Brugada Research Articles

The Role of Electrophysiological Study in the Risk Stratification of Brugada Syndrome.

Brugada syndrome (BrS) is a complex arrhythmogenic disease associated with an increased risk of sudden cardiac death (SCD). The role of electrophysiological study (EPS) for risk stratification purposes of asymptomatic BrS patients remains still controversial. This study aims to summarize the existing data about the role of electrophysiological study for arrhythmic risk stratification of BrS patients without a prior history of aborted SCD or fatal arrhythmic event. Two independent investigators (G.B. and G.T.) performed a systematic search in the MedLine database and Cochrane library from their inception until April 2022 without any limitations. The reference lists of the relevant research studies as well as the relevant review studies and meta-analyses were manually searched. Nineteen studies were included in the final analysis. The included studies enrolled 6218 BrS patients (mean age: 46.9 years old, males: 76%) while 4265 (68.6%) patients underwent an EPS. The quantitative synthesis showed that a positive EPS study was significantly associated with arrhythmic events in BrS patients (RR, 1.74 [1.23-2.45]; P = 0.002; I2 = 63%]. By including the studies that provided data on the association of EPS with arrhythmic events during follow-up in patients without a prior history of aborted SCD or fatal arrhythmic event, the association between positive EPS study and future arrhythmic events remained significant (RR, 1.60 [1.08-2.36]; P = 0.02; I2 = 19%). In conclusion, EPS is a useful invasive tool for the risk stratification of BrS patients and can be used to identify the population of BrS patients who may be candidates for primary prevention of SCD with implantable cardioverter-defibrillator (ICD) implantation.

Trigger and Substrate Mapping and Ablation for Ventricular Fibrillation in the Structurally Normal Heart.

Sudden cardiac death (SCD) represents approximately 50% of all cardiovascular mortality in the United States. The majority of SCD occurs in individuals with structural heart disease; however, around 5% of individuals have no identifiable cause on autopsy. This proportion is even higher in those <40 years old, where SCD is particularly devastating. Ventricular fibrillation (VF) is often the terminal rhythm leading to SCD. Catheter ablation for VF has emerged as an effective tool to alter the natural history of this disease among high-risk individuals. Important advances have been made in the identification of several mechanisms involved in the initiation and maintenance of VF. Targeting the triggers of VF as well as the underlying substrate that perpetuates these lethal arrhythmias has the potential to eliminate further episodes. Although important gaps remain in our understanding of VF, catheter ablation has become an important option for individuals with refractory arrhythmias. This review outlines a contemporary approach to the mapping and ablation of VF in the structurally normal heart, specifically focusing on the following major conditions: idiopathic ventricular fibrillation, short-coupled ventricular fibrillation, and the J-wave syndromes-Brugada syndrome and early-repolarization syndrome.

PO-05-083 ICD THERAPY CHARACTERISTICS IN PATIENTS WITH ARRHYTHMOGENIC RIGHT VENTRICULAR CARDIOMYOPATHY, BRUGADA SYNDROME, AND LONG QT SYNDROME

Arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome (BrS) and Long QT syndrome (LQTS) are inherited arrhythmia disorders that frequently result in sudden death in young populations. ARVC often presents with monomorphic VT amenable to antitachycardia pacing (ATP) therapy, whereas BrS and LQTS are traditionally characterized by polymorphic VT or torsade de pointes requiring shock therapy. To assess the rates of ATP and shock therapies in a cohort of patients with ARVC, BrS and LQTS. Patients with ICD indications for ARVC, BrS and LQTS from the ALTITUDE database (Boston Scientific, Natick, MA) between 2011-2021 were identified. ATP and shock-free survival rates were calculated by Kaplan-Meier (KM) method and hazard ratios were analyzed by the Cox proportional hazards model. A total of 103 ARVC, 218 BrS, and 509 LQTS patients were identified. Mean age (46.6 ± 17.4) did not differ between groups while LQT had more female (69.2%) compared to ARVC (34%) and BrS (30.7%). Mean follow-up was 3.7 years. A total of 47 (45.6%) ARVC patients, 39 (17.9%) BrS, and 136 (26.7%) LQTS patients received any therapies during the follow-up period. Episodes in ARVC patients were significantly less likely to result in a shock (71/402, 18%) compared to BrS (73/157, 47%) and LQTS (333/581, 57%) (p<0.001). Per 100 patient-years (Figure 1a), ARVC patients had arrhythmia episodes requiring only ATP therapy 4.7 times the frequency of episodes requiring shock therapy (89.0 versus 19.1), whereas BrS and LQTS patients had comparable rates of ATP only vs shocks (10.2 versus 8.8 in BrS and 13.5 vs 18.2 in LQTS). From KM curve analysis, three-year rate of an ATP-only event was 43.0% in ARVC, 10.3% in BrS and 18.0% in LQTS. As compared with ARVC, risk of ATP only events in BrS (HR 0.20, 95% CI 0.12-0.33, p<0.001) and LQTS (HR 0.44, 95% CI 0.30-0.65, p<.0.001) were significantly lower. Three-year rate of events requiring shock therapy was 33.4% in ARVC, 14.5% in BrS and 20.0% in LQTS. As compared with ARVC, risk of a shock in BrS (HR 0.33, 95% CI 0.20-0.54, p<0.001) and LQTS (HR 0.62, 95% CI 0.41-0.93, p=0.020) were also significantly lower. ARVC patients had more ventricular arrhythmia episodes. When therapy was triggered, episodes in ARVC were less likely to be treated with a shock compared to BrS and LQTS. This may help guide programming parameters and device type selection in high-risk individuals with inherited arrhythmia syndromes.

PO-04-212 BRUGADA EKG CLASSIFICATION WITH SELF-SUPERVISED VICREG PERTAINING: A NOVEL ARTIFICIAL INTELLIGENCE ARCHITECTURE FOR RARE ARRHYTHMIA

Brugada Syndrome (BrS) is a rare inherited arrhythmia syndrome with an estimated prevalence of 2-5 per 10,000. Despite its low prevalence, it is responsible for an estimated 4% to 12% of all sudden deaths and in patients with structurally normal hearts. Because of the resulting limited number of available BrS EKG examples, Convoluted Neural Networks (CNNs), which require a large labeled dataset for training, are limited in their ability to automate the detection of BrS. To develop an Artificial Intelligence (AI) model for classifying BrS in the surface EKG by applying a novel self-supervised pretraining architecture on unrelated, unlabeled EKGs, compensating for the limited number of BrS examples. We pretrained a convolutional neural network on randomly selected 3,500,000 EKGs from adult NYU patients utilizing VICReg (Variance-Invariance-Covariance Regularization), a novel regularization architecture for self-supervised training allowing the model to learn embedding (vectors) for each EKG that are informative and unique (Figure 1). We then fine tuned the original model on a set containing 200 adult NYU patients with a MUSE (GE, USA) diagnosis of BrS not included in the pretraining phase, and evaluated the fine-tuned model for binary classification of Brugada. We used a modification of a multilayered convolutional neural network architecture similar to ConvNext, with a Softmax activation function for classification. Of the 200 EKGs with a MUSE label of BrS, 114 (57%) were confirmed as BrS by an expert Electrophysiologist and blended with a cohort of randomly selected 200 EKGs with non – BrS label. The combined cohort was divided into 80% training (with 20% validation) and 20% test sub-sets. The CNN classified BrS with an AUC of 0.953 (sensitivity 0.87, specificity 0.87), outperforming all previously described supervised learning models trained on larger datasets. We developed a novel deep learning model using VICReg architecture for self-supervised pretraining. We demonstrate the classification of BrS EKGs with high performance achieved with a remarkably small labeled dataset. Supervised pretraining with VICReg may be a useful architecture for improving model classification performance on rare conditions with limited availability of labeled data.

Sinus rate and QRS duration on lead V1 predict major events in Brugada syndrome

Arrhythmic risk stratification remains a major challenge in asymptomatic Brugada patients. Many studies have evaluated risk stratification performance based on a single or a few ECG-derived parameters measured manually. We aimed to assess the predictive value of multi ECG parameters measured automatically in patients with Brugada syndrome. We performed a prospective, multicenter cohort study including Brugada patients with at least one available type 1 ECG (either spontaneously or drug-induced) between January 2005 and November 2021. Paper ECG were first digitized and then automatically analyzed using the Glasgow and Bravo algorithms. Clinical data and Brugada related events at diagnosis and during follow-up were collected. Sudden cardiac death, aborted cardiac arrest and appropriate ICD therapy in the VF zone were considered as major events. The predictive value of clinical and ECG parameters for symptomatic status was assessed using uni- and multivariate Cox models. ECGs from 301 Brugada patients (77% male, mean age at diagnosis 43 years, follow-up 7 ± 5.6 years) were analyzed. Brugada-related symptoms (cardiac syncope or major event) occurred in 23% of patients before diagnosis and 11% during follow-up (incidence: 1.57%/year). After multivariate Cox analysis, only 2 baseline ECG (without Ajmaline) parameters were significantly associated with major events: RR interval (HR = 1.01 [95%CI 1–1.01], P = 0.002) and QRS duration in lead V1 (HR = 1.04 [95%CI 1–1.07], P = 0.038). QRS prolongation in lead V1 > 113 ms (AUROC 0.69 [95%CI 0.58–0.8], Fig. 1) was associated with a 3-fold increased risk of major event [Fig. 2] (HR = 3.49 (95% CI [1.72–7.09], P = 0.001). Many previously described ECG parameter were not associated with major event in Brugada patients after multi-parameter evaluation. Sinus rate and QRS duration on lead V1 were independent risk factor. These results need to be validated on an external cohort. The predictive value of ECG parameters in Brugada syndrome should be assessed using automated multiparametric quantification.

Concise compilation of the most clinically relevant current knowledge on Brugada Syndrome

Brugada syndrome (BrS) is a rare disease in which arrhythmogenic changes occur in patients without any structural heart pathology. Its most dangerous complication is sudden cardiac arrest, which most often occurs around the 3rd and 4th decade of life of patients. In a similar period of time (20 - 40 years of age), the first clinical manifestations of the disease also most often occur, although this can happen even sooner in the case of a malignant form of the disease. Brugada syndrome is significantly (about 8 times) more frequently diagnosed in men, and the main factors that strongly worsen its prognosis are incidents of syncope noted in the patient's history and observed spontaneous changes in ECG studies [1][2].

EN-452414-5 VENTRICULAR CONDUCTION IS A MARKER FOR ARRHYTHMIC RISK IN OVERLAP SODIUM CHANNEL DISEASE

SCN5A-E1784K (c.5350G>A) is the most common variant associated with LQT3 long QT syndrome (LQTS) and Brugada syndrome (BrS). It exhibits incomplete penetrance and can manifest variably as a mixed clinical phenotype of LQTS, BrS and/or conduction disorders. This presents a challenge for risk stratification and optimal management. The objective was to describe the clinical characteristics and risk markers in a large, well characterised international cohort of SCN5A-E1784K subjects and identify parameters that associate with event-free survival. Comprehensive clinical data, including initial presentation and follow-up data, were collected from SCN5A-E1784K subjects. Outcome ‘events’ included sudden death, aborted cardiac arrest, and documented VF, VT or arrhythmic syncope. ‘Lethal events’ included sudden death, aborted cardiac arrest or documented VF or VT. The effect of age at the time of ECG acquisition on ECG parameters was modelled and the residual, i.e. difference between observed value and the predicted age-corrected value, was calculated for each individual and referred to as residual PR, residual QRS, residual QTc and residual RR. Common variants previously associated with QRS duration were genotyped and a polygenic risk score (PRSQRS) derived. Associations between clinical characteristics, PRSQRS and event free survival were investigated using Cox proportional hazard regression models. SCN5A-E1784K subjects (n=231) were recruited from 20 centres. Four (1.7%) had a mixed phenotype of BrS, LQT3 and cardiac conduction disease. A mixed phenotype of BrS and LQT3 without any evidence of conduction disease was seen in 18.1% and a mixed phenotype of BrS and conduction disease, and Long QT and conduction disease, were seen in 3.1% and 12.9% respectively. There was no phenotype in 9.9% (includes n=6 with missing data). Median follow-up was 24.9 years (15.2 - 43.3) and 45 (19%) experienced an event. Lethal events were observed in n=14 (6%). Longer QRS duration (corrected for age) and PRSQRS were both associated with shorter event-free intervals (Table 1 and Figure 1). QTc and BrS phenotype were not associated with risk. Ventricular myocardial conduction, as evidenced by QRS duration and its genomic associations, appears to play a role in the risk of arrhythmic events in patients with SCN5A-E1784K. This provides an important opportunity for the personalisation of risk stratification of these patients, whose management can otherwise be challenging.Tabled 1Hazard ratios estimated in SCN5A-E1784K using Cox regression with adjustment for ethnicityVariableEventsLethal eventsHR [95% CI]p-valueHR [95% CI]p-valueMale1.33 [0.71-2.48]0.3692.22 [0.77-6.44]0.142Residual PR (ms)1.01 [1.00-1.02]0.2261.02 [1.00-1.03]0.012Residual QRS (ms)1.04 [1.02-1.06]<0.001*1.05 [1.02-1.09]0.002*Abnormal QRS-axis1.48 [0.69-3.19]0.3160.89 [0.19-4.10]0.879Residual QTc (ms)1.00 [0.99-1.01]0.9691.00 [0.97-1.02]0.664Long QTc0.87 [0.38-2.00]0.7461.05 [0.28-3.90]0.943Residual RR (s)1.10 [0.18-6.80]0.9159.51 [0.37-242]0.173Spontaneous type 1 Brugada ECG1.60 [0.50-5.11]0.4262.25 [0.50-10]0.290HR: hazard ratio; 95% CI: 95% confidence interval; ∗p-value reaching Bonferroni corrected significance threshold Open table in a new tab

Inhibition of Wnt/β-catenin signaling upregulates Nav 1.5 channels in Brugada syndrome iPSC-derived cardiomyocytes.

The voltage-gated Nav 1.5 channels mediate the fast Na+ current (INa ) in cardiomyocytes initiating action potentials and cardiac contraction. Downregulation of INa , as occurs in Brugada syndrome (BrS), causes ventricular arrhythmias. The present study investigated whether the Wnt/β-catenin signaling regulates Nav 1.5 in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). In healthy male and female iPSC-CMs, activation of Wnt/β-catenin signaling by CHIR-99021 reduced (p < 0.01) both Nav 1.5 protein and SCN5A mRNA. In iPSC-CMs from a BrS patient, both Nav 1.5 protein and peak INa were reduced compared to those in healthy iPSC-CMs. Treatment of BrS iPSC-CMs with Wnt-C59, a small-molecule Wnt inhibitor, led to a 2.1-fold increase in Nav 1.5 protein (p = 0.0005) but surprisingly did not affect SCN5A mRNA (p = 0.146). Similarly, inhibition of Wnt signaling using shRNA-mediated β-catenin knockdown in BrS iPSC-CMs led to a 4.0-fold increase in Nav 1.5, which was associated with a 4.9-fold increase in peak INa but only a 2.1-fold increase in SCN5A mRNA. The upregulation of Nav 1.5 by β-catenin knockdown was verified in iPSC-CMs from a second BrS patient. This study demonstrated that Wnt/β-catenin signaling inhibits Nav 1.5 expression in both male and female human iPSC-CMs, and inhibition of Wnt/β-catenin signaling upregulates Nav 1.5 in BrS iPSC-CMs through both transcriptional and posttranscriptional mechanisms.

PO-02-153 THE ROLE OF MAPRE2 AND MICROTUBULES IN CARDIAC ELECTROPHYSIOLOGY AND BRUGADA SYNDROME

Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), yet these are only found in ∼20% of probands. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), which implicates microtubule (MT) involvement in BrS. To understand the role of MAPRE2 and MT in cardiac electrophysiology and BrS pathogenesis. Using CRISPR/Cas9, a mapre2 knock-out (KO) mutant was generated in zebrafish and validated by western blot. Morpholino targeting ttl (encoding tubulin tyrosine ligase) was injected into zebrafish embryos at the one-cell stage. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. 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<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<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<0.0001). Immunohistochemistry showed a 23% decrease of detyrosinated (glu) tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MT plus-end tracking experiments (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<0.001), distance (5.8±0.2 vs. 7.1±0.2 μm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001) with MAPRE2 KD, suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of glu-tubulin (46% increase; 16 controls vs. 16 ttl KDs; p<0.01) and Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Genetic ablation of mapre2 led to a decrease in NaV function, a hallmark of BrS. This is associated with a decrease in glu-tubulin as a marker of MT stability and changes in MT dynamics. Restoration of glu-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 via a novel paradigm of disrupting MT stability and dynamics.

PO-03-198 A NOVEL ELECTROCARDIOGRAPHIC MODEL FOR DEFINITE DRUG INDUCED BRUGADA SYNDROME.

Clinical, electrocardiographic, and genomic factors associated with the development of the drug induced type 1 Brugada pattern (DI-T1BP), in response to sodium channel blocker provocation (SCBP) have been investigated. These prior analyses have mostly been concerned with the prediction of the DI-T1BP rather than the strength of the diagnosis of Brugada syndrome (BrS). To analyse and compare the ECG response to SCBP with Ajmaline in a cohort of healthy subjects (Healthy Controls) and clinical patients with a DI-T1BP and a Shanghai Score (SS) ≥3.5 (Definite-BrS group). From an existing clinical cohort of consecutive patients investigated at our centre between 2010 - 2022, we identified those with a DI-T1BP and a SS ≥3.5. Respondents to a national advertisement for healthy volunteers completed an online medical questionnaire. Those fulfilling inclusion criteria were invited to undergo further evaluation with eligible subjects undergoing a diagnostic ajmaline challenge. Digital ECG acquisition facilitated the development of automated measurements. A univariable analysis following by a multivariable analysis were undertaken to compare ECG characteristics between the two groups. A logistic regression analysis was performed to identify ECG characteristics that accurately associated with the definite drug induced BrS patient group. Two-hundred-and-forty-eight volunteers completed an online questionnaire, 103 accepted an invitation to undergo further screening and 100 were recruited into the healthy control (HC) group including three with a DI-T1BP. From a cohort of 1241 consecutive patients undergoing ajmaline provocation, 372 had a DI-T1BP and 166 had a DI-T1BP and SS ≥3.5. Of the ECG characteristics investigated, ajmaline induced changes (delta) in precordial QRS area (mv/ms), Anterior STJ point amplitude (μV) and S wave duration in lead II (ms), were independent predictors of definite BrS, Table 1.0. A model incorporating these 3 ECG characteristics was an outstanding discriminator with an area under the curve of 0.937 [95% confidence interval 0.907 – 0.966], P<0.01, Figure 1. This novel predictive model demonstrates excellent discrimination between patients with definite BrS and healthy controls. Further refinement of this model in clinical patients with DI-T1BP and SS <3.5 will identify additional clinical characteristics that can be incorporated into a clinical-ECG diagnostic model.

BS-452757-4 ANTI-ARRHYTHMIC EFFECTS OF ANDROGEN DEPRIVATION THERAPIES IN A SCN5A LOSS-OF-FUNCTION MOUSE MODEL

Sex is a major determinant of the incidence and clinical presentation in arrhythmia syndromes such as Brugada syndrome (BrS). We generated a novel SCN5A-G1408R/+ knock-in mouse model mimicking key features of human BrS regarding male predominance and susceptibility to ventricular tachycardias. This study aimed to explore the effect and molecular mechanisms of androgen deprivation on cardiac arrhythmogenicity in a mouse model of BrS. Animals were studied using surface ECG with and without flecainide challenge (10 mg/kg body weight), telemetric recordings, molecular, biochemical as well as histological analyses. To study the effect of antiandrogenic treatments on the rhythmogenic state and its influence on transcriptional alterations of cardiac ion channel genes, male G1408R/+ mice were either chronically treated with the androgen receptor (AR) blocker flutamide by subcutaneously implanted osmotic pumps or orchiectomized. Sex-dependent analysis showed that 81% (25/31 animals) of male G1408R/+ and only 14% (2/14 animals) of their female littermates developed flecainide-induced polymorphic ventricular tachycardias (VT). Only male G1408R/+ mice, previously 100% inducible for VT underwent androgen deprivation (flutamide for 4 weeks vs. orchiectomy). The arrhythmogenic phenotype was rescued in both groups of antiandrogenic therapy. VT incidence in the flutamide group was reduced to 13% (2/16), while VT in the orchiectomy group declined to 33% (3/9). At baseline, (untreated) male G1408R/+ mice exhibited reduced cardiac expression of NaV1.5 protein compared to male wild type (wt) mice, particularly in the right ventricular myocardium. Treatment with flutamide for 4 weeks resulted in increased NaV1.5 expression in male G1408R/+ mice to levels similar to male wt mice. Interestingly, orchiectomized G1408R/+ mice revealed levels of NaV1.5 protein even higher compared to wt mice, which were particularly abundant in the right ventricular free wall. We observed a significant anti-arrhythmic effect induced by androgen deprivation in the G1408R/+ mouse model mimicking human BrS. Our data indicate that the anti-arrhythmic effect is potentially driven by increasing right ventricular NaV1.5 membrane expression as a result of anti-androgenic treatment.

PO-04-164 MOLECULAR MECHANISMS OF BRUGADA SYNDROME RELATED TO P.R211H MUTATION IN RRAD GENE

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of sudden cardiac death. More than 20% of BrS cases have been linked to mutations in SCN5A gene, which encodes the voltage-gated Na+ channel Nav1.5. We have previously identified a novel variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) monomeric GTPase. We have shown that p.R211H RRAD variant induces a reduction of the Na+ current (INa) amplitude and morphological defects in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). To characterize the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent (p.R210H) mutation and identify the mechanisms linking RRAD mutation to INa decrease and conduction defects. Mouse phenotype was determined using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and iPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type or p.R211H-Rad. Homozygous KI mice (12 weeks old) showed longer QRS complexes (12.3±0.2 ms; n=22) than wildtype (WT; 11.1±0.2 ms; n=13; P<0.01) and heterozygous KI (11.7±0.2 ms; n=22) mice, a defect persisting in older mice. Homozygous KI mice showed a 38% decrease in INa density versus WT mice (P<0.01; n=22 & 19). This was associated with a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01) and Connexin 43 (Cx43; by 34%; P = 0.06) in homozygous KI mice versus WT mice. Rad expression was also decreased by 33% (P < 0.05). Similar results were obtained in the BrS index case hiPS-CMs, in which an altered location of Rad (more nuclear than cytoplasmic) and Cx43 (lower clusters, but of larger size) was also observed, compared to control hiPS-CMs. Coimmunoprecipitation studies on left ventricular tissue of 30-week-old WT mice showed an interaction of Rad with Nav1.5, and of Rad with Cx43. Nav1.5 interaction with Rad has been confirmed in HEK cells, which are used to determine the effect of the mutation on this interaction. To conclude, BrS-related RRAD p.R211H mutation affects Rad expression and leads to a decrease of Nav1.5 and Cx43 cardiac expression, explaining both sodium current lower amplitude and conduction defects.

Functional identification of hot-spot mutations in cardiac calcium channel genes associated with the J wave syndromes.

J wave syndrome (JWS) is an inherited cardiac channelopathy associated with malignant ventricular arrhythmias and sudden cardiac death (SCD), which comprises early repolarization syndrome and Brugada syndrome. Here, we explore the association between variants in the L-type calcium channel gene subunits, α1C (CACNA1C) and β2b (CACNB2b), and the JWS phenotype. Using next-generation genetic sequencing of 402 JWS probands and their family members, we identified a CACNA1C-G37R (p.Gly37Arg) mutation in five individuals in four families, two of which had a family history of SCD as well as a CACNB2b-S143F (p.Ser143Phe) mutation in seven individuals in three families, two of which had a family history of SCD. The variants were located in exon 2 in CACNA1C and exon 5 in CACNB2b; both were in highly conserved amino acid residues. Whole-cell patch-clamp results showed that compared with the wild-type group, calcium current density of CACNB2b-S143F and CACNA1C-G37R were significantly lower displaying a dominant-negative effect. Our findings provide further support for the hypothesis that variants in CACNA1C and CACNB2b are associated with JWS. The results suggest that mutations in these two genes lead to loss-of-function of the cardiac calcium channel current warranting their inclusion in genetic screening protocols. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

SCN5A variants as genetic arrhythmias triggers for familial bileaflet mitral valve prolapse

Mitral valve prolapse (MVP) is a common valvulopathy with variable outcomes. Several studies reported MVP as an underestimated cause of life-threatening arrhythmias and sudden cardiac death (SCD), mostly in young adult women. The association of MVP with ventricular arrhythmias as a substrate of SCD has been documented in patients with MVP. Genetic variations in DCHS1, DZIP1, FLNA and PLD1 genes have been associated with MVP. The index case, a 42-year-old woman, was diagnosed with a bileaflet myxomatous MVP after a resuscitated SCD at the age of 27 years. MVP was the only abnormality found to explain the occurrence of life-threatening ventricular arrhythmia. Structural cardiac diseases and Brugada syndrome were excluded. Family screening revealed one symptomatic daughter with a history of two syncopes at 11 years old during physical effort. Her echocardiography showed a bileaflet MVP and a mildly dilated left ventricle. The MVP is complicated by moderate mitral regurgitation (MR). The oldest sister, 23 years old is currently asymptomatic. Her echocardiography revealed a prodromal MVP with a mild holosystolic MR. The father had normal echocardiography. The aim of this study was to identify the likely causative genetic etiology of the familial MVP and particularly the arrhythmias substrate linked to this phenotype. Whole exome sequencing (WES) was performed for the family. WES data were analyzed with a primary focus on genes related to MVP but also on genes implicated in valvular and aortic defects. Using family-based exome sequencing, no variants were found in known MVP genes. However, we identified two missense variants in the SCN5A gene. A rare variant SCN5A: p.Ala572Asp and the functional modifying SCN5A:p.His558Arg polymorphism. Both variants are shared between the mother and her daughter with a history of resuscitated SCD and syncopes, respectively. The second daughter with prodromal MVP as well as her healthy father carried only the SCN5A:p.His558Arg polymorphism. Our study is highly suggestive of the contribution of SCN5A mutations as a potential genetic cause of the electric instability leading to ventricular arrhythmias in familial MVP cases with syncope and/or SCD history.

PO-02-232 CARDIONEURAL ABLATION IN THE MANAGEMENT OF LONG QT SYNDROME, TYPE 7

Vasovagal syncope is a complex interaction of neurologic and cardiovascular mechanisms resulting in cardiac inhibition by the parasympathetic nervous system via the vagus nerve and associated ganglionic plexi. Some types of tachycardia may be induced by bradycardia, LQT type 3 being the most common example. In these patients, a pacemaker is sometimes indicated to avoid the bradycardia trigger for tachycardia. Cardioneural ablation (CNA) is an emerging therapy that offers the potential of abolishing vagal-induced bradycardia. It is most commonly used to treat vasovagal syncope but it may also provide benefit to patients with bradycardia-induced tachyarrhythmias. We present a case of a patient with Long QT type 7 with vasovagal syncope who had an asystolic pause which induced a run of nonsustained VT. Cardioneural ablation was utilized to avoid bradycardia, with the intention of avoiding future triggering of VT. An 8-year-old female with a past-medical history of Andersen-Tawil Syndrome (Long QT Syndrome type 7). She had two syncopal events which suggested a vagal etiology. After loop monitor placement, she had a third syncopal event, which recorded a period of sinus bradycardia, culminating in a 6.3 second asystolic pause, followed by a 6 beat run of VT, with cycle length of 200-260 msec. Cardioneural ablation was considered as a means of avoiding future bradycardic events. Procedure was performed under general anesthesia. Electroanatomic mapping (Ensite Precision) was used to create 3D cardiac geometry. The same system, coupled with transthoracic ultrasound, was also used to guide transseptal puncture. Sinus bradycardia could be induced at baseline with vagal stimulation. Areas of fractionated signals were mapped to the high atrial septum between the right upper pulmonary vein and the fossa ovalis; and also in the roof of the coronary sinus. Patient was known to have a persistent left SVC to the coronary sinus. These areas were targeted for ablation. Post ablation, she had no bradycardia inducible with vagal stimulation. She tolerated the procedure well, without complication. CNA shows potential promise in the treatment of vasovagal syncope. However, there may be other clinical settings in which it finds utility. Conditions which might benefit include Andersen-Tawil syndrome, Long QT type 3, Brugada syndrome, and possibly Congenital Central Hypoventilation syndrome. Further investigation is needed to elucidate the safety and efficacy of CNA in these settings.

ST-segment slope change in V1 predict major events in Brugada syndrome

Arrhythmic risk stratification remains a major challenge in asymptomatic Brugada patients. Many studies have evaluated risk stratification performance based on a single or a few ECG-derived parameters measured manually on baseline and/or after drug challenge ECGs. We aimed to assess the predictive value of ECG changes induced by pharmacological challenge in patients with drug induced type 1 Brugada pattern. We performed a prospective, multicenter cohort study including Brugada patients with one available drug-induced type 1 ECG and one baseline ECG between January 2005 and November 2021. Paper ECG were first digitized and then automatically analyzed using the Glasgow and Bravo algorithms. A non-normalized ECG change (ΔECG) was calculated between the parameters of the drug-induced and baseline ECG. Clinical data and Brugada related events at diagnosis and during follow-up were collected. Sudden cardiac death, aborted cardiac arrest and appropriate ICD therapy in the VF zone were considered as major events. The predictive value of clinical and ECG parameters for symptomatic status was assessed using uni- and multivariate Cox models. Off- and on-drug ECGs from 164 Brugada patients (72% male, mean age at diagnosis 43 years, follow-up 5.9 ± 4.9 years) were analyzed. Brugada-related symptoms (cardiac syncope or major event) occurred in 26% of patients before diagnosis and 6% during follow-up (incidence: 1.57%/year). After multivariate Cox analysis, 2 ΔECG parameters were also significantly associated with major events: ΔTpeak-Tend V1 (HR = 0.99, CI 95% [0.97–1], P = 0.017) and ΔST-segment Slope V1 (HR = 1, CI 95% [1–1.01], P = 0.014). ST-segment slope change in V1 > 219.5 μV/ms (AUROC 0.75 (95% CI [0.76–0.8]), Fig. 1) was associated with an increased risk of major event [Fig. 2] (HR = 11.59 (CI 95% [2.45–54.86]), P = 0.002). The magnitude of drug-induced ECG changes is associated with major event in Brugada patients. These results need to be validated on an external cohort but our results support using automated multiparametric ECG quantification for risk stratification in Brugada patients.

Molecular mechanisms of Brugada syndrome related to p.R211H variant in RRAD gene

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of sudden cardiac death. More than 20% of BrS cases have been linked to mutations in SCN5A gene, which encodes the voltage-gated Na+ channel Nav1.5. We have previously identified a variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) monomeric GTPase. RRAD p.R211H variant induces a reduction of the Na+ current (INa) amplitude and morphological defects in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). To determine the mechanisms linking RRAD variant to INa reduction and the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent p.R210H variant. Mouse phenotyping was performed using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and hiPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type (WT) or p.R211H-Rad. Twelve-week-old homozygous KI mice showed longer QRS complexes (12.3 ± 0.2 ms; n = 22) than WT (11.1 ± 0.2 ms; n = 13; P < 0.01) and heterozygous KI (11.7 ± 0.2 ms; n = 22) mice, a defect persisting in older mice. In homozygous KI mice INa density was decreased by 38% compared to WT mice (P < 0.01; n = 22 & 19). This was linked to a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01), Connexin 43 (Cx43; by 34%; P = 0.06) and Rad (by 33%; P < 0.01) in homozygous KI mice versus WT mice. Similar results were obtained in the hiPS-CMs of the BrS index case, in which an altered location of Rad (more nuclear than cytoplasmic) and Cx43 (lower clusters, but of larger size) was also observed, compared to control hiPS-CMs. Coimmunoprecipitation studies on left ventricular tissue of 30-week-old WT mice showed an interaction of Rad with Nav1.5, a result that was confirmed in HEKs, and of Rad with Cx43. Our results suggest that BrS-related RRAD p.R211H variant affects Rad expression and leads to a decrease of Nav1.5 and Cx43 expression, explaining both sodium current lower amplitude and conduction defects. Our results also show that Rad interacts with Nav1.5, and possibly Cx43.

Sodium channel protein alpha subunit genomics: building an integrated knowledgebase to translate between family members

The sodium channel alpha proteins consist of ten homologous human proteins (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, SCN11A), many involved in various human physiological pathways and clinical disorders/syndromes. Many human variants connected to these genes remain uncertain for human disease. To better support the understanding of challenging human variants, we have built an ortholog database of the alpha subunit proteins for the sodium channel. We combined 1,680 vertebrate species sequences of family members, protein modeling, molecular dynamic simulation of lipid embedded protein, screening of human variants (gnomAD, ClinVar, Geno2MP), and analysis of a large number of human RNAseq and single-cell RNA-seq to understand the function of each amino acid in the human proteins and how the amino acids are conserved across family members to contribute to sodium channel functions within diverse cells of human. There are 610 amino acids conserved across the alpha subunit family, representing the most conserved amino acids for each member. These amino acids drive the structural fold, membrane interactions, and channel opening. A cluster of amino acids is conserved between a subset of family members that defines subclusters of alpha subunit proteins. Expression analysis of each member reveals clusters of members co-expressed in various cells, where heart and brain datasets show the specificity of cell types that contribute to genotype-to-phenotype associations of each member. Further, to support variant characterizations, the ortholog alignment data provides a unique ability to discover loss vs. gain of function variants found between members, allowing for known pathogenic variants of one member to aid in the characterization of another member's uncertain variants. For example, a clinical variant of SCN8A linked to seizures was supported as loss-of-function through variants within SCN1A (epilepsy), SCN2A (seizures), SCN4A (Familial hyperkalemic periodic paralysis), and SCN5A (Brugada syndrome). Overall, we show an exciting and promising new tool for ortholog genomic mapping that can support clinical variant insights into physiological outcomes (genotype-to-phenotype relationship), which should be applied to other cellular ion channels and transporters. This research was funded by the Gerber Foundation (to JWP), National Institutes of Health (K01ES025435 to JWP), and Michigan State University. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PO-01-055 USING MACHINE LEARNING TECHNIQUES TO PREDICT WHETHER A PAEDIATRIC PATIENT WITH BRUGADA SYNDROME SHOULD RECEIVE AN ICD

Brugada Syndrome (BrS) is a lethal cardiac rhythm disorder that can result in life threatening arrhythmias and carries high risk of sudden cardiac death. The gold standard treatment is implantation of an ICD. Statistical models have been developed to ascertain if a patient should receive an ICD. Previously, we compared a machine learning approach to existing statistical models (Siddiqui and Gonzalez Corcia, 2021). The statistical model was designed to stratify the risk of lethal cardiac events in a young population (<20 years old). Our research found that using additional variables improved the accuracy of our model as compared to the statistical approaches. We now explore further variables to determine whether the accuracy of the predictions can be improved. The initial project undertook a study that compared a statistical model developed by Gonzalez Corcia et al. (2017) with machine learning models using a 2-Step methodology. The statistical model used four clinical variables (Symptoms, Spontaneous Type I ECG, Sinus Node Dysfunction/Atrial (Tachycardia and Conduction Abnormality). We found that adding four more variables (Genotype for Brugada, Cardiac Electrophysiology Study, Ajmaline Challenge and patient’s gender) improved the accuracy of the predictions (from 0.93 to 0.988). Here we add two further steps to explore further combinations of variables. In Step 3, in addition to the original variables we add Baseline PR interval, , Baseline QRS in V1, Baseline QTc, Genotype and Gender. In Step 4, we add Ajmaline Challenge, EP Study, PR interval, QTc and QRS after the Ajmaline challenge along with the original four variables. In Step 3, we trained a Random Forest classifier resulting in a prediction accuracy of 0.966. In Step 4, a Random Forest classifier resulted in a prediction accuracy of 0.931. This confirms that Step 2 from the original research is the optimal combination of variables that can help clinicians decide if a paediatric patient should receive an ICD. This research further confirms that Machine learning can play an integral role in improving clinical practice and patient outcomes, particularly given the psychological implications of having an ICD at a young age. This is true even though it is a life saving device in case of sudden cardiac arrest/lethal ventricular arrhythmias. The importance of this research is further proven by the fact that implanting an ICD in a young person remains controversial.

PO-01-240 UTILITY OF IN VITRO FUNCTIONAL CHARACTERIZATION IN THE PROMOTION OR DEMOTION OF CLINICALLY ENCOUNTERED VARIANTS OF UNCERTAIN SIGNIFICANCE IN THE THREE MOST COMMON LONG QT SYNDROME-CAUSATIVE GENES: KCNQ1, KCNH2, AND SCN5A

Genetic testing for cardiac channelopathies such as long QT syndrome (LQTS) and Brugada syndrome (BrS) is standard of care. Although the American College of Medical Genetics (ACMG) guidelines for variant interpretation have provided a framework for distinguishing pathogenic variants from benign, many rare genetic variants remain classified as a variant of uncertain significance (VUS) due to lack of epidemiologic or functional data to either promote or demote the variant to pathogenic or benign status, respectively. To functionally characterize clinically encountered VUSs in the three most common LQTS-causative genes: KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3 and BrS1) in vitro by patch clamp technique to provide additional evidence for variant promotion/demotion. A total of 89 rare nonsynonymous VUS (15 KCNQ1, 19 KCNH2, and 55 SCN5A) were engineered by site directed mutagenesis and expressed in either HEK293 cells or TSA201 cells. Whole cell patch-clamp technique was used to functionally characterize these 89 variants to determine gain-of-function (GOF), loss-of-function (LOF), or normal channel function. Overall, the majority of VUS (50/89; 56.2%) studied displayed a wild-type-like electrophysiological phenotype and enabled demotion to likely benign status. The remaining VUS (39/89; 43.8%) were promoted to likely pathogenic status based on either a GOF or LOF electrophysiological signature following patch-clamp analysis. Among 15 KCNQ1 variants, 11 (73.3%) had normal channel function, 3 (20.0%) LOF, and 1 (6.7%) GOF. Of the 19 KCNH2 variants, 11 (57.9%) displayed normal channel function, 5 (26.3%) LOF, and 3 (15.8%) GOF. Finally, among the 55 SCN5A variants, 28 (50.9%) exhibited normal channel function, 19 (34.5%) LOF, and 8 (14.5%) GOF. Here, we provide functional characterization of 89 nonsynonymous VUSs identified during the course of clinically indicated genetic testing. Consistent with the clinical index of suspicion in the VUS-positive subject, 44% of the VUS were promoted to a likely pathogenic variant while 56% were demoted to a likely benign variant. Whether genetic test companies will incorporate this functionally characterized compendium of VUS and issue amended genetic test reports awaits their review and incorporation into their internal framework for variant annotation.