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Home Office ethical approval was granted under project license 30/3070 and mice were euthanized by Home Office Schedule 1 methods.
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Materials and methods 2.1 MiceĪll animals were housed and maintained in the Mary Lyon Centre at MRC Harwell, under specific pathogen-free conditions in individually ventilated cages, with environmental conditions as outlined in the Home Office Code of Practice. Our findings thus demonstrate a causative link between elevated BCAAs and arrhythmia, which has implications for furthering our understanding of mechanisms underlying lethal arrhythmias, particularly those occurring in disease states associated with elevated BCAA levels such as diabetes and metabolic syndrome. 14 Studies in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) furthermore confirmed a direct pro-arrhythmic effect of elevated BCAAs and demonstrated the crucial involvement of the mammalian target of rapamycin (mTOR) activation in mediating these effects. In support of a role for BCAAs in modulation of cardiac electrical function, plasma BCAA levels were positively correlated to electrocardiogram (ECG) indices of conduction and repolarization in individuals from the general population. Affected mice displayed a drastic increase of plasma branched chain amino acid (BCAA-valine, isoleucine, leucine) levels due to the incomplete catabolism of BCAAs, enhanced arrhythmia susceptibility, cardiac conduction and repolarization disturbances, and excessive pro-arrhythmic intracellular calcium dysregulation in isolated cardiomyocytes. 13 Employing such a phenotype-driven ENU screen, we here identified a mutant mouse line presenting with sudden death and a homozygous nonsense mutation in the Bcat2 gene. 11, 12 In N-ethyl- N-nitrosourea (ENU) mutagenesis screens, pedigrees of mice with randomly induced point mutations are screened for a range of phenotypes and affected mice are subsequently used to map and clone the causative allele. 9, 10 Phenotype driven screens have been very successful in identifying novel genes and alleles associated with disease, and resolving gene function. Over the last decade, we and others have employed several novel approaches to identify new disease mechanisms and pathways regulating cardiac electrical (dys)function, including genomic studies in rodents. Development of efficient preventive and therapeutic strategies is essential but is as yet hampered by incomplete knowledge of disease mechanisms. 3–8 Despite decades of research, few anti-arrhythmic therapeutic options exist due to the complexity of underlying pathologies. 1, 2 Furthermore, patients suffering from metabolic disorders (diabetes, obesity) and heart failure are at increased risk for arrhythmias and SCD. 1 Arrhythmias typically occur in the setting of an underlying pathology, including myocardial ischaemia, structural derangements, and co-morbidities such as hypertension. SCD remains a leading cause of mortality in the Western world, accounting for up to 20% of all natural deaths, and up to 50% of all cardiovascular deaths. IntroductionĬardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation.Īrrhythmia, Electrophysiology, Sudden death, Metabolism, BCAA 1. Isolated cardiomyocytes from Bcat2 p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. At the age of 4–5 weeks, Bcat2 p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs-leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. Affected mice were found to be homozygous for the nonsense mutation Bcat2 p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. We employed a phenotype-driven N-ethyl- N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms.