PITX2c impairment electrical remodeling increases susceptibility to atrial fibrillation

FUNDING ACKNOWLEDGEMENTS: Type of funding sources: Public grant(s) – National budget only. Main funding source(s): the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT), the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government BACKGROUND: Functional mutations in the PITX2 (Paired-like homeodomain transcription factor 2) gene cause genetically inherited atrial fibrillation (AF). PITX2 is known to plays a pivotal role in regulating the expression of distinct ion channels, beta-adrenergic stimulation, cell-cell coupling and resulting in AF due to abnormal calcium abnormalities and electrical remodeling. However, the pathophysiological mechanisms linking impaired PITX2 with AF are still to be explored. This study was performed to generate PITX2c knockout human iPSC atrial cardiomyocytes (hiPSC-atrial CM), which are useful tools for arrhythmia modeling, and evaluate the mechanism of PITX2 deficiency leading to AF. METHODS: We generated a PITX2 knock-out iPSC line using a CRISPR/Cas9-based genome editing system. hiPSCs were electroporated with Cas9/sgRNA ribonucleoprotein (RNP) complexes. PITX2c knock-out hiPSC-CMs differentiated into atrial cardiomyocytes based on the manipulation of retinoic acid signaling. For the three-dimensional (3D) culture model, cells were plated in the spherical plate 5D. Electrophysiological properties and oxidative stress were assessed in PITX2c-deficient cardiomyocytes. Proarrhythmic effects of the PITX2c knock-out were quantified with AP morphology, AP duration (APD) restitution, wavelength (WL) and conduction velocity (CV) restitution using microelectrode arrays (MEA). RESULTS: We demonstrated that atrial-specific differentiated hiPSC-CMs expressed atrial-specific genes and the properties of action potentials were like those of human atrial cardiomyocytes. In addition, both 2D and 3D PITX2c knock-out atrial hiPSC-CMs showed signs of APD shortening and reduced wavelength (WL) and increased conduction velocity (CV) restitution (P<0.05). Next, a functional assessment of calcium homeostasis has shown that PITX2c causes atrial arrhythmias by impairing calcium handling genes (SERCA2a, RyR2, PLN). We then demonstrated that miR-374a-5p and miR-374b-5p could be involved in pathophysiological functions as therapeutic candidates when affinity for PITX2 was based on the upper context score percentile (> 99). CONCLUSIONS: This study provides an understanding of electrical remodeling by PITX2c loss-of-function mutations and suggests the potential therapeutic candidates by miR-374a-5p and miR-374b-5p, thus helping to understand the mechanism of PITX2 deficiency leading to atrial fibrillation.