Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression data of postnatal Pitx2 deficient heart

ABSTRACT: Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation locus. Pitx2 haploinsufficient mice are prone to pacing induced atrial fibrillation indicating that reduced Pitx2 promotes an arrhythmogenic substrate within the atrium. Here, we inactivated Pitx2 in postnatal heart and discovered that unstressed adult Pitx2 mutant mice had sinus node dysfunction with impaired atrial conduction, an arrhythmia closely associated with atrial fibrillation. A genome-wide search for Pitx2 transcriptional targets using ChIP-sequencing and RNA expression profiling shows that Pitx2 represses target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators many of which have been implicated in human atrial fibrillation by genome wide association studies. Pitx2 control and mutant hearts were collected from 3-, 6- and 12-week-old mice. At each time point, three cotrols and three mutants were collected as biological replicates. cDNA microarray analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA).

ORGANISM(S): Mus musculus

SUBMITTER: Min Zhang

PROVIDER: E-GEOD-50399 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Pitx2, an atrial fibrillation predisposition gene, directly regulates ion transport and intercalated disc genes.

Tao Ye Y Zhang Min M Li Lele L Bai Yan Y Zhou Yuefang Y Moon Anne M AM Kaminski Henry J HJ Martin James F JF

Circulation. Cardiovascular genetics 20140106 1

<h4>Background</h4>Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation (AF) locus. When deleted in the mouse germline, Pitx2 haploinsufficiency predisposes to pacing-induced AF, indicating that reduced Pitx2 promotes an arrhythmogenic substrate. Previous work focused on Pitx2 developmental functions that predispose to AF. Although Pitx2 is expressed in postnatal left atrium, it is unknown whether Pitx2 has distinct postnatal and developmental functions. ...[more]

PMID: 24395921

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Project description:Introduction: Atrial fibrillation (AF) is the most common arrhythmia and affects around 1% of the population with increasing incidence and substantial morbidity. Functional effects of genetic variants associated with AF should help to identify targets involved in AF initiation and/or subsequent remodeling. Gene variants next to the gene of the transcription factor PITX2 (paired-like homeodomain transcription factor 2) showed the strongest association with AF. A significant number of AF patients showed reduced PITX2 levels in the left atrium. Mouse models have been used to study functional aspects of PITX2 gene deletion (“knock out”), but there are substantial differences in atrial electrophysiology between mouse and human . HiPSC-CM should be able to bridge the gap, provided the cells reproduce typical characteristics of human atrium. Methods: PITX2 knock out samples from a control cell line of human induced pluripotent stem cells (hiPSC) were obtained. Differentiation and generation of Atrial-like engineered heart tissue (aEHT) was performed. RNA-sequencing (RNA-Seq), Quantitative Real-time PCR (RT-qPCR), Protein analysis by Western Blot, Contraction and force analysis, action potential measurements, Calcium current measurements and differential gene expression analysis were run on the samples. Results: Effective knock out of PITX2 was confirmed by mRNA sequencing and WB. It was shown that PITX2 deficiency reduces contraction force. PITX2 deficiency induces action potential triangulation with more negative maximum diastolic potential. Activation of muscarinic receptors shortens APD in both PITX2 knockout and controls but hyperpolarization is lost in PITX2 knock out. It was shown that Ca2+ current density is smaller in PITX2 knock out than controls. We found that more negative MDP in PITX2 knock out is not associated with higher IK1. Conclusion: We improved atrial differentiation, coming very close to human atrial electrophysiology. We demonstrate that PITX2 deficiency induces key characteristics known from persistent AF.

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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Gene expression data of postnatal Pitx2 deficient heart

Publications

Pitx2, an atrial fibrillation predisposition gene, directly regulates ion transport and intercalated disc genes.

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