Novel Gain-of-Function Mutation in the Kv11.1 Channel Found in the Patient with Brugada Syndrome and Mild QTc Shortening.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter IKr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on IKr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the IKr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

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.

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.

Functional Characterisation of the Rare SCN5A p.E1225K Variant, Segregating in a Brugada Syndrome Familial Case, in Human Cardiomyocytes from Pluripotent Stem Cells.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Pathogenic rare mutations in the SCN5A gene, encoding the alpha-subunit of the voltage-dependent cardiac Na+ channel protein (Nav1.5), are identified in 20% of BrS patients, affecting the correct function of the channel. To date, even though hundreds of SCN5A variants have been associated with BrS, the underlying pathogenic mechanisms are still unclear in most cases. Therefore, the functional characterization of the SCN5A BrS rare variants still represents a major hurdle and is fundamental to confirming their pathogenic effect. Human cardiomyocytes (CMs) differentiated from pluripotent stem cells (PSCs) have been extensively demonstrated to be reliable platforms for investigating cardiac diseases, being able to recapitulate specific traits of disease, including arrhythmic events and conduction abnormalities. Based on this, in this study, we performed a functional analysis of the BrS familial rare variant NM_198056.2:c.3673G>A (NP_932173.1:p.Glu1225Lys), which has been never functionally characterized before in a cardiac-relevant context, as the human cardiomyocyte. Using a specific lentiviral vector encoding a GFP-tagged SCN5A gene carrying the specific c.3673G>A variant and CMs differentiated from control PSCs (PSC-CMs), we demonstrated an impairment of the mutated Nav1.5, thus suggesting the pathogenicity of the rare BrS detected variant. More broadly, our work supports the application of PSC-CMs for the assessment of the pathogenicity of gene variants, the identification of which is increasing exponentially due to the advances in next-generation sequencing methods and their massive use in genetic testing.

Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome.

The development of high-throughput automated patch-clamp technology is a recent breakthrough in the field of Brugada syndrome research. Brugada syndrome is a heart disorder marked by abnormal electrocardiographic readings and an elevated risk of sudden cardiac death due to arrhythmias. Various experimental models, developed either in animals, cell lines, human tissue or computational simulation, play a crucial role in advancing our understanding of this condition, and developing effective treatments. In the perspective of the pathophysiological role of ion channels and their pharmacology, automated patch-clamp involves a robotic system that enables the simultaneous recording of electrical activity from multiple single cells at once, greatly improving the speed and efficiency of data collection. By combining this approach with the use of patient-derived cardiomyocytes, researchers are gaining a more comprehensive view of the underlying mechanisms of heart disease. This has led to the development of more effective treatments for those affected by cardiovascular conditions.

Bioinformatics Insights on the Physicochemical Properties of SCN5A Mutant Proteins Associated with the Brugada Syndrome.

BACKGROUND: The Brugada syndrome (BrS) is a heart rhythm condition that is commonly associated with a strong predisposition for sudden cardiac death. Malignant ventricular arrhythmias could occur secondary to the dysfunction of the cardiac sodium voltage-gated Na(v)1.5 channel (SCN5A). OBJECTIVE: This study aimed to perform a multiparametric computational analysis of the physicochemical properties of SCN5A mutants associated with BrS using a set of bioinformatics tools. METHODS: In-house algorithms were calibrated to calculate, in a double-blind test, the Polarity Index Method (PIM) profile and protein intrinsic disorder predisposition (PIDP) profile of each sequence, and computer programs specialized in the genomic analysis were used. RESULTS: Specific regularities in the charge/polarity and PIDP profile of the SCN5A mutant proteins enabled the re-creation of the taxonomy, allowing us to propose a bioinformatics method that takes advantage of the PIM profile to identify this group of proteins from their sequence. CONCLUSION: Bioinformatics programs could reproduce characteristic PIM and PIDP profiles of the BrS-related SCN5A mutant proteins. This information can contribute to a better understanding of these altered proteins.

Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model.

BACKGROUND: A missense mutation in the α1c subunit of voltage-gated L-type Ca2+ channel-coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes. OBJECTIVE: We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). METHODS: We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model. RESULTS: Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the in vitro electrophysiological properties. CONCLUSION: Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

Gene therapy targeting protein trafficking regulator MOG1 in mouse models of Brugada syndrome, arrhythmias, and mild cardiomyopathy.

Brugada syndrome (BrS) is a fatal arrhythmia that causes an estimated 4% of all sudden death in high-incidence areas. SCN5A encodes cardiac sodium channel NaV1.5 and causes 25 to 30% of BrS cases. Here, we report generation of a knock-in (KI) mouse model of BrS (Scn5aG1746R/+). Heterozygous KI mice recapitulated some of the clinical features of BrS, including an ST segment abnormality (a prominent J wave) on electrocardiograms and development of spontaneous ventricular tachyarrhythmias (VTs), seizures, and sudden death. VTs were caused by shortened cardiac action potential duration and late phase 3 early afterdepolarizations associated with reduced sodium current density (INa) and increased Kcnd3 and Cacna1c expression. We developed a gene therapy using adeno-associated virus serotype 9 (AAV9) vector-mediated MOG1 delivery for up-regulation of MOG1, a chaperone that binds to NaV1.5 and traffics it to the cell surface. MOG1 was chosen for gene therapy because the large size of the SCN5A coding sequence (6048 base pairs) exceeds the packaging capacity of AAV vectors. AAV9-MOG1 gene therapy increased cell surface expression of NaV1.5 and ventricular INa, reversed up-regulation of Kcnd3 and Cacna1c expression, normalized cardiac action potential abnormalities, abolished J waves, and blocked VT in Scn5aG1746R/+ mice. Gene therapy also rescued the phenotypes of cardiac arrhythmias and contractile dysfunction in heterozygous humanized KI mice with SCN5A mutation p.D1275N. Using a small chaperone protein may have broad implications for targeting disease-causing genes exceeding the size capacity of AAV vectors.

Generation of two induced pluripotent stem cell (iPSC) lines (BBANTWi006-A, BBANTWi007-A) from Brugada syndrome patients carrying an SCN5A mutation.

Brugada syndrome (BrS) is an inherited primary electrical disorder of the heart. 25% of BrS patients carry a mutation in the SCN5A gene, encoding the cardiac specific voltage-gated sodium channel Nav1.5. Here we report two iPSC lines (BBANTWi006-A, BBANTWi007-A) of a brother and a sister carrying an SCN5A mutation (c.4813 + 3_4813 + 6dupGGGT) causing BrS. iPSCs were generated from dermal fibroblasts and reprogrammed with the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). The generated iPSCs showed a normal karyotype, expressed pluripotency markers, were differentiated into cells of the three germ layers and carried the original genotype.

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.

Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome.

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Why Is Only Type 1 Electrocardiogram Diagnostic of Brugada Syndrome? Mechanistic Insights From Computer Modeling.

BACKGROUND: Three types of characteristic ST-segment elevation are associated with Brugada syndrome but only type 1 is diagnostic. Why only type 1 ECG is diagnostic remains unanswered. METHODS: Computer simulations were performed in single cells, 1-dimensional cables, and 2-dimensional tissues to investigate the effects of the peak and late components of the transient outward potassium current (Ito), sodium current, and L-type calcium current (ICa,L) as well as other potassium currents on the genesis of ECG morphologies and phase 2 reentry (P2R). RESULTS: Although a sufficiently large peak Ito was required to result in the type 1 ECG pattern and P2R, increasing the late component of Ito converted type 1 ECG to type 2 ECG and suppressed P2R. Increasing the peak Ito promoted spiral wave breakup, potentiating the transition from tachycardia to fibrillation, but increasing the late Ito prevented spiral wave breakup by flattening the action potential duration restitution and preventing P2R. A sufficiently large ICa,L conductance was needed for P2R to occur, but once above the critical conductance, blocking ICa,L promoted P2R. However, selectively blocking the window and late components of ICa,L suppressed P2R, countering the effect of the late Ito. Blocking either the peak or late components of sodium current promoted P2R, with the late sodium current blockade having the larger effect. As expected, increasing other potassium currents potentiated P2R, with ATP-sensitive potassium current exhibiting a larger effect than rapid and slow component of the delayed rectifier potassium current. CONCLUSIONS: The peak Ito promotes type 1 ECG and P2R, whereas the late Ito converts type 1 ECG to type 2 ECG and suppresses P2R. Blocking the peak ICa,L and either the peak or the late sodium current promotes P2R, whereas blocking the window and late ICa,L suppresses P2R. These results provide important insights into the mechanisms of arrhythmogenesis and potential therapeutic targets for treatment of Brugada syndrome. Graphic Abstract: A graphic abstract is available for this article.

Transcriptional profiles of genes related to electrophysiological function in Scn5a+/- murine hearts.

The Scn5a gene encodes the major pore-forming Nav 1.5 (α) subunit, of the voltage-gated Na+ channel in cardiomyocytes. The key role of Nav 1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss-of-function Nav 1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a+/- genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein-coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria-specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/- animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS.

Transmembrane protein 168 mutation reduces cardiomyocyte cell surface expression of Nav1.5 through αB-crystallin intracellular dynamics.

Transmembrane protein 168 (TMEM168) was found to be localized on the nuclear membrane. A heterozygous mutation (c.1616G>A, p. R539Q) in TMEM168 was identified in patients with Brugada syndrome. This mutation reduced expression of cardiomyocyte sodium channel Nav1.5 via Nedd4-2 E3 ubiquitin ligase-induced ubiquitination and degradation. However, the detailed molecular mechanism provoked by the TMEM168 mutant remains unclear. Here, we demonstrated that small heat shock protein αB-crystallin, which can bind to Nav1.5 and Nedd4-2 and interfere with the association of both proteins, was strongly recruited from the cell surface to the perinuclear region because of the much higher affinity of αB-crystallin with the TMEM168 mutant than with wild-type TMEM168. Following knockdown of αB-crystallin in HL-1 cardiomyocytes, the interaction of Nav1.5 with Nedd4-2 was increased, despite the reduced expression of Nav1.5. Moreover, reduction of Nav1.5 expression by αB-crystallin knockdown was rescued in the presence of a proteasome inhibitor MG-132, suggesting the importance of the αB-crystallin-modulated ubiquitin-proteasome system for the stability of Nav1.5 expression. Collectively, the balance of molecular interactions among Nav1.5, Nedd4-2 and αB-crystallin plays a role in the regulation of cardiomyocyte cell surface expression of Nav1.5, and the TMEM168 mutant disturbs this balance, resulting in a decrease in Nav1.5 expression.

SCN5A compound heterozygosity mutation in Brugada syndrome: Functional consequences and the implication for pharmacological treatment.

AIMS: SCN5A gene encodes the α-subunit of Nav1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Nav1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na+ channels in vitro. MATERIALS AND METHODS: The functional properties of human wild-type and Nav1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells. KEY FINDINGS: Both variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na+ current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na+ channel. SIGNIFICANCE: De novo compound heterozygosis mutations in SNC5A disrupt the Na+ macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na+ current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na+ channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Nav1.5.

Novel SCN5A p.Val1667Asp Missense Variant Segregation and Characterization in a Family with Severe Brugada Syndrome and Multiple Sudden Deaths.

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.

iPSC-Cardiomyocyte Models of Brugada Syndrome-Achievements, Challenges and Future Perspectives.

Brugada syndrome (BrS) is an inherited cardiac arrhythmia that predisposes to ventricular fibrillation and sudden cardiac death. It originates from oligogenic alterations that affect cardiac ion channels or their accessory proteins. The main hurdle for the study of the functional effects of those variants is the need for a specific model that mimics the complex environment of human cardiomyocytes. Traditionally, animal models or transient heterologous expression systems are applied for electrophysiological investigations, each of these models having their limitations. The ability to create induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), providing a source of human patient-specific cells, offers new opportunities in the field of cardiac disease modelling. Contemporary iPSC-CMs constitute the best possible in vitro model to study complex cardiac arrhythmia syndromes such as BrS. To date, thirteen reports on iPSC-CM models for BrS have been published and with this review we provide an overview of the current findings, with a focus on the electrophysiological parameters. We also discuss the methods that are used for cell derivation and data acquisition. In the end, we critically evaluate the knowledge gained by the use of these iPSC-CM models and discuss challenges and future perspectives for iPSC-CMs in the study of BrS and other arrhythmias.

Single-cell transcriptomics trajectory and molecular convergence of clinically relevant mutations in Brugada syndrome.

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. To evaluate the molecular convergence of clinically relevant mutations and to identify developmental cardiac cell types that are associated with BrS etiology, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, and regulatory and structural genes related to BrS. Among the clinically relevant mutations, 266 are unique singletons and 88 mutations are recurrent. We observed an over-representation of clinically relevant mutations (∼80%) in SCN5A gene and also identified several candidate genes, including GPD1L, TRPM4, and SCN10A. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion transport domains in multiple genes, including SCN5A, TRPM4, and SCN10A. A comparative protein domain analysis of SCN5A further established a significant (P = 0.04) enrichment of clinically relevant mutations within ion transport domain, including a significant (P = 0.02) mutation hotspot within 1321-1380 residue. The enrichment of clinically relevant mutations within SCN5A ion transport domain is stronger (P = 0.00003) among early onset of BrS. Our spatiotemporal cellular heart developmental (prenatal to adult) trajectory analysis applying single-cell transcriptome identified the most frequently BrS-mutated genes (SCN5A and GPD1L) are significantly upregulated in the prenatal cardiomyocytes. A more restrictive cellular expression trajectory is prominent in the adult heart ventricular cardiomyocytes compared to prenatal. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.NEW & NOTEWORTHY Brugada syndrome is a rare inherited arrhythmia with high risk of sudden cardiac death. We present the findings for a molecular convergence of clinically relevant mutations and identification of a single-cell transcriptome-derived cardiac cell types that are associated with the etiology of BrS. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.

Specific decreasing of Na+ channel expression on the lateral membrane of cardiomyocytes causes fatal arrhythmias in Brugada syndrome.

Reduced cardiac sodium (Na+) channel current (INa) resulting from the loss-of-function of Na+ channel is a major cause of lethal arrhythmias in Brugada syndrome (BrS). Inspired by previous experimental studies which showed that in heart diseases INa was reduced along with expression changes in Na+ channel within myocytes, we hypothesized that the local decrease in INa caused by the alteration in Na+ channel expression in myocytes leads to the occurrence of phase-2 reentry, the major triggering mechanism of lethal arrhythmias in BrS. We constructed in silico human ventricular myocardial strand and ring models, and examined whether the Na+ channel expression changes in each myocyte cause the phase-2 reentry in BrS. Reducing Na+ channel expression in the lateral membrane of each myocyte caused not only the notch-and-dome but also loss-of-dome type action potentials and slowed conduction, both of which are typically observed in BrS patients. Furthermore, the selective reduction in Na+ channels on the lateral membrane of each myocyte together with spatial tissue heterogeneity of Na+ channel expression caused the phase-2 reentry and phase-2 reentry-mediated reentrant arrhythmias. Our data suggest that the BrS phenotype is strongly influenced by expression abnormalities as well as genetic abnormalities of Na+ channels.