Abstract 4138278: A Titin Missense Variant Causes Atrial Fibrillation

Background: Rare loss-of-function variants in TTN , the gene encoding the sarcomeric protein titin, have been causally associated with atrial fibrillation (AF). Missense TTN variants ( TTN mv) are generally regarded as benign in relation to dilated cardiomyopathy, but their role in AF is unknown. Hypothesis: TTN mv are associated with clinical outcomes and AF pathogenesis. Aims: We aimed to determine if TTN mv are associated with AF/heart failure (HF) hospitalizations and investigate if a rare missense variant ( TTN -T32756I) caused AF using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). Methods: Whole exome sequencing was performed in 131 adults at an urban academic healthcare system who identified as non-Hispanic Black (NHB) or Hispanic/Latinx (HL). Multivariable Cox proportional hazard models were used to assess cumulative risk of AF/HF hospitalization. Isogenic TTN -T32756I iPSC-aCM mutants were generated using the CRISPR-Cas9 technique. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping and patch-clamping to assess the electrophysiological (EP) properties. Results: We identified 137 TTN mvs in 76/131 (58.0%) of subjects, which were most commonly in the TTN A-band. 64 AF-related and 110 HF-related hospitalizations occurred after a median follow-up time of 4.16 years. Hospitalization risk was higher in TTN mv carriers (HR 1.81, Fig. 1A ). We also identified three patients with early-onset AF who shared a rare missense variant T32756I. TTN-T32756I -iPSC-aCMs displayed aberrant contractility ( Fig. 1B-D ), shortened action potential duration ( Fig. 1G-H ), increased activity of cardiac K + channel ( Fig. 1I ), and dysregulated Ca 2+ homeostasis ( Fig. 1J ) without compromising sarcomeric integrity ( Fig. 1E-F ). The titin-binding protein, Four-and-a-Half Lim domains 2 (FHL2), also has increased binding with KCNQ1 and its modulatory subunit KNCE1 in TTN-T32756I -iPSC-aCMs, which may enhance the slow delayed rectifier K + current ( I ks ). Suppression of FHL2 normalized I ks , supporting FHL2 as a mediator of the increased I ks ( Fig. 1K) . Conclusion: In a single-center multi-ethnic AF cohort, TTN mv were associated with increased AF/HF hospitalization, and the T32756I- TTN mv causes ion channel remodeling and an AF-like EP phenotype. These findings establish a mechanistic link between TTN mv, K + ion channels, and sarcomeric regulatory proteins that may represent a novel therapeutic target.