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. 2014 Feb;7(1):23-32.
doi: 10.1161/CIRCGENETICS.113.000259. Epub 2014 Jan 6.

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

Ye Tao  1 , Min Zhang, Lele Li, Yan Bai, Yuefang Zhou, Anne M Moon, Henry J Kaminski, James F Martin
Affiliations

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

Ye Tao et al. Circ Cardiovasc Genet. 2014 Feb.

Abstract

Background: 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.

Methods and results: To investigate Pitx2 postnatal function, we conditionally inactivated Pitx2 in the postnatal atrium while leaving its developmental function intact. Unstressed adult Pitx2 homozygous mutant mice display variable R-R interval with diminished P-wave amplitude characteristic of sinus node dysfunction, an AF risk factor in human patients. An integrated genomics approach in the adult heart revealed Pitx2 target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators. Importantly, many Pitx2 target genes have been implicated in human AF by genome-wide association studies. Immunofluorescence and transmission electron microscopy studies in adult Pitx2 mutant mice revealed structural remodeling of the intercalated disc characteristic of human patients with AF.

Conclusions: Our findings, revealing that Pitx2 has genetically separable postnatal and developmental functions, unveil direct Pitx2 target genes that include channel and calcium handling genes, as well as genes that stabilize the intercalated disc in postnatal atrium.

Keywords: atrial fibrillation; genetics; genome-wide association analysis.

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Figures

Figure 1
Figure 1
Pitx2 conditional knock out mice have abnormal heart function – sinus node dysfunction with impaired atrial conduction. By telemetry ECG tracing in awake mice, heart function of the adult control (Pitx2 Flox/Flox) and Pitx2 CKO (Pitx2 Flox/Flox MCK-Cre) mice were tested. (A) In control mice, normal sinus rhythm was observed. (B) In the Pitx2 CKO mice, mutant heart function including irregular R-R intervals and low voltage of P waves (open arrowheads) were observed. Tracings from one control and two Pitx2 CKO mice are shown. All mice shown were 3-month-old. The unit of R-R interval is millisecond (ms).
Figure 2
Figure 2
Age-related gene expression profiling in Pitx2 mutants. (A) Clustering analysis of gene expression profile across three time points in control and Pitx2 mutant hearts. Expression patterns of five representative groups are shown in heat map. The color bar represents relative expression level for each gene across different samples. (B) Differential expression between Pitx2 mutant and control across three time points. Mean value for each group was used to calculate fold change between mutants and controls. Box plots for selected clusters show median (middle line), third and first quartile (top and bottom hinges of the boxes), whiskers and outliers (black dots). (C) Gene ontology analysis of genes in each group. GO terms related to biological process and molecular function categories.
Figure 3
Figure 3
Overlay of Pitx2 ChIP-Seq and gene expression profiling assays for Pitx2 CKO heart. (A) Gene ontology (GO) analysis for genes (n=653) from overlay of Pitx2 ChIP-Seq and Pitx2 CKO microarray assays (genes in close proximity to Pitx2 binding chromosomal regions identified by Pitx2 ChIP-Seq assay that also show significant change in microarray assay for Pitx2 CKO heart). Both assays performed on 3-month-old adult mouse hearts. (B) Heat map of gene expression profiling obtained by Pitx2 CKO microarray assay. The color bar represents relative expression level of log-transformed value for each gene across different samples. Ctrl1-Ctrl3: control mouse heart (Pitx2 Flox/Flox); CKO1-CKO3: Pitx2 CKO mouse heart (Pitx2 Flox/Flox MCK-Cre). (C) Motif analysis for peaks from Pitx2 ChIP-Seq (n=11,280). Eriched DNA binding motifs and best matched factors are shown.
Figure 4
Figure 4
Validation of potential Pitx2 targets from ChIP-Seq and microarray assays. (A) Genome browser tracks for potential targets of Pitx2 in adult heart identified by ChIP-Seq. Peaks from ChIP-Seq, conservation with human genome and the chromosomal regions used in reporter assay are shown. Normalized ChIP-Seq tag numbers are shown on the Y-axis. (B) Alteration in expression levels for the potential Pitx2 targets were detected by realtime RT-PCR in the left atrium of 3 to 4-month-old control and Pitx2 conditional knock out mice. (n=6) (C) Reporter assay for ChIP-Seq identified potential targets of Pitx2. Chromosomal regions were named after the genes in closest proximity. Values and error bars represent mean and standard deviation (n=6).
Figure 5
Figure 5
Pitx2 CKO mouse heart has altered expression pattern for β-catenin and disrupted intercalated discs (IDs). (A-D) β-catenin immunostaining on left atrium sections of 3-month-old control (Pitx2 Flox/Flox) (A-B) and Pitx2 CKO (C-D) mice shows enhanced expression of β-catenin on cardiomyocytes lateral aspect (big arrows in D) and in cytoplasm (big arrows in C). Small arrows point to intercalated discs. (E-J) Electron microscopy shows disrupted IDs in 1-year-old Pitx2 CKO heart. (E-G) in control left atrium, IDs (big arrows) and Mitochondia were intact (triangles). (H-J) in Pitx2 CKO left atrium, some IDs were disrupted (arrowheads). Large number of mitochondria were deteriorated (asterisks). Indistinct Z-lines were observed in Pitx2 CKO left atrium (small arrows).
Figure 6
Figure 6
Model of Pitx2’s function in postnatal heart. In postnatal heart, Pitx2 binds to its targets through DNA binding domain and regulates expression of targets. Three groups of genes were identified to be targets of Pitx2. The first group contains genes regulating cell adhesion/cell junction assembly, which is critical for preserving normal structure of intercalated disc. The second group contains genes that regulate ion transportation in and between cardiomyocytes, which are important for normal conduction of electrical signal that induces heart contraction. The third group contains transcription regulators that regulate a large number of downstream targets and involved in regulating cardiac function and maintaining homeostasis of cardiomyocytes.
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References

    1. Benjamin EJ, Chen PS, Bild DE, Mascette AM, Albert CM, Alonso A, et al. Prevention of atrial fibrillation: Report from a national heart, lung, and blood institute workshop. Circulation. 2009;119:606–618. - PMC - PubMed
    1. Mahida S, Ellinor PT. New advances in the genetic basis of atrial fibrillation. J Cardiovasc Electrophysiol. 2012;23:1400–1406. - PMC - PubMed
    1. Husser D, Adams V, Piorkowski C, Hindricks G, Bollmann A. Chromosome 4q25 variants and atrial fibrillation recurrence after catheter ablation. J Am Coll Cardiol. 2010;55:747–753. - PubMed
    1. Wang J, Klysik E, Sood S, Johnson RL, Wehrens XH, Martin JF. Pitx2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification. Proc Natl Acad Sci U S A. 2010;107:9753–9758. - PMC - PubMed
    1. Chinchilla A, Daimi H, Lozano-Velasco E, Dominguez JN, Caballero R, Delpon E, et al. Pitx2 insufficiency leads to atrial electrical and structural remodeling linked to arrhythmogenesis. Circ Cardiovasc Genet. 2011;4:269–279. - PubMed

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