Background: Titin as the largest human protein plays a crucial role for structural integrity and contractile function of the heart. Mutations in the TTN gene are associated with late-onset heart failure, often presenting with a dilated cardiomyopathy phenotype. This condition is frequently accompanied by various cardiac arrhythmias, with atrial fibrillation (AF) being the most common. Large-cohort studies have demonstrated a strong association between titin mutations and early-onset AF. However, the mechanistic basis of how titin influences atrial electrophysiology at the cellular level remains poorly understood. In this study, we aim to characterize the electrophysiological remodelling patterns that contribute to proarrhythmogenic alterations in the context of TTN mutations. Understanding these mechanisms may shed light on titin’s electrophysiological role in cardiomyocytes and provide novel insights into the pathogenesis of AF.
Methods: To investigate the electrophysiological remodelling associated with titin mutations, we performed patch-clamp experiments on genetically engineered induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying TTNtruncation mutations, and compared them to an isogenic control line. A cell line harbouring the heterozygous A-band mutation TTNtv Ex327 was used to model the disease phenotype, while an additional line with the homozygous I-band mutation TTNtv Ex49 was included to study the phenotypic contrast. Electrophysiological recordings included measurements of both spontaneous and stimulated action potentials (APs), as well as underlying potassium currents.
Results: Quantification of spontaneous APs revealed a significant reduction of the AP duration at 50% repolarization (APD50) in the iPSC-CMs carrying the A-band TTN mutation. These cells also exhibited a significantly depolarized resting membrane potential compared to the isogenic control. In contrast, AP amplitude and upstroke velocity remained unchanged. iPSC-CMs with the homozygous I-band mutation showed no significant differences in any of the measured electrophysiological parameters. Additionally, cell size analysis revealed no significant differences across the groups, ruling out hypertrophic remodelling as a confounding factor. Analysis of induced APs corroborated the findings observed in spontaneous AP recordings, showing a consistent reduction in APD50 in the A-band mutation line. To explore the mechanisms underlying these electrophysiological changes, we further measured potassium currents. Cells with the A-band mutation displayed significantly elevated sustained potassium currents, which include contributions from hERG, KVLQT1, and KV1.5 channels. These channels are primarily active during the early repolarization phase of the AP and likely account for the observed shortening of APD50. Additionally, inward rectifier potassium currents were significantly increased in the A-band mutant cells, providing a plausible explanation for the elevated resting membrane potential.
Conclusion: This study demonstrates that titin mutations, particularly those within the A-band, induce significant alterations in cardiac AP morphology, potentially mediated by dysregulation of potassium currents. These findings suggest that AF in titin-related cardiomyopathy may not simply be a secondary consequence of heart failure, but rather a distinct, mutation-driven characteristic of the disease phenotype.
