Abstract Body (Do not enter title and authors here): Background. Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in clinical practice, contributing to substantial morbidity and mortality. Thus, there is an urgent need to understand the molecular contributions to AF susceptibility. We identified a novel missense mutation (M527L) in the cardiac transcription factor NFATC1 in a multigenerational family with autosomal dominant young onset atrial fibrillation (AF). Germline deletion of nfatc1 in zebrafish (KO) causes atrial tachyarrhythmia and sudden death in the juvenile stage.
Objective. To define the electrophysiological and molecular phenotype of NFATC1 deletion in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs).
Methods. Using CRISPR/Cas9, we targeted NFATC1 exon 4 and generated a compound heterozygous iPSC line harboring 16 bp and 14 bp deletions in trans (NFATC1 KO), confirmed to lack detectable NFATC1 protein. Spontaneous optical action potentials (AP) were measured in 30-day post-differentiation WT and NFATC1 KO iPSC-CMs, using di-4-ANBDQBS. Single-cell RNA-seq was performed using the Chromium GEM-X Single Cell 3' kit and an Element Bioscience AVITI sequencer, with a 30,000 reads/cell depth. DeSeq differential gene analysis was performed, followed by Enrichr pathway analysis.
Results. APs in WT iPSC-CMs were mostly regular, with no delayed afterdepolarizations (DADs) observed. By contrast, APs in KO iPSC-CMs were mostly irregular, with DADs observed in 28% of records (p=6.52E-4, Table 1). Increased spontaneous beat rate and a wider beat to beat interval range were noted in KO cells (55±3 bpm WT [n=43] vs 73±7 bpm KO [n=33], p=0.015; and 128±21 ms WT [n=39] vs 345±70 ms KO [n=30], p=0.015). Analysis of differentially expressed genes identified perturbations in known AF pathways, including calcium, GPCR, cAMP, cGMP-PKG and MAPK signaling (Table 2).
Conclusions. Loss of NFATC1 in iPSC-CMs leads to spontaneous electrical instability (evidenced by increased spontaneous beating rate, beat-to-beat variability, and DADs), supporting its essential role in maintaining normal cardiac excitability. Differential gene expression analysis highlighted perturbations in key AF-related signaling cascades, illustrating how NFATC1 deficiency disrupts cardiomyocyte homeostatic networks. Taken together, these data establish NFATC1 as a key transcriptional regulator of cardiac excitability and that loss of NFATC1 is pro-arrhythmogenic.