Abstract 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 ( Playrr Ex1sj allele). Using qRT-PCR we detected upregulation of the cardiac isoform, Pitx2c , during visceral organ morphogenesis in Playrr Ex1sj mutant embryos. Surface ECG (AliveCor®) and 24-hour telemetry ECG detected bradycardia and irregular interbeat (R-R) intervals suggestive of SND in Playrr Ex1sj mutant adults. Programmed stimulation of Playrr Ex1sj mutant adults resulted in pacing-induced AF. Within the right atrium of Playrr Ex1sj 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 Playrr Ex1sj adult mice. Finally, transcriptomic analysis by chromatin run-on and sequencing (ChRO-seq) in atria of Playrr Ex1sj 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.

The Long Noncoding RNA Playrr Regulates Pitx2 Dosage and Protects Against Cardiac Arrhythmias