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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE291749 | GEO | 2025/11/11

REPOSITORIES: GEO

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Project description:Rationale: The most significantly associated atrial fibrillation (AF) risk loci in humans map to a noncoding gene desert upstream of the evolutionarily conserved left-right (LR) transcription factor Pitx2, a master regulator of LR asymmetric organ development. Pitx2 dosage is fundamentally linked to the development of sinus node dysfunction (SND) and AF, the most common cardiac arrhythmia affecting adults, but the mechanistic basis for this remains obscure. We identified a conserved long noncoding RNA (lncRNA), Playrr, which is exclusively transcribed on the embryo’s right side, opposite to Pitx2 on the left, that participates in mutually antagonistic transcriptional regulation with Pitx2. Objective: The objective of this study was to investigate a role of Playrr in regulating Pitx2 transcription and protecting against the development of cardiac rhythm disturbances. Methods and Results: Playrr expression in the developing heart was analyzed with RNA in situ hybridization. Playrr was expressed asymmetrically (on the right) to Pitx2 (on the left) in developing mouse embryos, including in mouse embryonic sinoatrial node cells. We utilized CRISPR/Cas9 genome editing in mice to target Playrr, generating mice lacking Playrr RNA transcript (PlayrrEx1sj allele). Using qRT-PCR we detected upregulation of the cardiac isoform, Pitx2c, during visceral organ morphogenesis in PlayrrEx1sj mutant embryos. Surface ECG (AliveCor®) and 24-hour telemetry ECG detected bradycardia and irregular interbeat (R-R) intervals suggestive of SND in PlayrrEx1sj mutant adults. Programmed stimulation of PlayrrEx1sj mutant adults resulted in pacing-induced AF. Within the right atrium of PlayrrEx1sj mutant hearts, Masson’s trichrome stain revealed increased collagen deposition indicative of fibrosis, and immunofluorescence demonstrated mis-localization of Connexin 43 in atrial cardiomyocytes. These findings suggested an altered atrial substrate in PlayrrEx1sj adult mice. Finally, transcriptomic analysis by chromatin run-on and sequencing (ChRO-seq) in atria of PlayrrEx1sj mutant mice compared to wild type controls revealed differential expression of genes involved in cell-cell adhesion and motility, fibrosis, and dysregulation of the key cardiac genes Tbx5 and Hcn1. Conclusions: Adult mice lacking functional Playrr lncRNA transcript have baseline bradyarrhythmia and increased susceptibility to AF. These cardiac phenotypes are similar to those observed in Pitx2 heterozygous mice. Interactions between Pitx2 and Playrr may provide a genetic mechanism for modulating Pitx2 dosage and susceptibility to SND and AF.

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

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