Essential Role of the RIα Subunit of cAMP-Dependent Protein Kinase in Regulating Cardiac Contractility and Heart Failure Development.

BACKGROUND: The heart expresses 2 main subtypes of cAMP-dependent protein kinase (PKA; type I and II) that differ in their regulatory subunits, RIα and RIIα. Embryonic lethality of RIα knockout mice limits the current understanding of type I PKA function in the myocardium. The objective of this study was to test the role of RIα in adult heart contractility and pathological remodeling. METHODS: We measured PKA subunit expression in human heart and developed a conditional mouse model with cardiomyocyte-specific knockout of RIα (RIα-icKO). Myocardial structure and function were evaluated by echocardiography, histology, and ECG and in Langendorff-perfused hearts. PKA activity and cAMP levels were determined by immunoassay, and phosphorylation of PKA targets was assessed by Western blot. L-type Ca2+ current (ICa,L), sarcomere shortening, Ca2+ transients, Ca2+ sparks and waves, and subcellular cAMP were recorded in isolated ventricular myocytes (VMs). RESULTS: RIα protein was decreased by 50% in failing human heart with ischemic cardiomyopathy and by 75% in the ventricles and in VMs from RIα-icKO mice but not in atria or sinoatrial node. Basal PKA activity was increased ≈3-fold in RIα-icKO VMs. In young RIα-icKO mice, left ventricular ejection fraction was increased and the negative inotropic effect of propranolol was prevented, whereas heart rate and the negative chronotropic effect of propranolol were not modified. Phosphorylation of phospholamban, ryanodine receptor, troponin I, and cardiac myosin-binding protein C at PKA sites was increased in propranolol-treated RIα-icKO mice. Hearts from RIα-icKO mice were hypercontractile, associated with increased ICa,L, and [Ca2+]i transients and sarcomere shortening in VMs. These effects were suppressed by the PKA inhibitor, H89. Global cAMP content was decreased in RIα-icKO hearts, whereas local cAMP at the phospholamban/sarcoplasmic reticulum Ca2+ ATPase complex was unchanged in RIα-icKO VMs. RIα-icKO VMs had an increased frequency of Ca2+ sparks and proarrhythmic Ca2+ waves, and RIα-icKO mice had an increased susceptibility to ventricular tachycardia. On aging, RIα-icKO mice showed progressive contractile dysfunction, cardiac hypertrophy, and fibrosis, culminating in congestive heart failure with reduced ejection fraction that caused 50% mortality at 1 year. CONCLUSIONS: These results identify RIα as a key negative regulator of cardiac contractile function, arrhythmia, and pathological remodeling.

Novel Therapeutics and Upcoming Clinical Trials Targeting Inflammation in Cardiovascular Diseases.

Cardiovascular disease (CVD) remains a major health burden despite significant therapeutic advances accomplished over the last decades. It is widely and increasingly recognized that systemic inflammation not only represents a major cardiovascular risk and prognostic factor but also plays pivotal roles in CVD development and progression. Despite compelling preclinical evidence suggesting large potential of anti-inflammatory pharmacological interventions across numerous CVDs, clinical translation remains incomplete, mainly due to (1) yet undefined molecular signaling; (2) challenges of safety and efficacy profile of anti-inflammatory drugs; and (3) difficulties in identifying optimal patient candidates and responders to anti-inflammatory therapeutics, as well as optimal therapeutic windows. Randomized controlled trials demonstrated the safety/efficacy of canakinumab and colchicine in secondary cardiovascular prevention, providing confirmation for the involvement of a specific inflammatory pathway (NLRP3 [NACHT, LRR, and PYD domain-containing protein 3] inflammasome/IL [interleukin]-1ß) in atherosclerotic CVD. Colchicine was recently approved by the US Food and Drug Administration for this indication. Diverse anti-inflammatory drugs targeting distinct inflammatory pathways are widely used for the management of other CVDs including myocarditis and pericarditis. Ongoing research efforts are directed to implementing anti-inflammatory therapeutics across a growing number of CVDs, through repurposing of available anti-inflammatory drugs and development of novel anti-inflammatory compounds, which are herein concisely discussed. This review also summarizes the main characteristics and findings of completed and upcoming randomized controlled trials directly targeting inflammation in CVDs and discusses the major challenges and future perspectives in the exciting and constantly expanding landscape of cardioimmunology.

Ablation Strategies for Repeat Procedures in Atrial Fibrillation Recurrences Despite Durable Pulmonary Vein Isolation: The Prospective Randomized ASTRO AF Multicenter Trial.

BACKGROUND: Ablation strategies for patients with symptomatic atrial fibrillation and isolated pulmonary veins vary and their effects on arrhythmia recurrence remain unclear. A prospective randomized German multicenter trial sought to compare 2 ablation strategies in this patient cohort. METHODS: Patients with atrial fibrillation despite durable pulmonary vein isolation were randomly assigned at 7 centers to undergo low-voltage area ablation using 3-dimensional mapping and irrigated radiofrequency current ablation (group A) or empirical left atrial appendage isolation (LAAI) using the cryoballoon followed by staged interventional left atrial appendage closure (group B). The primary end point was freedom from atrial tachyarrhythmias between 91 and 365 days after index ablation. The study was powered for superiority of LAAI compared with low-voltage area. RESULTS: Patients (40% women; mean age, 68.8±8 years) with paroxysmal (32%) or persistent atrial fibrillation (68%) were randomized to undergo low-voltage area ablation (n=79) or cryoballoon-guided LAAI (n=82). After a planned interim analysis, enrollment was halted for futility on January 10, 2023. In the LAAI group, 77 of 82 left atrial appendages were successfully isolated with subsequent left atrial appendage closure in 57 patients. Procedure-related complications occurred in 4 (5%) and 11 (13.5%) patients in group A and B, respectively (P=0.10). The median follow-up was 367 days (interquartile range, 359-378). The Kaplan-Meier point estimate for freedom from atrial tachyarrhythmias was 51.7% (CI, 40.9%-65.4%) for group A and 55.5% (CI, 44.4%-69.2%; P=0.8069) for group B. CONCLUSIONS: The current study did not detect superiority of cryoballoon-guided LAAI over low-voltage area ablation in patients with atrial fibrillation despite durable PVI. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04056390.

Circadian variation pattern of sudden cardiac arrest occurred in Chinese community.

BACKGROUND: The circadian variation pattern of sudden cardiac arrest (SCA) occurred in Chinese community including both community healthcare centres and primary hospitals remains unknown. This study analysed the circadian variation of SCA in the Chinese community. METHODS: Data between 2018 and 2022 from the remote ECG diagnosis system of Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine were analysed to examine the circadian rhythm of SCA, stratified by initial shockable (ventricular tachycardia or ventricular fibrillation) versus non-shockable (asystole or pulseless electrical activity) rhythm. RESULTS: Among 10 210 cases of SCA, major cases (8736, 85.6%) were non-shockable and 1474 (14.4%) cases were shockable. The circadian rhythm of SCA was as follows: peak time was from 08:00 to 11:59 (30.1%), while deep valley was from 00:00 to 03:59 (7.5%). The proportions of events by non-shockable and shockable events were similar and both reached their peak from 08:00 to 11:59, with a percentage of 29.0% and 36.4%, respectively. Multivariable analysis showed that the relative risk of shockable compared with non-shockable arrests was lower between 00:00 and 03:59 (adjusted OR (aOR): 0.72, 95% CI: 0.54 to 0.97, p=0.028) and 04:00 to 07:59 (aOR: 0.60, 95% CI: 0.46 to 0.79, p<0.001), but higher between 08:00 and 11:59 (aOR: 1.34, 95% CI: 1.09 to 1.64, p=0.005). CONCLUSIONS: In Chinese community, there is a distinct circadian rhythm of SCA, regardless of initial rhythms. Our findings may be helpful in decision-making, in that more attention and manpower should be placed on the morning hours of first-aid and resuscitation management in Chinese community.

Timing of oral anticoagulation in atrial fibrillation patients after acute ischaemic stroke and outcome after 3 months: results of the multicentre Berlin Atrial Fibrillation Registry.

BACKGROUND: Oral anticoagulation (OAC) is key in stroke prevention in patients with atrial fibrillation (AF) but there is uncertainty regarding the optimal timing of OAC (re)initiation after stroke, as recent large randomised controlled trials have methodological weaknesses and excluded stroke patients on therapeutic anticoagulation at stroke onset as well as patients started on a vitamin K antagonist after stroke. The '1-3-6-12 days rule', based on expert consensus and referring to stroke severity, was used in clinical practice to initiate OAC after acute ischaemic stroke or transient ischaemic attack (TIA) since publication in 2013. METHODS: We retrospectively assessed whether compliance to the '1-3-6-12 days rule' was associated with the composite endpoint (recurrent stroke, systemic embolism, myocardial infarction, major bleeding or all-cause death). RESULTS: Among 708 registry patients with known AF before stroke and hospitalisation within 72 hours after stroke, 432 were anticoagulated at stroke onset. OAC was started according to the '1-3-6-12 days rule' in 255 (39.2%) patients. Non-adherence to the '1-3-6-12 days rule' was not associated with the composite endpoint within 3 months in 661 patients who (re-)started on OAC (log-rank test: p=0.74).Results were similar for 521 patients (re)started on a non-vitamin K-dependent OAC. CONCLUSION: (Re)starting OAC after stroke followed the '1-3-6-12 days rule' in about 40% of all patients with AF, and more often in those anticoagulated at stroke onset. Adherence to the '1-3-6-12 days rule' did not reduce the composite clinical endpoint, if OAC was restarted within 3 months of stroke/TIA. TRIAL REGISTRATION NUMBER: NCT02306824.

Role of Calmodulin in Cardiac Disease: Insights on Genotype and Phenotype.

Calmodulin, a protein critically important for the regulation of all major cardiac ion channels, is the quintessential cellular calcium sensor and plays a key role in preserving cardiac electrical stability. Its unique importance is highlighted by the presence of 3 genes in 3 different chromosomes encoding for the same protein and by their extreme conservation. Indeed, all 3 calmodulin (CALM) genes are among the most constrained genes in the human genome, that is, the observed variants are much less than expected by chance. Not surprisingly, CALM variants are poorly tolerated and accompany significant clinical phenotypes, of which the most important are those associated with increased risk for life-threatening arrhythmias. Here, we review the current knowledge about calmodulin, its specific physiological, structural, and functional characteristics, and its importance for cardiovascular disease. Given our role in the development of this knowledge, we also share some of our views about currently unanswered questions, including the rational approaches to the clinical management of the affected patients. Specifically, we present some of the most critical information emerging from the International Calmodulinopathy Registry, which we established 10 years ago. Further progress clearly requires deep phenotypic information on as many carriers as possible through international contributions to the registry, in order to expand our knowledge about Calmodulinopathies and guide clinical management.

IRS2 Signaling Protects Against Stress-Induced Arrhythmia by Maintaining Ca2+ Homeostasis.

BACKGROUND: The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. METHODS: A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. RESULTS: The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. CONCLUSIONS: Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants.

BACKGROUND: The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which ß-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. METHODS: Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. RESULTS: By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. CONCLUSIONS: Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

Sex-specific association of cardiovascular drug doses with adverse outcomes in atrial fibrillation.

OBJECTIVES: Sex differences occur in atrial fibrillation (AF), including age at first manifestation, pathophysiology, treatment allocation, complication rates and quality of life. However, optimal doses of cardiovascular pharmacotherapy used in women with AF with or without heart failure (HF) are unclear. We investigated sex-specific associations of beta-blocker and renin-angiotensin system (RAS) inhibitor doses with cardiovascular outcomes in patients with AF or AF with concomitant HF. METHODS: We used data from the prospective Basel Atrial Fibrillation and Swiss Atrial Fibrillation cohorts on patients with AF. The outcome was major adverse cardiovascular events (MACEs), including death, myocardial infarction, stroke, systemic embolisation and HF-related hospitalisation. Predictors of interest were spline (primary analysis) or quartiles (secondary analysis) of beta-blocker or RAS inhibitor dose in per cent of the maximum dose (reference), in interaction with sex. Cox models were adjusted for demographics, comorbidities and comedication. RESULTS: Among 3961 patients (28% women), MACEs occurred in 1113 (28%) patients over a 5-year median follow-up. Distributions of RAS inhibitor and beta-blocker doses were similar in women and men. Cox models revealed no association between beta-blocker dose or RAS inhibitor dose and MACE. In a subgroup of patients with AF and HF, the lowest hazard of MACE was observed in women prescribed 100% of the RAS inhibitor dose. However, there was no association between RAS dose quartiles and MACE. CONCLUSIONS: In this study of patients with AF, doses of beta-blockers and RAS inhibitors did not differ by sex and were not associated with MACE overall.

Cardiac conduction system as a new organ at risk in radiotherapy.

BACKGROUND: Cardiovascular diseases represent the most common non-oncologic cause of death in patients following radiotherapy (RT) in the thoracic region. Radiation-induced heart disease (RIHD) can manifest as various heterogeneous clinical entities. However, the influence of RT on the cardiac conduction system has only recently gained more attention. Arrhythmogenic toxicity, i.e., conduction disorders and arrhythmias, constitutes a significant part of these adverse effects. The cardiac conduction system is not routinely monitored as an organ at risk (OaR). Its specific histological nature and function suggest different sensitivity and response to radiation. The heart is a highly heterogeneous organ, and the routinely monitored dose to the whole heart may not adequately characterize the risk of increased arrhythmogenic toxicity from RT. Cardiac structures, including the conduction system, appear to be additional OaRs for which dose distribution should be monitored. MATERIAL AND METHODS: For the systematic selection of studies, we utilized the PubMed database with keywords derived from the analysis of existing literature. The search was limited to English-language publications, and the selection criteria included relevance to the topic and the quality of methodology. PURPOSE: This article summarizes the impact of RT on the cardiac conduction system. CONCLUSION: Radiotherapy-induced cardiotoxicity significantly affects morbidity and mortality. The heart exhibits heterogeneity in terms of radiosensitivity. Certain cardiac subregions in the dose distribution show a higher correlation with poorer overall survival than routinely monitored doses to the whole heart and derived parameters (the volumes irradiated with the doses of 5 or 30 Gy - V5 or V30, respectively). The most radiosensitive subregions appear to be the base of the heart, including the beginning of the conduction system. Higher doses to the conduction system, especially the sinoatrial (SA) node, are associated with a higher incidence of a wide range of arrhythmias and poorer overall survival. However, dose limits (Dmean and Dmax) for the conduction system have not yet been established. Dosimetric studies have identified cutoff doses to the SA node, exceeding which there is a significant increase in mortality and the occurrence of arrhythmias.

Single Construct Suppression and Replacement Gene Therapy for the Treatment of All CALM1-, CALM2-, and CALM3-Mediated Arrhythmia Disorders.

BACKGROUND: CaM (calmodulin)-mediated long-QT syndrome is a genetic arrhythmia disorder (calmodulinopathies) characterized by a high prevalence of life-threatening ventricular arrhythmias occurring early in life. Three distinct genes (CALM1, CALM2, and CALM3) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy. METHODS: Five custom-designed CALM1-, CALM2-, and CALM3-targeting short hairpin RNAs (shRNAs) were tested for knockdown (KD) efficiency using TSA201 cells and reverse transcription-quantitative polymerase chain reaction. A dual-component suppression and replacement (SupRep) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1-, CALM2-, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 cDNA (replacement). CALM1-F142L, CALM2-D130G, and CALM3-D130G induced pluripotent stem cell-derived CMs were generated from patients with CaM-mediated long-QT syndrome. A voltage-sensing dye was used to measure action potential duration at 90% repolarization (APD90). RESULTS: Following shRNA KD efficiency testing, a candidate shRNA was identified for CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P<0.0001). Transfection with CALM-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P<0.0001). Additionally, transfection with CALM-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P<0.0001) and CALM3-D130G (978±81 to 446±6 ms; P<0.001). CONCLUSIONS: We provide the first proof-of-principle suppression-replacement gene therapy for CaM-mediated long-QT syndrome. The CALM-SupRep gene therapy shortened the pathologically prolonged APD90 in CALM1-, CALM2-, and CALM3-variant CaM-mediated long-QT syndrome induced pluripotent stem cell-derived CM lines. The single CALM-SupRep construct may be able to treat all calmodulinopathies, regardless of which of the 3 CaM-encoding genes are affected.

Retractable congestive heart failure due to sinus valsalva rupture with coexisting gerbode defect: a rare case report.

Sinus of Valsalva aneurysm (SoVA) is a rare disease with less than 1% prevalence in the population. Most cases are asymptomatic, however, significant clinical manifestations are possible due to fistula formation and sudden rupture resulting in cardiac shunt. Eventually it may develop into progressive heart failure with high morbidity. We report the case of a 33 year old female patient who presented with shortness of breath, ascites, and recurring hospitalisation. The cardiac examination revealed sinus tachycardia along with loud and continuous murmurs on the left parasternal border. Several standard diagnostic procedures could not be performed due to malignant arrhythmia in supine position. Echocardiography examination revealed SoV rupture with a gerbode defect, which was the underlying cause of severe retractable heart failure.

Wellens Syndrome in a patient with a history of hypertension and chronic obstructive pulmonary disease: a case report.

Wellens syndrome, an ST Elevation Myocardial Infarction (STEMI) equivalent, is also known as T-wave left anterior descending (LAD) coronary artery disease. Wellens syndrome is characterized by a unique electrocardiogram (ECG) pattern that suggests a significant stenosis in the left anterior descending coronary artery that warrants immediate intervention. Hereby, we present a case report of Wellens syndrome in a patient with a history of hypertension and chronic obstructive pulmonary disease (COPD) that may be potentially mistaken for pseudo- Wellens syndrome because the ECG pattern mimics left ventricular strain pattern (LVSP) in left ventricular hypertrophy (LVH). Thus, cautious examination of recent chest pain and ECG is important to differentiate Wellens syndrome and LVSP in patients with hypertension and COPD to perform early detection and aggressive intervention since they may help to lessen the adverse results.

Identifying pulmonary embolism - high risk patients after total knee replacement: systematic review.

OBJECTIVE: To determine the incidence, onset, risk factors and mortality of pulmonary embolism in total knee replacement patients. METHODS: The systematic review was conducted in September 2022, and comprised search on PubMed, ScienceDirect, Scopus and Crossref databases for studies published from 1977 till September 7, 2022, in the English language related to the incidence of pulmonary embolism after primary total knee replacement. Cochrane Handbook for Systematic Reviews of Interventions was used to assess risk of bias, and the Newcastle-Ottawa Scale was used to assess the quality of evidence. RESULTS: Of the 3,910 studies initially identified, 66(1.68%) were analysed in detail, which together had 13,258,455 total knee replacement patients. Pulmonary embolism was reported in 76,515(0.58%) cases. The onset of pulmonary embolism ranged 2-150 days post-surgery. Patients with older age, diabetes mellitus, higher body mass index, atrial fibrillation, previous venous thromboembolism, high Charlson Comorbidity Index score, hypertension, arrhythmia and chronic heart failure were at significantly higher risk (p<0.05). The overall mortality rate of pulmonary embolism in such cases ranged 10.53-100%. CONCLUSIONS: Pulmonary embolism is a rare complication after orthopaedic surgery, but it has a very high mortality rate. By recognising the risk factors, attending physicians can optimise the use of chemoprophylaxis, thus preventing pulmonary embolism.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Vigorous Exercise in Patients With Congenital Long QT Syndrome: Results of the Prospective, Observational, Multinational LIVE-LQTS Study.

BACKGROUND: Whether vigorous exercise increases risk of ventricular arrhythmias for individuals diagnosed and treated for congenital long QT syndrome (LQTS) remains unknown. METHODS: The National Institutes of Health-funded LIVE-LQTS study (Lifestyle and Exercise in the Long QT Syndrome) prospectively enrolled individuals 8 to 60 years of age with phenotypic and/or genotypic LQTS from 37 sites in 5 countries from May 2015 to February 2019. Participants (or parents) answered physical activity and clinical events surveys every 6 months for 3 years with follow-up completed in February 2022. Vigorous exercise was defined as ≥6 metabolic equivalents for >60 hours per year. A blinded Clinical Events Committee adjudicated the composite end point of sudden death, sudden cardiac arrest, ventricular arrhythmia treated by an implantable cardioverter defibrillator, and likely arrhythmic syncope. A National Death Index search ascertained vital status for those with incomplete follow-up. A noninferiority hypothesis (boundary of 1.5) between vigorous exercisers and others was tested with multivariable Cox regression analysis. RESULTS: Among the 1413 participants (13% <18 years of age, 35% 18-25 years of age, 67% female, 25% with implantable cardioverter defibrillators, 90% genotype positive, 49% with LQT1, 91% were treated with beta-blockers, left cardiac sympathetic denervation, and/or implantable cardioverter defibrillator), 52% participated in vigorous exercise (55% of these competitively). Thirty-seven individuals experienced the composite end point (including one sudden cardiac arrest and one sudden death in the nonvigorous group, one sudden cardiac arrest in the vigorous group) with overall event rates at 3 years of 2.6% in the vigorous and 2.7% in the nonvigorous exercise groups. The unadjusted hazard ratio for experience of events for the vigorous group compared with the nonvigorous group was 0.97 (90% CI, 0.57-1.67), with an adjusted hazard ratio of 1.17 (90% CI, 0.67-2.04). The upper 95% one-sided confidence level extended beyond the 1.5 boundary. Neither vigorous or nonvigorous exercise was found to be superior in any group or subgroup. CONCLUSIONS: Among individuals diagnosed with phenotypic and/or genotypic LQTS who were risk assessed and treated in experienced centers, LQTS-associated cardiac event rates were low and similar between those exercising vigorously and those not exercising vigorously. Consistent with the low event rate, CIs are wide, and noninferiority was not demonstrated. These data further inform shared decision-making discussions between patient and physician about exercise and competitive sports participation. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02549664.