Molecular Autopsy With Banked Cord Blood Reveals Brugada Syndrome in Past Sudden Death Case.

Molecular autopsy has recently been gaining attention as a means of postmortem diagnosis; however, it is usually performed using the victim's blood sample at the time of death. Here, we report the first case of a deceased infant with Brugada syndrome whose diagnosis was made with banked cord blood. A seemingly healthy 1-year-old male infant collapsed while having a fever; this collapse was witnessed by his mother. Despite cardiopulmonary resuscitation, he died of ventricular fibrillation. No abnormalities of cardiac structure were identified on autopsy. Genomic samples were not stored at the time because of a lack of suspicion for familial arrhythmia. Five years later, his sister showed Brugada electrocardiogram pattern while febrile from Kawasaki disease. Their father showed a spontaneous type 1 Brugada electrocardiogram pattern. A heterozygous SCN5A p.R893C variant was found by genetic testing in the proband's father and sister. Furthermore, the proband's genetic testing was performed using his banked cord blood, which identified the same variant. Family history of Brugada syndrome with an SCN5A-R893C variant and clinical evidence led to a postmortem diagnosis of Brugada syndrome in the proband. Identification of this variant in this case later contributed to verifying SCN5A-R893C as a pathogenic variant through data accumulation. Banked cord blood may prove useful for conducting molecular autopsies in previously undiagnosed cases of sudden death in which genomic samples were not stored.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and ßMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Pediatric and Familial Genetic Arrhythmia Syndromes: SCN5A-Related Disorders When It Is Not Long QT Type 3: Clinical Signs and Symptoms.

The following case series presents three different pediatric patients with SCN5A-related disease. In addition, family members are presented to demonstrate the variable penetrance that is commonly seen. Identifying features of this disease is important, because even in the very young, SCN5A disorders can cause lethal arrhythmias and sudden death.

Brugada Syndrome and Pulmonary Atresia with Intact Interventricular Septum: Fortuitous Finding or New Genetic Connection?

Brugada syndrome is a rare arrhythmogenic syndrome associated mainly with pathogenic variants in the SCN5A gene. Right ventricle outflow tract fibrosis has been reported in some cases of patients diagnosed with Brugada syndrome. Pulmonary atresia with an intact ventricular septum is characterized by the lack of a functional pulmonary valve, due to the underdevelopment of the right ventricle outflow tract. We report, for the first time, a 4-year-old boy with pulmonary atresia with an intact ventricular septum who harbored a pathogenic de novo variant in SCN5A, and the ajmaline test unmasked a type-1 Brugada pattern. We suggest that deleterious variants in the SCN5A gene could be implicated in pulmonary atresia with an intact ventricular septum embryogenesis, leading to overlapping phenotypes.

Variable Penetrance and Expressivity of a Rare Pore Loss-of-Function Mutation (p.L889V) of Nav1.5 Channels in Three Spanish Families.

A novel rare mutation in the pore region of Nav1.5 channels (p.L889V) has been found in three unrelated Spanish families that produces quite diverse phenotypic manifestations (Brugada syndrome, conduction disease, dilated cardiomyopathy, sinus node dysfunction, etc.) with variable penetrance among families. We clinically characterized the carriers and recorded the Na+ current (INa) generated by p.L889V and native (WT) Nav1.5 channels, alone or in combination, to obtain further insight into the genotypic-phenotypic relationships in patients carrying SCN5A mutations and in the molecular determinants of the Nav1.5 channel function. The variant produced a strong dominant negative effect (DNE) since the peak INa generated by p.L889V channels expressed in Chinese hamster ovary cells, either alone (-69.4 ± 9.0 pA/pF) or in combination with WT (-62.2 ± 14.6 pA/pF), was significantly (n ≥ 17, p < 0.05) reduced compared to that generated by WT channels alone (-199.1 ± 44.1 pA/pF). The mutation shifted the voltage dependence of channel activation and inactivation to depolarized potentials, did not modify the density of the late component of INa, slightly decreased the peak window current, accelerated the recovery from fast and slow inactivation, and slowed the induction kinetics of slow inactivation, decreasing the fraction of channels entering this inactivated state. The membrane expression of p.L889V channels was low, and in silico molecular experiments demonstrated profound alterations in the disposition of the pore region of the mutated channels. Despite the mutation producing a marked DNE and reduction in the INa and being located in a critical domain of the channel, its penetrance and expressivity are quite variable among the carriers. Our results reinforce the argument that the incomplete penetrance and phenotypic variability of SCN5A loss-of-function mutations are the result of a combination of multiple factors, making it difficult to predict their expressivity in the carriers despite the combination of clinical, genetic, and functional studies.

Phosphorylation of cardiac sodium channel at Ser571 anticipates manifestations of the aging myopathy.

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction.NEW & NOTEWORTHY We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy.

Exenatide reduces atrial fibrillation susceptibility by inhibiting hKv1.5 and hNav1.5 channels.

Exenatide, a promising cardioprotective agent, protects against cardiac structural remodeling and diastolic dysfunction. Combined blockade of sodium and potassium channels is valuable for managing atrial fibrillation (AF). Here, we explored whether exenatide displayed anti-AF effects by inhibiting human Kv1.5 and Nav1.5 channels. We used the whole-cell patch-clamp technique to investigate the effects of exenatide on hKv1.5 and hNav1.5 channels expressed in human embryonic kidney 293 cells and studied the effects of exenatide on action potential (AP) and other cardiac ionic currents in rat atrial myocytes. Additionally, an electrical mapping system was used to explore the effects of exenatide on electrical properties and AF activity in isolated rat hearts. Finally, a rat AF model, established using acetylcholine and calcium chloride, was employed to evaluate the anti-AF potential of exenatide in rats. Exenatide reversibly suppressed IKv1.5 with IC50 of 3.08 µM, preferentially blocked the hKv1.5 channel in its closed state, and positively shifted the voltage-dependent activation curve. Exenatide also reversibly inhibited INav1.5 with IC50 of 3.30 µM, negatively shifted the voltage-dependent inactivation curve, and slowed its recovery from inactivation with significant use-dependency at 5 and 10 Hz. Furthermore, exenatide prolonged AP duration and suppressed the sustained K+ current (Iss) and transient outward K+ current (Ito), but without inhibition of L-type Ca2+ current (ICa,L) in rat atrial myocytes. Exenatide prevented AF incidence and duration in rat hearts and rats. These findings demonstrate that exenatide inhibits IKv1.5 and INav1.5in vitro and reduces AF susceptibility in isolated rat hearts and rats.

Genetic Testing in Brugada Syndrome: A 30-Year Experience.

BACKGROUND: A pathogenic/likely pathogenic variant can be found in 20% to 25% of patients with Brugada syndrome (BrS) and a pathogenic/likely pathogenic variant in SCN5A is associated with a worse prognosis. The aim of this study is to define the diagnostic yield of a large gene panel with American College of Medical Genetics and Genomics variant classification and to assess prognosis of SCN5A and non-SCN5A variants. METHODS: All patients with BrS, were prospectively enrolled in the Universitair Ziekenhuis Brussel registry between 1992 and 2022. Inclusion criteria for the study were (1) BrS diagnosis; (2) genetic analysis performed with a large gene panel; (3) classification of variants following American College of Medical Genetics and Genomics guidelines. Patients with a pathogenic/likely pathogenic variant in SCN5A were defined as SCN5A+. Patients with a reported variant in a non-SCN5A gene or with no reported variants were defined as patients with SCN5A-. All variants were classified as missense or predicted loss of function. RESULTS: A total of 500 BrS patients were analyzed. A total of 104 patients (20.8%) were SCN5A+ and 396 patients (79.2%) were SCN5A-. A non-SCN5A gene variant was found in 75 patients (15.0%), of whom, 58 patients (77.3%) had a missense variant and 17 patients (22.7%) had a predicted loss of function variant. At a follow-up of 84.0 months, 48 patients (9.6%) experienced a ventricular arrhythmia (VA). Patients without any variant had higher VA-free survival, compared with carriers of a predicted loss of function variant in SCN5A+ or non-SCN5A genes. There was no difference in VA-free survival between patients without any variant and missense variant carriers in SCN5A+ or non-SCN5A genes. At Cox analysis, SCN5A+ or non-SCN5A predicted loss of function variant was an independent predictor of VA. CONCLUSIONS: In a large BrS cohort, the yield for SCN5A+ is 20.8%. A predicted loss of function variant carrier is an independent predictor of VA.

Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5.

BACKGROUND: 14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AIMS: In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. METHODS AND RESULTS: Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. CONCLUSIONS: Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.

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.

Decreasing microtubule detyrosination modulates Nav1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes.

AIMS: The microtubule (MT) network plays a major role in the transport of the cardiac sodium channel Nav1.5 to the membrane, where the latter associates with interacting proteins such as dystrophin. Alterations in MT dynamics are known to impact on ion channel trafficking. Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, is associated with an increase in MT detyrosination, decreased sodium current (INa), and arrhythmias. Parthenolide (PTL), a compound that decreases MT detyrosination, has shown beneficial effects on cardiac function in DMD. We here investigated its impact on INa and Nav1.5 subcellular distribution. METHODS AND RESULTS: Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL, 20 µM EpoY, or dimethylsulfoxide (DMSO) for 3-5 h, followed by patch-clamp analysis to assess INa and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Nav1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased INa and reduced AP upstroke velocity (Vmax) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased INa magnitude (without affecting INa gating properties) and AP Vmax in mdx CMs, but had no effect in WT CMs. Moreover, STORM analysis showed that in mdx CMs, Nav1.5 clusters were decreased not only in the grooves of the lateral membrane (LM; where dystrophin is localized) but also at the LM crests. PTL restored Nav1.5 clusters at the LM crests (but not at the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Nav1.5 cluster density was also reduced at the intercalated disc (ID) region of mdx CMs, which was restored to WT levels by PTL. Treatment of mdx CMs with EpoY, a specific MT detyrosination inhibitor, also increased INa density, while decreasing the amount of detyrosinated MTs, confirming a direct mechanistic link. CONCLUSION: Attenuating MT detyrosination in mdx CMs restored INa and enhanced Nav1.5 localization at the LM crest and ID. Hence, the reduced whole-cell INa density characteristic of mdx CMs is not only the consequence of the lack of dystrophin within the LM grooves but is also due to reduced Nav1.5 at the LM crest and ID secondary to increased baseline MT detyrosination. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution in pathophysiological conditions.

Polymorphic Variants of SCN5A Gene (rs41312433 and rs1805124) Associated with Coronary Artery Affliction in Patients with Severe Arrhythmias.

Several mutations in this gene for the α subunit of the cardiac sodium channel have been identified in a heterogeneous subset of cardiac rhythm syndromes, including Brugada syndrome, progressive cardiac conduction defect, sick sinus node syndrome, atrial fibrillation and dilated cardiomyopathy. The aim of our study was to associate some SCN5A polymorphic variants directly with confirmed coronary stenoses in patients with non-LQTS ventricular fibrillation/flutter treated by an implantable cardioverter defibrillator. MATERIALS AND METHODS: A group of 32 unrelated individuals, aged 63 ± 12 years, was included in the study. All the patients were examined, diagnosed and treated with an implantable cardioverter defibrillator at the Department of Internal Cardiology Medicine, Faculty Hospital Brno. The control group included 87 persons of similar age without afflicted coronary circulation, which was confirmed coronagraphically. Genomic DNA was extracted from samples of peripheral blood according to the standard protocol. Two SCN5A polymorphisms-IVS9-3C/A (rs41312433) and A1673G (rs1805124, H558R)-were examined in association with coronary artery stenosis in the patients. RESULTS: In the case-control study, no significant differences in genotype distribution/allelic frequencies were observed for IVS9-3c>a and A1673G gene polymorphisms between patients with severe arrhythmias and healthy persons. The distribution of SCN5A double genotypes was not significantly different among different types of arrhythmias according to their ejection fraction in arrhythmic patients (p = 0.396). The ventricular arrhythmias with an ejection fraction below 40% were found to be 10.67 times more frequent in patients with multiple coronary stenosis with clinically valid sensitivity, specificity and power tests. In the genotype-phenotype study, we observed a significant association of both SCN5A polymorphisms with the stenosis of coronary vessels in the patients with severe arrhythmia. The double genotype of polymorphisms IVS9-3C/A together with A1673G (CCAA) as well as their simple genotypes were associated with significant multiple stenosis of coronary arteries (MVS) with high sensitivity and specificity (p = 0.05; OR = 5 (95% CI 0.99-23.34); sensitivity 0.70; specificity 0.682; power test 0.359) Moreover, when a concrete stenotic coronary artery was associated with SCN5A genotypes, the CCAA double genotype was observed to be five times more frequent in patients with significant stenosis in the right coronary artery (RCA) compared to those without affliction of this coronary artery (p = 0.05; OR = 5 (95% CI 0.99-23.34); sensitivity 0.682; specificity 0.700; power test 0.359). The CCAA genotype was also more frequent in patients without RCA affliction with MVS (p = 0.008); in patients with ACD affliction but without MVS (p = 0.008); and in patients with both ACD affliction and MVS compared to those without ACD affliction and MVS (p = 0.005). CONCLUSIONS: Our study presents a highly sensitive and specific association of two polymorphisms in SCN5A with significant coronary artery stenoses in patients with potentially fatal ventricular arrhythmias. At the same time, these polymorphisms were not associated with arrhythmias themselves. Thus, SCN5A gene polymorphic variants may form a part of germ cell gene predisposition to ischemia.

The Beneficial Role of Telemedicine for Arrhythmic Risk Stratification in Asymptomatic Brugada Syndrome: An Exemplary Case Report.

Telemedicine and remote monitoring devices, including implantable loop recorders (ILR), are increasingly adopted in the cardiologic setting. These are valuable tools in the arrhythmic stratification of patients at risk of sudden cardiac death, providing a tailored therapeutic management to prevent lethal arrhythmias. We report a case of an asymptomatic 18-year-old boy with a family history of syncope and cardiac arrest, who had a diagnosis of Brugada syndrome with an inducible type 1 pattern and carrier of a missense mutation of the SCN5A gene. In light of the risk factors, although not recommended by current guidelines, we decided to proceed with the implantation of an ILR with remote monitoring service. A few months later, an episode of asymptomatic sustained polymorphic ventricular tachycardia was promptly observed by the remote monitoring, leading to a timely implantation of a subcutaneous cardiac implantable defibrillator.

Kir2.1-NaV1.5 channelosome and its role in arrhythmias in inheritable cardiac diseases.

Sudden cardiac death in children and young adults is a relatively rare but tragic event whose pathophysiology is unknown at the molecular level. Evidence indicates that the main cardiac sodium channel (NaV1.5) and the strong inward rectifier potassium channel (Kir2.1) physically interact and form macromolecular complexes (channelosomes) with common partners, including adapter, scaffolding, and regulatory proteins that help them traffic together to their eventual membrane microdomains. Most important, dysfunction of either or both ion channels has direct links to hereditary human diseases. For example, certain mutations in the KCNJ2 gene encoding the Kir2.1 protein result in Andersen-Tawil syndrome type 1 and alter both inward rectifier potassium and sodium inward currents. Similarly, trafficking-deficient mutations in the gene encoding the NaV1.5 protein (SCN5A) result in Brugada syndrome and may also disturb both inward rectifier potassium and sodium inward currents. Moreover, gain-of-function mutations in KCNJ2 result in short QT syndrome type 3, which is extremely rare but highly arrhythmogenic, and can modify Kir2.1-NaV1.5 interactions in a mutation-specific way, further highlighting the relevance of channelosomes in ion channel diseases. By expressing mutant proteins that interrupt or modify Kir2.1 or NaV1.5 function in animal models and patient-specific pluripotent stem cell-derived cardiomyocytes, investigators are defining for the first time the mechanistic framework of how mutation-induced dysregulation of the Kir2.1-NaV1.5 channelosome affects cardiac excitability, resulting in arrhythmias and sudden death in different cardiac diseases.

Overexpression of SCN5A overcomes ABC transporter-mediated multidrug resistance in acute myeloid leukemia through promoting apoptosis.

BACKGROUND: This study aimed to explore the effect and mechanism of SCN5A overcoming ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR) in acute myeloid leukemia (AML) through promoting apoptosis. RESEARCH DESIGN AND METHODS: The tissues derived from AML patients were divided into Sensitive group and Resistance group according to the presence of drug-resistance. Human AML cell line HL-60 and drug-resistant strain HL-60/ADR were divided into HL-60/ADR-vector group, HL-60/ADR-SCN5A group, HL-60-vector group and HL-60-SCN5A group. RT-qPCR was used to detect the mRNA expression level of SCN5A; MTT assay to assess the survival rate and proliferation level of cells; flow cytometry to determine the apoptosis level; and western blot to check the levels of SCN5A, P-glycoprotein (P-gp), MDR protein 1 (MRP1), MDR gene 1 (MDR1), breast cancer resistance protein (BCRP), Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) proteins in cells. RESULTS: SCN5A expressed lowly in drug-resistant AML tissues and cells. Up-regulation of SCN5A inhibited MDR in HL-60 cells, enhanced the chemosensitivity of HL-60/ADR, and increased the apoptosis levels of HL-60 and HL-60/ADR cells. However, over-expression of SCN5A inhibited the expression of MDR-related proteins. CONCLUSIONS: SCN5A may overcome ABC transporter-mediated MDR in AML through enhancing the apoptosis and inhibiting the expression of MDR proteins.

Errata: Rare Compound Heterozygous Missense Mutation of the SCN5A Gene with Childhood-Onset Sick Sinus Syndrome in Two Chinese Sisters: A Case Report.

An error appeared in the article entitled "Rare Compound Heterozygous Missense Mutation of the SCN5A Gene with Childhood-Onset Sick Sinus Syndrome in Two Chinese Sisters: A Case Report" by Yanyun Wang, Siyu Long, Chenxi Wei, and Xiaoqin Wang (Vol. 64 No.2, 299-305, 2023). The name of the first affiliation on page 299 was wrong. It should be "Laboratory of Molecular Translational Medicine, Center for Translational Medicine, West China Second University Hospital, Sichuan University, Chengdu, China" and not "Laboratory of Molecular Translational Medicine, Center for Translational Medicine, Sichuan University, Chengdu, China".

Mannitol and hyponatremia regulate cardiac ventricular conduction in the context of sodium channel loss of function.

Scn5a heterozygous null (Scn5a+/-) mice have historically been used to investigate arrhythmogenic mechanisms of diseases such as Brugada syndrome (BrS) and Lev's disease. Previously, we demonstrated that reducing ephaptic coupling (EpC) in ex vivo hearts exacerbates pharmacological voltage-gated sodium channel (Nav)1.5 loss of function (LOF). Whether this effect is consistent in a genetic Nav1.5 LOF model is yet to be determined. We hypothesized that loss of EpC would result in greater reduction in conduction velocity (CV) for the Scn5a+/- mouse relative to wild type (WT). In vivo ECGs and ex vivo optical maps were recorded from Langendorff-perfused Scn5a+/- and WT mouse hearts. EpC was reduced with perfusion of a hyponatremic solution, the clinically relevant osmotic agent mannitol, or a combination of the two. Neither in vivo QRS duration nor ex vivo CV during normonatremia was significantly different between the two genotypes. In agreement with our hypothesis, we found that hyponatremia severely slowed CV and disrupted conduction for 4/5 Scn5a+/- mice, but 0/6 WT mice. In addition, treatment with mannitol slowed CV to a greater extent in Scn5a+/- relative to WT hearts. Unexpectedly, treatment with mannitol during hyponatremia did not further slow CV in either genotype, but resolved the disrupted conduction observed in Scn5a+/- hearts. Similar results in guinea pig hearts suggest the effects of mannitol and hyponatremia are not species specific. In conclusion, loss of EpC through either hyponatremia or mannitol alone results in slowed or disrupted conduction in a genetic model of Nav1.5 LOF. However, the combination of these interventions attenuates conduction slowing.NEW & NOTEWORTHY Cardiac sodium channel loss of function (LOF) diseases such as Brugada syndrome (BrS) are often concealed. We optically mapped mouse hearts with reduced sodium channel expression (Scn5a+/-) to evaluate whether reduced ephaptic coupling (EpC) can unmask conduction deficits. Data suggest that conduction deficits in the Scn5a+/- mouse may be unmasked by treatment with hyponatremia and perinexal widening via mannitol. These data support further investigation of hyponatremia and mannitol as novel diagnostics for sodium channel loss of function diseases.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

GPD1L-A306del modifies sodium current in a family carrying the dysfunctional SCN5A-G1661R mutation associated with Brugada syndrome.

Loss-of-function variants of SCN5A, encoding the sodium channel alpha subunit Nav1.5 are associated with high phenotypic variability and multiple cardiac presentations, while underlying mechanisms are incompletely understood. Here we investigated a family with individuals affected by Brugada Syndrome (BrS) of different severity and aimed to unravel the underlying genetic and electrophysiological basis.Next-generation sequencing was used to identify the genetic variants carried by family members. The index patient, who was severely affected by arrhythmogenic BrS, carried previously uncharacterized variants of Nav1.5 (SCN5A-G1661R) and glycerol-3-phosphate dehydrogenase-1-like protein (GPD1L-A306del) in a double heterozygous conformation. Family members exclusively carrying SCN5A-G1661R showed asymptomatic Brugada ECG patterns, while another patient solely carrying GPD1L-A306del lacked any clinical phenotype.To assess functional mechanisms, Nav1.5 channels were transiently expressed in HEK-293 cells in the presence and absence of GPD1L. Whole-cell patch-clamp recordings revealed loss of sodium currents after homozygous expression of SCN5A-G1661R, and reduction of current amplitude to ~ 50% in cells transfected with equal amounts of wildtype and mutant Nav1.5. Co-expression of wildtype Nav1.5 and GPD1L showed a trend towards increased sodium current amplitudes and a hyperpolarizing shift in steady-state activation and -inactivation compared to sole SCN5A expression. Application of the GPD1L-A306del variant shifted steady-state activation to more hyperpolarized and inactivation to more depolarized potentials.In conclusion, SCN5A-G1661R produces dysfunctional channels and associates with BrS. SCN5A mediated currents are modulated by co-expression of GDP1L and this interaction is altered by mutations in both proteins. Thus, additive genetic burden may aggravate disease severity, explaining higher arrhythmogenicity in double mutation carriers.