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

View ORCID ProfileFrances L. Chen, Eva M. Oxford, Shao-Pei Chou, Na Li, John P. Leach, Sienna K. Perry, View ORCID ProfileBhargav D. Sanketi, Christina Cong, Sophie A. Kupiec-Weglinski, Rebecca Dubowitz, Erin Daugherity, View ORCID ProfileJames F. Martin, View ORCID ProfileCharles G. Danko, View ORCID ProfileNatasza A. Kurpios
doi: https://doi.org/10.1101/2022.09.20.508562
Frances L. Chen
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
DVM, PhD
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  • ORCID record for Frances L. Chen
Eva M. Oxford
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
DVM, PhD, DACVIM
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Shao-Pei Chou
2Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
PhD
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Na Li
3Department of Medicine- Cardiovascular Research, Baylor College of Medicine, Houston, TX 77030, USA
PhD
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John P. Leach
3Department of Medicine- Cardiovascular Research, Baylor College of Medicine, Houston, TX 77030, USA
PhD
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Sienna K. Perry
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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Bhargav D. Sanketi
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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Christina Cong
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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Sophie A. Kupiec-Weglinski
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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Rebecca Dubowitz
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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Erin Daugherity
4Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
5Center for Animal Resources and Education, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
DVM, MS, DACLAM
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James F. Martin
6Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
7Texas Heart Institute, Houston, TX 77030, USA
MD, PhD
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Charles G. Danko
2Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
PhD
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Natasza A. Kurpios
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
PhD
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  • ORCID record for Natasza A. Kurpios
  • For correspondence: natasza.kurpios@cornell.edu
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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 (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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213514

  • Abbreviations

    AF
    atrial fibrillation
    GWAS
    genome-wide association study
    lncRNA
    long noncoding RNA
    ISH
    in situ hybridization
    qRT-PCR
    quantitative real time polymerase chain reaction
    SAN
    sinoatrial node
    SND
    sinus node dysfunction
    SNP
    single nucleotide polymorphism
    ChRO-seq
    chromatin run-on & sequencing
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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    The Long Noncoding RNA Playrr Regulates Pitx2 Dosage and Protects Against Cardiac Arrhythmias
    Frances L. Chen, Eva M. Oxford, Shao-Pei Chou, Na Li, John P. Leach, Sienna K. Perry, Bhargav D. Sanketi, Christina Cong, Sophie A. Kupiec-Weglinski, Rebecca Dubowitz, Erin Daugherity, James F. Martin, Charles G. Danko, Natasza A. Kurpios
    bioRxiv 2022.09.20.508562; doi: https://doi.org/10.1101/2022.09.20.508562
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    The Long Noncoding RNA Playrr Regulates Pitx2 Dosage and Protects Against Cardiac Arrhythmias
    Frances L. Chen, Eva M. Oxford, Shao-Pei Chou, Na Li, John P. Leach, Sienna K. Perry, Bhargav D. Sanketi, Christina Cong, Sophie A. Kupiec-Weglinski, Rebecca Dubowitz, Erin Daugherity, James F. Martin, Charles G. Danko, Natasza A. Kurpios
    bioRxiv 2022.09.20.508562; doi: https://doi.org/10.1101/2022.09.20.508562

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