants significantly reduced the expression level of Navβ4. Based on 2015 ACMG Standards and Guidelines, p.Gly8Ser and p.Ala145Ser can be classified as the pathogenic and likely pathogenic variant, respectively. Our data suggest that SCN4B is a susceptibility gene for common VT and idiopathic VT and link rare SCN4B variants with large effects (OR = 6.17-7.27) to common VT.

Significant association of rare variant p.Gly8Ser in cardiac sodium channel β4-subunit SCN4B with atrial fibrillation.

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Nav 1.5 (encoded by the SCN5A gene) and one or more auxiliary β-subunits, including Nav β1 to Nav β4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p=4.16×10-4 , odds ratio [OR]=3.14) between p.Gly8Ser and common AF as well as lone AF (p=0.018, OR=2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.

AMPK-mediated degradation of Nav1.5 through autophagy.

The voltage-gated cardiac sodium channel, Nav1.5, is the key component that controls cardiac excitative electrical impulse and propagation. However, the dynamic alterations of Nav1.5 during cardiac ischemia and reperfusion (I/R) are seldom reported. We found that the protein levels of rat cardiac Nav1.5 were significantly decreased in response to cardiac I/R injury. By simulating I/R injury in cells through activating AMPK by glucose deprivation, AMPK activator treatment, or hypoxia and reoxygenation (H/R), we found that Nav1.5 was down-regulated by AMPK-mediated autophagic degradation. Furthermore, AMPK was found to phosphorylate Nav1.5 at threonine (T) 101, which then regulates the interaction between Nav1.5 and the autophagic adaptor protein, microtubule-associated protein 1 light chain 3 (LC3), by exposing the LC3-interacting region adjacent to T101 in Nav1.5. This study highlights an instrumental role of AMPK in mediating the autophagic degradation of Nav1.5 during cardiac I/R injury.-Liu, X., Chen, Z., Han, Z., Liu, Y., Wu, X., Peng, Y., Di, W., Lan, R., Sun, B., Xu, B., Xu, W. AMPK-mediated degradation of Nav1.5 through autophagy.

Биофизические механизмы натриевых каналопатий в миокарде: cиндром удлиненного интервала QT и синдром Бругада

Mutations in gene SCN5A, which encodes the α-subunit of cardiac voltage-gated sodium channel Nav1.5, are often associated with long QT syndrome type 3 (LQT3) and Brugada syndrome type 1 (BrS1). Recently large progress was achieved in an understanding of biophysical mechanisms of these arrhythmias. The LQT3 syndrome is associated with the gain-of-function due to accelerated activation, decelerated inactivation, appearance of sustained current, and accelerated recovery from inactivation. In contrast, The BrS1 syndrome is associated with loss-of-function due to defective channel trafficking, impaired activation; enhanced fast and slow inactivation, and decelerated recovery from inactivation. Mutations associated with inherited arrhythmias, can also disturb interactions of Nav1.5 with different proteins and/or ligands and cause abnormal reactions in response to pharmacological agents. Furthermore, mutations can affect post-translational modifications and sensitivity to pH and temperature. Here we review current knowledge on biophysical mechanisms of Brugada and long QT syndromes. We focus on limitations of studies that use heterologous expression systems and causes hampering our understanding of genotype-phenotype relations of SCN5A mutations.

Phosphorylation of cardiac voltage-gated sodium channel: Potential players with multiple dimensions.

Cardiomyocytes are highly coordinated cells with multiple proteins organized in micro domains. Minor changes or interference in subcellular proteins can cause major disturbances in physiology. The cardiac sodium channel (NaV1.5) is an important determinant of correct electrical activity in cardiomyocytes which are localized at intercalated discs, T‐tubules and lateral membranes in the form of a macromolecular complex with multiple interacting protein partners. The channel is tightly regulated by post‐translational modifications for smooth conduction and propagation of action potentials. Among regulatory mechanisms, phosphorylation is an enzymatic and reversible process which modulates NaV1.5 channel function by attaching phosphate groups to serine, threonine or tyrosine residues. Phosphorylation of NaV1.5 is implicated in both normal physiological and pathological processes and is carried out by multiple kinases. In this review, we discuss and summarize recent literature about the (a) structure of NaV1.5 channel, (b) formation and subcellular localization of NaV1.5 channel macromolecular complex, (c) post‐translational phosphorylation and regulation of NaV1.5 channel, and (d) how these phosphorylation events of NaV1.5 channel alter the biophysical properties and affect the channel during disease status. We expect, by reviewing these aspects will greatly improve our understanding of NaV1.5 channel biology, physiology and pathology, which will also provide an insight into the mechanism of arrythmogenesis at molecular level.

Abstract 17384: Cardiac-Specific Overexpression of Human Mutant Desmoplakin in Mice Disrupts Cardiac Voltage-Gated Sodium Channel Expression

Introduction: Arrhythmogenic cardiomyopathy (AC) is characterized by bi-ventricular dilation, fibro-fatty infiltration and life-threatening arrhythmias. Disruptions in cardiac voltage-gated sodium channel (Nav1.5) expression and function are known to cause arrhythmias. We have demonstrated that cardiac-specific overexpression of human mutant desmoplakin (DSP, Tg-R2834H) in mice leads to AC. However, whether mutant DSP expression in the heart affects the Nav1.5 distribution and function are unknown Hypothesis: Here, we tested whether Nav1.5 localization and expression are altered in the R2834H-Tg mouse hearts. Methods: Primary cardiomyocytes and frozen myocardial sections from non-transgenic (NTg), wild-type DSP (Tg-DSP) and Tg-R2834H mice were used for immunofluorescence studies to assess subcellular localization of DSP, desmin, Nav1.5, Cx43, plakoglobin and β-catenin. Western blot and qPCR were used for quantitative analysis. Results: Double staining of cardiomyocytes from NTg mice with DSP and Nav1.5 revealed that Nav1.5 was colocalized with DSP at the intercalated discs (IDs). In contrast, Tg-R2834H cardiomyocytes exhibited marked increase of mutant DSP expression at the IDs concomitant with a reduction in Nav1.5 immunoreactive signals. Tg-R2834H cardiomyocytes also revealed an aberration of DSP and desmin colocalizations at the IDs. There were not obvious differences in Cx43 expression between the genotypes, although the redistribution of Cx43 from the IDs to the sarcolemma was evident in Tg-R2834H cardiomyocytes. qPCR results correlated with reduced Nav1.5 mRNA expression in the Tg-R2834H mouse hearts. Conclusions: Defective DSP protein expression in the heart disrupts Nav1.5 localization and expression, implying an interaction between DSP and Nav1.5 to orchestrate normal mechanical and electrical coupling. Further electrophysiology studies to assess whole-cell Na + currents in these cardiomyocytes will provide insight into DSP and Nav1.5 interaction.

Cardiac voltage-gated sodium channel expression and electrophysiological characterization of the sodium current in the zebrafish (Danio rerio) ventricle

Na+ channel α-subunit composition of the zebrafish heart and electrophysiological properties of Na+ current (INa) of zebrafish ventricular myocytes were examined. Eight Na+ channel α-subunits were expressed in both atrium and ventricle of the zebrafish heart. Nav1.5Lb, an orthologue to the human Nav1.5, was clearly the predominant isoform in both chambers representing 65.2 ± 4.1% and 83.1 ± 2.1% of all Na+ channel transcripts in atrium and ventricle, respectively. Nav1.4b, an orthologue to human Nav1.4, formed 34.1 ± 4.1 and 16.2 ± 2.0% of the Na+ channel transcripts in atrium and ventricle, respectively. The density of INa and the rate of action potential upstroke in zebrafish ventricular myocytes at 28 °C were similar to those of human ventricles at the comparable temperature. Na+ channel isoforms and the main electrophysiological characteristics of the INa are largely similar in zebrafish and human hearts indicating evolutionary conservation of Na+ channel composition and function. The zebrafish INa differs from the human cardiac INa in terms of higher tetrodotoxin sensitivity (IC50-value = 5.3 ± 0.1 nM) and slower inactivation kinetics. The zebrafish INa was inhibited with tricaine (MS-222) with an IC50-value of 1.2 ± 0.18 mM (336 mg l−1), suggesting some care in the use of MS-222 as an anesthetic.

Cardiac voltage-gated sodium channel mutations associated with left atrial dysfunction and stroke in children

Cardiac atrial arrhythmias are the most common type of heart rhythm disorders. Its genetic elucidation remains challenging with poor understanding of cellular and molecular processes. These arrhythmias usually affect elderly population but in rare cases, young children may also suffer from such electrical diseases. Severe complications, including stroke, are commonly age related. This study aims to identify a genetic link between electro-mechanic atrial dysfunction and stroke in children. In two unrelated boys of 11 and 14 years with both stroke and atrial arrhythmias, the clinical phenotype was determined through a complete physical examination, electrocardiogram (ECG), Holter ECG, and computed tomography. The genetic testing was performed on a large 95 genes panel implicated in myocardial electrical imbalance, using the next generation sequencing method. The panel also includes the genes usually associated with the development of cardiomyopathies. In one child, a left atrial dilation was observed. The 2nd boy suffered from atrial standstill. Both suffered from atrial bradycardia, flutter, and fibrillation. The complete genetic testing revealed the SCN5A c.3823G>A (p.D1275N) mutation in the first family, c.1141-2A>G and c.3157G>A (p.E1053K) mutations in the second family. Our results strengthen the association between Nav1.5 mutations and the occurrence of stroke in young patients. It emphasizes the need to look for atrial myopathy in the decision process for anticoagulation in young patients with atrial arrhythmic events.

O-GlcNAcylation of cardiac Nav1.5 contributes to the development of arrhythmias in diabetic hearts

BackgroundCardiovascular complications are major causes of mortality and morbidity in diabetic patients. The mechanisms underlying the progression of diabetic heart (DH) to ventricular arrhythmias are unclear. O-linked GlcNAcylation (O-GlcNAc) is a reversible post-translational modification for the regulation of diverse cellular processes. The purpose of this study was to assess whether the cardiac voltage-gated sodium channel (Nav1.5) is subjected to O-linked GlcNAcylation (O-GlcNAc), which plays an essential role in DH-induced arrhythmias. Methods and resultsIn this study, Sprague-Dawley rats (male, 200-230 g) were treated with a single high-dose of streptozotocin (STZ, 80 mg/kg) to generate a rat model of diabetes. STZ-induced 3-month diabetic rats displayed increased susceptibility to ventricular arrhythmias. The elevated O-GlcNAc modification was correlated with decreases in both total and cytoplasmic Nav1.5 expression in vivo and in vitro. In addition, both co-immunoprecipitation and immunostaining assays demonstrated that hyperglycemia could increase the O-GlcNAc-modified Nav1.5 levels and decrease the interaction between Nav1.5 and Nav1.5-binding proteins Nedd4–2/SAP-97. Furthermore, patch-clamp measurements in HEK-293 T cells showed that Nav1.5 current densities decreased by 30% after high-glucose treatment, and the sodium currents increased via O-GlcNAc inhibition. ConclusionOur data suggested that hyperglycemia increased the O-GlcNAc modification of Nav1.5 expression and decreased the interaction between Nav1.5 and Nedd4–2/SAP-97, which led to the abnormal expression and distribution of Nav1.5, loss of function of the sodium channel, and prolongation of the PR/QT interval. Excessive O-GlcNAc modification of Nav1.5 is a novel signaling event, which may be an underlying contributing factor for the development of the arrhythmogenesis in DH.