small molecules

2026-09-18 | Unmasking Brugada Syndrome With Oral Propafenone in a Resource-Limited Setting: A Case Report

Brugada syndrome (BrS) is a cardiac channelopathy predisposing patients to malignant ventricular arrhythmias and sudden cardiac death (SCD) in the absence of structural heart disease.The diagnosis requires documentation of a type 1 Brugada electrocardiographic pattern, which can be unmasked using sodium channel-blocking agents in symptomatic patients presenting with non-diagnostic baseline patterns.We report a case of a 24-year-old male patient with a history of unexplained syncope who exhibited a nondiagnostic type 2 Brugada pattern on a baseline electrocardiogram (ECG).Due to the local unavailability of standard intravenous antiarrhythmics, a pharmacological provocation test was conducted using a single 600 mg oral dose of propafenone.Under continuous cardiac monitoring, the patient safely developed a diagnostic type 1 "coved" electrocardiographic pattern starting at the third hour, confirming the diagnosis of BrS.The ECG returned to its baseline pattern eight hours after the initiation of the test, and no cardiovascular complications occurred.This case highlights the clinical utility and safety of oral propafenone as an effective alternative diagnostic challenge for unmasking BrS in resource-limited environments.Furthermore, it emphasizes the necessity of utilizing extended monitoring protocols due to the specific pharmacokinetic profile and delayed onset of action of oral agents.

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2026-09-10 | Management of Brugada storm in a district general hospital.

A 61-year-old man with Brugada syndrome (BrS) (SCN5A mutation) presented with chest pain to a district general hospital (DGH). There was no history of syncope or ventricular arrhythmias. A subcutaneous implantable cardioverter-defibrillator (S-ICD) was in situ for primary prevention following extraction of a previous transvenous ICD due to infection. Investigations suggested a non-ST-elevation myocardial infarction. While awaiting coronary angiography, he developed a ventricular tachycardia (VT) storm with recurrent cardiac arrests over 30 minutes due to polymorphic VT, receiving seven S-ICD shocks. Defibrillation, isoprenaline and quinidine were employed to manage the polymorphic VT storm, permitting transfer to a tertiary centre for further investigations. Coronary angiography demonstrated an occluded marginal artery, which was managed medically. Isoprenaline was weaned following quinidine initiation, the S-ICD reactivated, and he was discharged. A year later, there have been no further ventricular arrhythmias while on quinidine. This case report demonstrates the effectiveness of isoprenaline, which is easily accessible and readily available in DGHs, in stabilising an acute Brugada storm. Quinidine allowed successful weaning from isoprenaline, but lack of availability limits its use. Finally, this case report highlights both the benefits and risks of primary prevention ICDs in BrS.

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2026-08-13 | Expression Defects of SCN5A Common Polymorphisms S524Y and H558R in the Q1077 Splice Variant Can Be Rescued by Mexiletine.

The cardiac sodium channel NaV1.5, encoded by SCN5A, generates the inward sodium current required for myocardial excitability and impulse conduction. Loss-of-function mutations of NaV1.5 have been implicated in inherited arrhythmia syndromes, including Brugada syndrome, progressive cardiac conduction disease, and congenital sick sinus syndrome. The common SCN5A polymorphism H558R has reported minor allele frequencies ranging from 9.2% to 29% across ethnic groups, whereas S524Y has been described in individuals of African ancestry with a minor allele frequency of approximately 3.3%. Two splice variants of human SCN5A, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077, exist in every human in a 2:1 mRNA transcript ratio. We engineered these two polymorphisms in both backgrounds and reported that when S524Y and H558R were expressed in the Q1077del variant, current densities were normal. In the Q1077 variant, however, the current densities showed a dramatic reduction compared to those in the Q1077del variant or WT-Q1077. We previously reported that incubation with the antiarrhythmic drug mexiletine "rescued" expression deficiencies in the Brugada syndrome. Cells expressing S524Y/Q1077 and H558R/Q1077 were incubated for 48 h with or without mexiletine (500 μM), followed by drug washout before electrophysiological assessment. Mexiletine significantly increased current density for both S524Y/Q1077 and H558R/Q1077 compared with untreated cells, restoring current density to levels comparable to WT-Q1077. Flow cytometry using a FLAG-tagged channel demonstrated that mexiletine-mediated rescue was associated with increased cell-surface expression. The magnitude of the expression defects caused by H558R and S524Y in the Q1077 splice background is similar to that observed with arrhythmia-associated SCN5A mutations, and we show for the first time that the defects for both polymorphisms can be rescued with mexiletine. Although it is unknown whether they result in heightened arrhythmia susceptibility in patients homozygous for the minor allele, our result may have implications for therapy for mutations with loss-of-function phenotypes modified by these common polymorphisms.

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2026-08-04 | Islands of Fractionation for Automated Fractionation Mapping in Epicardial Brugada Syndrome Substrate Ablation.

Brugada syndrome epicardial RVOT ablation reduces VF, but procedural endpoints remain debated. Automated fractionation maps are often interpreted as scattered points, which may reflect artifacts. We propose Islands of Fractionation (IOF), a framework comparing EnSite X fractionation count settings against voltage-defined lesion-effect reference region. This case series included six Brugada syndrome patients undergoing epicardial substrate ablation. Mapping was performed in sinus rhythm using EnSite X with an Advisor HD Grid catheter. Automated fractionation maps were generated with the EnSite X CFE-count algorithm using refractory settings of 14 and 20 ms. Abnormal points were defined as fractionation count ≥3; lesion-effect reference region was defined by voltage change (pre >0.5 mV, post <0.3 mV). Island overlap was quantified using DBSCAN clustering and mesh-based area estimation. IOF revealed a consistent coverage-parsimony trade-off between refractory settings. The 14-ms setting produced broader islands with higher reference-region coverage (median overlap 84.6% [IQR 80.8-93.7]) but larger extraneous mapped area (up to 72.9 cm2). The 20-ms setting produced more compact islands with markedly reduced extraneous area (0.0-34.8 cm2) but lower coverage (59.9% [IQR 37.2-74.7]). In parallel, fractionation burden within the ablated region decreased substantially after ablation in both settings (median total burden reduction 86% vs 93%), supporting lesion-effect concordance of automated fractionation metrics. IOF provides an island-based framework for interpreting automated fractionation mapping and describing its spatial concordance with lesion-effect regions. In this retrospective series, 14 ms favored broader coverage, whereas 20 ms favored greater parsimony.

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2026-07-17 | Late INa as a Therapeutic Target: New Strategies, Computational Modelling, Drug Development, and Clinical Translation.

The Nav1.5 channel, a major isoform of voltage-gated sodium ion channel, is mainly found in ventricular cardiomyocytes, playing a key role in generating essential cardiac action potentials for normal heart rhythms. Mutations in Nav1.5 have been associated with severe heart conditions such as long QT syndrome, Brugada syndrome, cardiac conduction disorders, atrial fibrillation, and dilated cardiomyopathy. Recent research has linked Nav1.5 to cardiac fibrosis and proposed its role in non-cardiac illnesses, including specific neurological disorders and cancers, subjects that will be reviewed in this paper. On the other hand, sodium-glucose cotransporter 2 inhibitors (SGLT2i), initially designed to manage diabetes by facilitating glucose excretion through urine, have demonstrated unexpected and encouraging cardioprotective benefits in clinical trials. This review compares the important SGLT2 inhibitors empagliflozin, dapagliflozin, and canagliflozin in terms of their interactions with Nav1.5 and their therapeutic effects on the heart. We also investigate new medications and compounds being developed to regulate Nav1.5 function, providing a preview of potential future treatments. Past attempts to develop late INa inhibitors and difficulties in transitioning from the research phase to clinical trials have raised doubts about the optimal design of such trials. In addition, we cover the applications of molecular dynamics simulations in understanding the mechanism of action of these drugs within the Nav1.5 channel computationally. We hope that this review identifies new opportunities to generate more effective inhibitors using novel methods and advanced multiscale molecular modelling techniques.

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gene therapies

2026-03-09 | Characterization of the R893C NaV1.5 mutation in Brugada syndrome.

Brugada syndrome (BrS) is a genetically determined cardiac arrhythmogenic syndrome with increased risk of sudden cardiac death. BrS is mostly caused by mutations in SCN5A gene encoding the primary ɑ-subunit of the cardiac sodium channel NaV1.5. We aimed at characterizing the functional alterations caused by the R893C mutation, identified in a proband diagnosed with BrS, and establishing whether the mutation is associated with BrS. Although several mutations have been reported in the close vicinity of R893, the functional role of this region remains unknown and, in addition, exploring SCN5A mutations in patients with inherited arrhythmogenic syndromes is critical for understanding the pathogenesis of arrhythmias. The mutations were introduced by site-directed mutagenesis. The variants were transiently expressed in CHO cells and potassium currents were measured using the whole-cell patch clamp technique. Patch clamp recordings have demonstrated that R893C almost completely abolished the sodium current, INa, though the mutation did not exert dominant-negative effect on wild-type NaV1.5 channels. We also observed significant decrease in channel activation and a depolarized shift of steady-state inactivation curve, however, the kinetics of inactivation and recovery from fast inactivation were not changed by the mutation. Moreover, the reducing agent Dithiotreitol partially restored the normal function of NaV1.5 in the R893C mutant highlighting a likely mechanism for loss of conduction via formation of disulphide bridges. We showed that R893H channels also failed to produce any detectable INa that confirms the importance of the highly conserved R893 in gating. Our study reveals R893C is a loss-of-function mutation with altered electrophysiological characteristics of NaV1.5. Thus, R893C may contribute to the BrS phenotype of the proband. Our findings may facilitate the understanding of the mechanisms of arrhythmogenesis in BrS, as it helps to identify mutational hotspots in BrS. Moreover, our work may improve novel gene therapy and new therapeutic drug design targeting NaV1.5 channelopathies.

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2026-01-23 | Brugada Syndrome: New Implications for Heterozygous Carriers of the Pathogenic SCN5A c.689T>C(p.Ile230Thr) Variant.

Pathogenic variants in the SCN5A gene and its subunits have been identified in individuals with Brugada Syndrome. One such SCN5A variant, c.689T>C(p.Ile230Thr), was previously reported as disease-causing only in homozygous individuals, with heterozygous carriers being unaffected. Presently, we discuss the case of an adolescent patient who presented for further evaluation of pre-syncopal episodes with ECG findings consistent with Brugada pattern who on further genetic evaluation was found to be a heterozygous carrier of the pathogenic SCN5A c.689T>C(p.Ile230Thr) variant. This unique case allows us to think differently about heterozygous carriers for this specific mutation, and while the risk for developing a life-threatening arrhythmia may be low, heterozygous carriers may benefit from clinical monitoring to reduce the potential for adverse cardiac outcomes. A 17-year-old male presented after a pre-syncopal episode whose ECG demonstrated sinus bradycardia and type 1 Brugada pattern. Genetic testing revealed this patient to be a heterozygous carrier of a pathogenic SCN5A variant (c.689T>C(p.Ile230Thr)) which was also found in his father and brother, neither of whom had symptoms but did have ECG changes. He was diagnosed with Brugada Syndrome and advised to avoid known triggers. This case highlights the potential risk of severe cardiac arrhythmias in heterozygous carriers of the SCN5A c.689T>C (p.Ile230Thr) variant, previously thought to be benign. The 17-year-old patient, along with his asymptomatic father and brother who also carried the variant, exhibited ECG changes consistent with Brugada pattern. This finding suggests that heterozygous carriers may require closer monitoring and early intervention to prevent future life-threatening cardiac events.

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2025-10-30 | Gene Therapy for Inherited Cardiac Arrhythmias.

Inherited cardiac arrhythmias are genetic disorders that impact the heart's electrical circuitry. These disorders are caused by inherited mutations and markedly increase the mortality risk of affected individuals. Conventional treatments, such as pharmacological agents for rate and rhythm control or surgical interventions, are often not efficacious and cause long-term complications. Gene therapy has recently emerged as a feasible and potentially curative approach for treating inherited cardiac arrhythmia syndromes. Advancements in elucidating the molecular mechanisms, pinpointing essential candidate genes, and improving gene delivery have rendered gene therapy as a feasible treatment alternative. This review examines recent studies on gene therapy applications in Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, long QT syndrome, familial atrial fibrillation, and arrhythmogenic cardiomyopathy. Additionally, we analyze current advancements in viral and non-viral gene delivery methods, highlighting their therapeutic potential and related challenges.

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2025-10-22 | CRISPR/Cas9-Based Gene Editing for Correcting Inherited Channelopathies.

Inherited cardiac channelopathies, including long QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia, are major causes of arrhythmic morbidity and sudden cardiac death in young individuals. Current therapies, such as pharmacologic agents, implantable cardioverter-defibrillators, and lifestyle modifications, reduce risk but fail to correct the underlying genetic substrate, creating an urgent need for curative strategies. CRISPR/Cas9 genome editing has emerged as a transformative platform with the potential to directly repair pathogenic variants. Recent advances in base and prime editing, together with novel viral and nonviral delivery platforms, have enabled precise correction of disease-causing mutations in preclinical models. Proof-of-concept studies using animal models and patient-derived iPSC-cardiomyocytes demonstrate restoration of electrophysiologic stability, suppression of arrhythmias, and durable functional benefit. Nevertheless, translational challenges remain, including off-target effects, delivery barriers, immune responses, scalability, and ethical considerations. Ongoing innovations-such as engineered nucleases, improved delivery vectors, immunogenicity mitigation strategies, and integration of artificial intelligence for personalized guide design-are expected to accelerate clinical translation. This review synthesizes current knowledge on CRISPR-based strategies for inherited channelopathies, highlighting both the promise and limitations of gene editing as a path toward durable, disease-modifying therapies capable of preventing sudden cardiac death.

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2025-08-26 | Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications.

Cardiac arrhythmias remain a major source of morbidity and mortality, often stemming from molecular and structural abnormalities that are insufficiently addressed by current pharmacologic and interventional therapies. Gene therapy has emerged as a transformative approach, offering precise and durable interventions that directly target the arrhythmogenic substrate. Across the spectrum of inherited and acquired arrhythmias-including long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, atrial fibrillation, and post-infarction ventricular tachycardia-gene-based strategies such as allele-specific silencing, gene replacement, CRISPR-mediated editing, and suppression-and-replacement constructs are showing growing translational potential. Advances in delivery platforms, including cardiotropic viral vectors, lipid nanoparticle-encapsulated mRNA, and non-viral reprogramming tools, have further enhanced the specificity and safety of these approaches. Additionally, innovative applications such as biological pacemaker development and mutation-agnostic therapies underscore the versatility of genetic modulation. Nonetheless, significant challenges remain, including vector tropism, immune responses, payload limitations, and the translational gap between preclinical models and human electrophysiology. Integration of patient-derived cardiomyocytes, computational simulations, and large-animal studies is expected to accelerate clinical translation. This review provides a comprehensive synthesis of the mechanistic rationale, therapeutic strategies, delivery platforms, and translational frontiers of gene therapy for cardiac arrhythmias.

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proteins

2026-09-09 | Molecular basis of dual-site engagement and selectivity of CcoTx3-synth for hNaV1.5.

Voltage-gated sodium channels (NaVs) are critical membrane proteins in excitable cells, with NaV1.5 playing a pivotal role in cardiac electrophysiology. Mutations of human NaV1.5 (hNaV1.5) are linked to severe cardiac channelopathies, including atrial fibrillation and Brugada syndrome, making it a prime therapeutic target. Natural peptide toxins from venomous animals, such as tarantulas, offer valuable molecular tools for exploring NaV function. Among these, CcoTx3, a toxin from the straight-horned baboon tarantula, selectively inhibits hNaV1.5/β1 (IC50 = 447 nM), while other NaV subtypes are unaffected (hNaV1.1/β1-1.4/β1) or only partially blocked at higher concentrations (hNaV1.8/β1). Despite its pharmacological potential, the structural basis of CcoTx3 interaction with hNaV1.5 remains unresolved, particularly for the resting-state voltage-sensing domains II (VSDII) and IV (VSDIV), for which no experimental structure exists. This study combined molecular modeling and site-directed mutagenesis to elucidate the binding mechanism of the synthetic CcoTx3-hNaV1.5. Models of CcoTx3 bound to VSDII and VSDIV were generated, and molecular interactions were analyzed. Key binding residues were validated through site-directed mutagenesis at neurotoxin sites 3 and 4, followed by two-electrode voltage clamp assays. S743, E746, E747, R800, and S802 were identified as critical hotspots for inhibition of channel activation in VSDII, while D1610 and K1614 were key for fast inactivation inhibition. Synthetic CcoTx3 toxin (CcoTx3-synth) also interacted with zwitterionic and negatively charged liposomes, supporting a reduction-of-dimensionality mechanism. These findings provide the first structural and functional insights into the selective multisite engagement of CcoTx3-synth with hNaV1.5 and may guide the design of peptide therapeutics targeting cardiac channelopathies.

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2022-06-30 | Trafficking and Gating Cooperation Between Deficient Nav1.5-mutant Channels to Rescue INa

Pathogenic variants in SCN5A, the gene encoding the cardiac Na+ channel α-subunit Nav1.5, result in life-threatening arrhythmias, e.g., Brugada syndrome, cardiac conduction defects and long QT syndrome. This variety of phenotypes is underlied by the fact that each Nav1.5 mutation has unique consequences on the channel trafficking and gating capabilities. Recently, we established that sodium channel α-subunits Nav1.5, Nav1.1 and Nav1.2 could dimerize, thus, explaining the potency of some Nav1.5 pathogenic variants to exert dominant-negative effect on WT channels, either by trafficking deficiency or coupled gating.The present study sought to examine whether Nav1.5 channels can cooperate, or transcomplement each other, to rescue the Na+ current (INa). Such a mechanism could contribute to explain the genotype-phenotype discordance often observed in family members carrying Na+-channel pathogenic variants.Patch-clamp and immunocytochemistry analysis were used to investigate biophysical properties and cellular localization in HEK293 cells and rat neonatal cardiomyocytes transfected respectively with WT and 3 mutant channels chosen for their particular trafficking and/or gating properties.As previously reported, the mutant channels G1743R and R878C expressed alone in HEK293 cells both abolished INa, G1743R through a trafficking deficiency and R878C through a gating deficiency. Here, we showed that coexpression of both G1743R and R878C nonfunctioning channels resulted in a partial rescue of INa, demonstrating a cooperative trafficking of Nav1.5 α-subunits. Surprisingly, we also showed a cooperation mechanism whereby the R878C gating-deficient channel was able to rescue the slowed inactivation kinetics of the C-terminal truncated R1860X (ΔCter) variant, suggesting coupled gating.Altogether, our results add to the evidence that Nav channels are able to interact and regulate each other's trafficking and gating, a feature that likely contributes to explain the genotype-phenotype discordance often observed between members of a kindred carrying a Na+-channel pathogenic variant.

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2022-03-24 | 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.

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2021-06-09 | Commentary: Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the CACNA1 Gene Associated With Brugada Syndrome

GENERAL COMMENTARY article Front. Physiol., 09 June 2021Sec. Cardiac Electrophysiology https://doi.org/10.3389/fphys.2021.682567

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2021-01-08 | Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the CACNA1 Gene Associated With Brugada Syndrome

Brugada syndrome (BrS) is an inherited arrhythmogenic disease that may lead to sudden cardiac death in young adults with structurally normal hearts. No pharmacological therapy is available for BrS patients. This situation highlights the urgent need to overcome current difficulties by developing novel groundbreaking curative strategies. BrS has been associated with mutations in 18 different genes of which loss-of-function (LoF) CACNA1C mutations constitute the second most common cause. Here we tested the hypothesis that BrS associated with mutations in the CACNA1C gene encoding the L-type calcium channel (LTCC) pore-forming unit (Ca v α1.2) is functionally reverted by administration of a mimetic peptide (MP), which through binding to the LTCC chaperone beta subunit (Ca v β2) restores the physiological life cycle of aberrant LTCCs. Two novel Ca v α1.2 mutations associated with BrS were identified in young individuals. Transient transfection in heterologous and cardiac cells showed LoF phenotypes with reduced Ca 2+ current (I Ca ). In HEK293 cells overexpressing the two novel Ca v α1.2 mutations, Western blot analysis and cell surface biotinylation assays revealed reduced Ca v α1.2 protein levels at the plasma membrane for both mutants. Nano-BRET, Nano-Luciferase assays, and confocal microscopy analyses showed (i) reduced affinity of Ca v α1.2 for its Ca v β2 chaperone, (ii) shortened Ca v α1.2 half-life in the membrane, and (iii) impaired subcellular localization. Treatment of Ca v α1.2 mutant-transfected cells with a cell permeant MP restored channel trafficking and physiologic channel half-life, thereby resulting in I Ca similar to wild type. These results represent the first step towards the development of a gene-specific treatment for BrS due to defective trafficking of mutant LTCC.

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oligonucleotides

2025-10-20 | Characterization of a Splice-Altering Variant in SCN5A Associated With Brugada Syndrome　- Insights Into Splice Error Correction.

Brugada syndrome (BrS) is an arrhythmic disease associated with SCN5A loss-of-function variants. We identified a novel single nucleotide substitution, SCN5A c.1338G>A, in the last codon of exon10 in a patient with drug-induced BrS. The aim of this study was to investigate the impact of this splice-altering variant and examine whether antisense oligonucleotides (ASOs) could correct the splice alteration. Genomic DNA was extracted from the patient's blood lymphocytes. Coding exons of inherited arrhythmia genes were screened and SCN5A c.1338G>A was identified. SpliceAI predicted its prominent potential to alter splicing among 168 single nucleotide variants in the SCN5A region including 10 variants with allele frequency (AF) <0.01, and the usage of a cryptic splice donor site 4 bp downstream from the authentic splice donor site. Minigene splicing reporter assays were performed using HEK-293 cells and induced pluripotent stem cells-cardiomyocytes, and successfully demonstrated a dominant selection of the predicted splice site. Three different ASOs were tested in the same platform. Although the ASOs reduced the production of splice error products, they did not succeed in increasing authentically spliced products. We confirmed a splice site alteration by SCN5A c.1338G>A and propose extended use of SpliceAI for screening a target genomic region. The attempts to correct mis-splicing near the canonical splice site were not entirely successful, so further development of technology is awaited.

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2024-01-11 | The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

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

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2023-11-01 | The role of MAPRE2 and microtubules in Brugada syndrome pathogenesis

Abstract Background Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), which are found in only ∼20% of patients. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), implicating microtubule (MT) involvement in BrS. Purpose To understand the role of MAPRE2 and MT in BrS pathogenesis. Methods A mapre2 knock-out (KO) zebrafish model was generated using CRISPR/Cas9 and validated by western blot. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry with confocal imaging. Morpholino targeting mapre2 or ttl (encoding tubulin tyrosine ligase) were injected into zebrafish embryos at the one-cell stage. A transgenic zebrafish line with cdh2 (encoding N-cadherin) tandem fluorescent timer (tFT) was used to study adherens junction. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes (CM) with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Results Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p&lt;0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p&lt;0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p&lt;0.0001). Confocal imaging of KO hearts showed disorganized adherens junction based on N-cadherin staining. Similarly, morpholino KD of mapre2 in the cdh2 tFT line showed ∼40% mislocalization of mature N-cadherin relative to ZO-1 staining in larval ventricles (8 KDs vs 9 control hearts; p&lt;0.05), suggesting disruption of adherens junction. In terms of MT, immunohistochemistry showed a 23% decrease of detyrosinated tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p&lt;0.01). MAPRE2 KD in iPSC-CM (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 µm/min; p&lt;0.001), distance (5.8±0.2 vs. 7.1±0.2 µm; p&lt;0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p&lt;0.001), suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of detyrosinated tubulin (46% increase; 16 controls vs. 16 KDs; p&lt;0.01) as well as Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p&lt;0.01) to WT levels. Conclusion Genetic ablation of mapre2 led to a decrease in cardiac NaV function, a hallmark of BrS. This is associated with disruption of adherens junction, a decrease of detyrosinated tubulin as a marker of MT stability, and changes in MT dynamics. Restoration of detyrosinated tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis by disruption of adherens junction via changes in MT dynamics.

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2016-09-28 | Embryonic type Na+ channel β-subunit, SCN3B masks the disease phenotype of Brugada syndrome

SCN5A is abundant in heart and has a major role in INa. Loss-of-function mutation in SCN5A results in Brugada syndrome (BrS), which causes sudden death in adults. It remains unclear why disease phenotype does not manifest in the young even though mutated SCN5A is expressed in the young. The aim of the present study is to elucidate the timing of the disease manifestation in BrS. A gain-of-function mutation in SCN5A also results in Long QT syndrome type 3 (LQTS3), leading to sudden death in the young. Induced pluripotent stem cells (iPSCs) were generated from a patient with a mixed phenotype of LQTS3 and BrS with the E1784K SCN5A mutation. Here we show that electrophysiological analysis revealed that LQTS3/BrS iPSC-derived cardiomyocytes recapitulate the phenotype of LQTS3 but not BrS. Each β-subunit of the sodium channel is differentially expressed in embryonic and adult hearts. SCN3B is highly expressed in embryonic hearts and iPSC-derived cardiomyocytes. A heterologous expression system revealed that INa of mutated SCN5A is decreased and SCN3B augmented INa of mutated SCN5A. Knockdown of SCN3B in LQTS3/BrS iPSC-derived cardiomyocytes successfully unmasked the phenotype of BrS. Isogenic control of LQTS3/BrS (corrected-LQTS3/BrS) iPSC-derived cardiomyocytes gained the normal electrophysiological properties.

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2015-10-01 | Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias.

Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human. Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations. IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.

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2026-04-22 | Brugada phenocopy induced by Russell's viper envenomation: resolution following anti-snake venom therapy.

Russell's viper envenomation is known to cause complications such as coagulopathy, acute kidney injury and haemolysis. Cardiac involvement, though rare, has been reported in the form of arrhythmias, myocardial infarction and ECG changes such as ST-segment elevation or T-wave inversions. We report a case of a middle-aged farmer who presented to our centre with Russell's viper envenomation, characterised by local envenomation, coagulopathy, haemolysis and ocular manifestations. His ECG showed a Brugada pattern, with no evidence of any structural cardiac abnormalities or elevated cardiac enzymes. A repeat ECG performed 72 hours after anti-snake venom therapy showed resolution of the ECG findings. A diagnosis of Brugada phenocopy secondary to Russell's viper envenomation was made. The most likely mechanism causing a Brugada phenocopy in Russell's viper envenomation may be due to phospholipase A2 present in the venom, causing alteration in the cardiac voltage-gated sodium channels.

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2026-02-13 | [Cardiac arrhythmias-Cellular mechanisms, consequences and challenges in the diagnostics using human models].

Cardiac arrhythmias significantly contribute to morbidity and mortality in cardiovascular diseases. Hereditary ion channel disorders, including Brugada syndrome, long/short QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT), represent critical entities with elevated risk of malignant ventricular arrhythmias. Although beta-blockers, ion channel modulators and implantable cardioverter defibrillators (ICD) are available and the indications for them are increasingly based on genetic analyses, variants of uncertain significance (VUS) substantially impair the risk stratification. Atrial tachyarrhythmias, particular atrial fibrillation, can cause an often-reversible arrhythmia-induced cardiomyopathy (AIC). Pathophysiologically, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays an important role in atrial and ventricular arrhythmias. It can be activated by various pathological triggers, such as high heart rate, neuroendocrine activation and oxidative stress, the latter often resulting from mechano-energetic uncoupling. The development of effective treatment options for arrhythmia-associated cardiovascular diseases requires the use of human model systems in addition to animal experimental approaches, due to the genetic heterogeneity and systemic comorbidities. Patient-specific cardiac stem cell-models enable the classification of VUS and personalised drug testing. The integration of new methodological approaches into existing animal experimental approaches thus paves the way for functionally validated precision medicine and could fundamentally transform the treatment of both disease patterns.

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2025-06-19 | Safe administration of venetoclax-obinutuzumab in a chronic lymphocytic leukemia patient with Brugada syndrome.

Brugada syndrome (BS) is a genetic condition that predisposes individuals to life-threatening arrhythmias, posing a challenge in the management of chronic lymphocytic leukemia (CLL). While BTK inhibitors have been associated with ventricular arrhythmias, data on the cardiac safety of venetoclax and obinutuzumab in BS patients remain limited. We describe the case of a 60-year-old CLL patient with a baseline type 2 Brugada ECG pattern who was treated with venetoclax plus obinutuzumab due to concerns over BTK inhibitor-associated cardiac risks. During the first obinutuzumab infusion, the patient experienced an infusion-related reaction with hypotension, hypokalemia, and transient conversion to a type 1 Brugada ECG pattern. Supportive measures, including electrolyte correction, led to ECG normalization. Venetoclax was well tolerated, and the patient achieved a complete response with undetectable measurable residual disease. This case underscores the importance of cardiac monitoring in CLL patients with BS and suggests that venetoclax plus obinutuzumab may represent a safe and effective therapeutic alternative when BTK inhibitors are contraindicated.

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2025-01-01 | Phenotypes in Brugada syndrome with different genotypes triggered by fever or inflammation using gene-edited iPSCs

Abstract Background Fever or inflammation state may enhance the Brugada syndrome (BrS) phenotype in some but not all patients. However, the underlying mechanism in human cardiomyocytes has not yet been clarified. Methods Human induced pluripotent stem cell (hiPSC) lines generated from fibroblasts of three BrS patients harboring variants in SCN10A (abbreviated as BrS1) and CACNB2 (abbreviated as BrS2), SCN5A (abbreviated as BrS3) and one healthy donor (abbreviated as WT) and a site-corrected (using CRISPR/Cas9) hiPSC line of each BrS patient (abbreviated as isogenic1, isogenic2 and isogenic3) were used for differentiation into cardiomyocytes (hiPSC-CMs). Western blot, patch clamp and calcium transient analyses were carried out. Results All 3 BrS cell lines showed a significantly reduced peak sodium current (INa) compared with isogenic or WT cells at baseline. Hyperthermia challenge (40 °C) significantly decreased INa and enhanced arrhythmogeneity in BrS1 and BrS3 but not in BrS2 cells. The hyperthermia effects involved PKA reduction. The lipopolysaccharide (LPS) challenge exacerbated the phenotype in electrophysiological characteristics in all 3 BrS cell lines. ROS-Blocker abolished the LPS effects in all BrS hiPSC-CMs, while an interleukin-6 receptor blocker abolished the proarrhythmic effect of LPS in BrS1 and BrS3 hiPSC-CMs but not in hiPSC-CMs of BrS2. Conclusions Hyperthermia exacerbated the BrS phenotype in hiPSC-CMs carrying SCN10A and SCN5A variants, whereas LPS aggravated the phenotype in all three BrS variants through distinct mechanisms; Hyperthermia and LPS effects on BrS phenotype may be genotype-dependent. Graphical abstract

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2024-11-27 | The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies.

Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype-phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.

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