PITX2 dosage-dependent pacemaker cell state changes underlie sinus node dysfunction and atrial arrhythmia-susceptibility II

Reduced spatiotemporal transcription factor levels have been shown to affect traits, organism development and disease predisposition. Here, we investigated the impact of physiologically relevant increases in transcription factor dosage, which has remained largely unexplored. Patients with genomic deletions far upstream of PITX2 present with sinus node dysfunction (SND) and atrial fibrillation. We show that delB mice, which recapitulate the deletion-driven arrhythmia in patients, ectopically express PITX2 in sinus node pacemaker cardiomyocytes (PCs) in a highly heterogeneous pattern from early embryonic development onwards. During development, delB sinus nodes form subdomains of mildly affected PC and strongly affected PCs that have lost their PC identity and switched towards an atrial cardiomyocyte-like state in a PITX2 dosage-dependent manner. Compared to heterozygous delB mice, homozygous delB mice have more PITX2+ PCs that express on average more PITX2, form proportionally larger subdomains of strongly affected PCs and have much more severe SND and atrial arrhythmia susceptibility. Ectopic expression of PITX2 in human induced pluripotent stem cell-derived PCs resulted in PITX2 dosage-dependent transcriptional changes reminiscent of those observed in the sinus node subdomains of delB mice. These results show that genetically driven ectopic expression of PITX2 in PCs causes SND and increased susceptibility for atrial arrhythmias, illustrating how spatiotemporally-defined increases in transcription factor dosage translates into developmental defects and disease predisposition. Overall design: Wild-type (n=3), delB/+ (n=6), delB/delB (n=3) fetal (E17.5) hearts were analysed using spatial transcriptomics. Regions of interest were segmented based on HCN4 and TNNI3 expression, including the TNNI3+ right atrium (RA), HCN4+ wild-type sinus node, and delB/+ and delB/delB HCN4-high and HCN4-low sinus node.