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A Cellular Atlas of Pitx2-Dependent Cardiac Development

ABSTRACT: The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation (AF), however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2-null embryos. We uncovered that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 biology and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease, and arrhythmogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE131181 | GEO | 2019/06/03

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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A Cellular Atlas of Pitx2-Dependent Cardiac Development

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