Characterizing an arrhythmia-related titin mutation using patient stem cell-derived atrial cardiomyocytes

Atrial fibrillation (AF) is a common arrhythmia that is linked to a greater risk of ischemic stroke and heart failure. Multiple genetic studies have established an association between protein truncating variants in the titin gene and increased risk of AF in the presence or absence of cardiomyopathy. Titin truncating variants are a known cause of dilated cardiomyopathy, thus existing studies have focused on the effects of these variants in ventricular cardiomyocytes. The structural and functional consequences of titin truncating variants in atrial cardiomyocytes and how such variants lead to arrhythmias such as AF are unclear. Our objective was to investigate the cellular effects of titin truncating variants identified in patients with unexplained AF. We identified a heterozygous titin truncating variant in a patient with early-onset atrial fibrillation and generated induced pluripotent stem cell lines (iPSCs) of the patient. We used CRISPR/Cas9 homology-directed repair to perform genome-editing of the patient iPSCs and corrected the titin truncating variant to wildtype. We differentiated patient iPSCs with the titin truncating variant and wildtype iPSCs into ventricular-like and atrial-like cardiomyocytes. We characterized cellular electrophysiology by optically mapping voltage and calcium transients and assessed the organization of sarcomere structures within cardiomyocytes through immunofluorescent staining of sarcomere proteins and confocal microscopy. iPSC-derived atrial cardiomyocytes displayed cell type-specific characteristics including faster beat rates (mean ± sem, beats/minute: 150.9 ± 21.8 vs. 21.9 ± 3.6; p = 0.01) and shorter rate-corrected action potentials (cAPD80: 195.9 ± 23.8 vs. 394.7 ± 42.8 ms; p = 0.048) compared to ventricular cardiomyocytes. Analysis of sarcomere organization showed poorer structural alignment in iPSC-derived atrial cardiomyocytes with the titin truncating variant compared to wildtype (% organization: 66.3 ± 6.8 vs. 88.0 ± 2.9; p = 0.03; Figure 1A and 1B). Similarly, iPSC-derived ventricular cardiomyocytes with the titin truncating variant showed poorer sarcomere organization compared to wildtype (62.0 ± 3.9 vs. 82.9 ± 2.9; p = 0.008; Figure 1C). Titin truncating variants lead to abnormal sarcomere organization in both atrial and ventricular iPSC-derived cardiomyocytes, and this phenotype can be reverted through CRISPR/Cas9 correction of the titin truncating variant to wildtype. These findings further our understanding of the role of titin in the atria and provide insight to the mechanisms by which titin truncating variants may promote arrhythmogenesis.