Abstract 4147856: A TTN Frameshift Mutation Leads to Dysfunctional Sarcomere and Calcium Handling in iPSC-derived Atrial Cardiomyocytes

Background: Increasingly, rare variants in genes encoding sarcomeric proteins like titin, have been associated with early-onset atrial fibrillation (EOAF) in part by the development of an atrial myopathy. Although TTN truncating variants (TTNtvs) have been causally linked with cardiomyopathy syndromes, their role in the pathogenesis of atrial myopathy and EOAF is poorly understood. Hypothesis: Atrial myopathy due to a TTNtv creates an arrhythmogenic substrate for EOAF by disrupting sarcomere structure and calcium handling. Aims: We investigated electrophysiologic (EP) mechanisms by which a TTNtv (TTN-Val23653 frameshift [FS]) in the A-band of titin causes an atrial myopathy and EOAF utilizing mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) generated from a large Caucasian kindred with a strong cardiac phenotype. Methods: We performed clinical phenotyping and genetic testing of the proband and family members. hiPSC-aCMs generated from the proband and wild-type (WT) were characterized by immunofluorescence (IF) and calcium transients to determine contractility and calcium handling properties. Results: The proband presented with EOAF at age 57 years. Transthoracic echocardiography revealed moderate left atrial enlargement and reduced left ventricular ejection fraction (25-30%). Cascade screening of the TTN-FS kindred confirmed co-segregation of the variant with EOAF, dilated cardiomyopathy, heart failure, stroke, and ventricular arrhythmias (Fig. 1A-B). IF staining revealed disrupted sarcomere structures and shortened sarcomere length in TTN-FS iPSC-aCMs vs. WT (Fig. 1C). Calcium transient analysis revealed that TTN-FS iPSC-aCMs had a shorter latency to peak and smaller peak calcium transient amplitudes as compared to WT (Fig. 1D). However, the TTN-FS iPSC-aCMs exhibited a similar time to baseline vs. WT iPSC-aCMs which suggested that the TTNtv was critically involved in the contractile amplitude and peak formation kinetics, but not the relaxation phase. Conclusions: Our results suggest that a TTNtv disrupts sarcomere structure and calcium handling leading to an atrial myopathy and EOAF. Therapeutic targeting of the atrial myopathy may prevent the development of EOAF.