Atrial identity is determined by a COUP-TFII regulatory network - PubMed (original) (raw)

Atrial identity is determined by a COUP-TFII regulatory network

San-pin Wu et al. Dev Cell. 2013.

Abstract

Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, the factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP-TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T tubules. Changes in atrial characteristics are accompanied by alterations of 2,584 genes, of which 81% were differentially expressed between atria and ventricles, suggesting that a major function of myocardial COUP-TFII is to determine atrial identity. Chromatin immunoprecipitation assays using E13.5 atria identified classic atrial-ventricular identity genes Tbx5, Hey2, Irx4, MLC2v, MLC2a, and MLC1a, among many other cardiac genes, as potential COUP-TFII direct targets. Collectively, our results reveal that COUP-TFII confers atrial identity through direct binding and by modulating expression of a broad spectrum of genes that have an impact on atrial development and function.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Morphology of the ventricularized atria

(A–J) Hearts of 2 months old adult mice staining for MLC2a and MLC2v in denoted genotypes. Hematoxylin was utilized for counterstaining. Scale bars indicate 500 µm. (K–L) Bar graphs showing mean length (K) and width (L) of isolated cardiomyocytes. (M–O) Confocal fluorescent images of representative di-8-ANEPPS-stained myocytes with genotypes denoted at side. (P) Index of the spatial integrity of T-Tubules (TT-power). Each genotype comprised of 3 animals at 2 months old. Error bars denote standard error of mean. ***, p<0.001. ra, right atria; rv, right ventricles; la, left atria; lv, left ventricles. See also Figure S1.

Figure 2

Figure 2. Molecular profile of ventricularized atria in CKO hearts

(A) Venn diagram to compare the genes up-regulated in CKO atria (black circle) and ventricular genes (grey circle). (B) Venn diagram to compare the genes down-regulated in CKO atria (black circle) and atrial genes (grey circle). (C–H) Relative mRNA levels of marker genes in adult atria (n=4 for each genotype). Error bars denote standard error of mean. ***, p<0.001. See also Tables S1, S2 and S3.

Figure 3

Figure 3. Electric properties of adult CKO myocytes and hearts

(A–C) Representative diagrams of action potential recording from isolated CTRLA (A), CKOA (B) and CTRLV (C) myocytes. (D) Mean action potential durations of isolated cardiomyocytes at APD90. (E&F) Representative surface and intra-atrial ECG during programmed induction of AF in CTRL (E) and CKO (F) mice. N (numbers of cells) =15 for CTRLA, 15 for CKOA and 7 for CTRLV within each bar from 3 animals per group. Error bars denote standard error of mean. ***, p<0.001; mV, millivolt; ms, millisecond. See also Figure S2 and Tables S4 and S5.

Figure 4

Figure 4. Temporal analysis of identity switch

(A–H) Heart regions of CKO and control mice stained for MLC2v with age denoted on the side. (I) qRTPCR for MLC2v mRNA levels in atria from E9.5 to 2 months old adult mice. Fold changes of CKO are normalized to the corresponding control at the same age. Asterisks denote significant differences between genotypes of the same age. (J–M) Heart regions E18.5 of CKOMyh6-MCM and corresponding control mice stained for MLC2v. E18.5TamE12.5 and E18.5TamE15.5, embryos received tamoxifen at E12.5 and E15.5, respectively. Inserts in (K&M), double staining of MLC2v (red) and β-gal (green) with DAPI for nuclei staining on an adjacent section of (K&M), respectively. Hematoxylin was utilized for counterstaining in all sections except inserts in (K& M). Error bars denote standard error of mean. ***, p<0.001. ra, right atria; rv, right ventricles; la, left atria; lv, left ventricles. See also Figure S3.

Figure 5

Figure 5. Regulation of atrial/ventricular identity transcription factors by COUP-TFII

(A–I) Relative mRNA levels of denoted genes from pooled E14.5 CTRL (solid bar) and CKO (open bar) atria measured by qPCR. Each group comprised two independent pools of atrial samples. Levels were shown as relative folds over control. (J–L) ChIP-PCR assays on pooled E13.5 atria using anti-COUP-TFII antibody (solid bar) or IgG (open bar). Diagrams on the top indicated COUP-TFII binding sites. Bar graphs show enrichment of DNA fragments pulled down by antibodies. N indicates a region in the Hbb locus without COUP-TFII or Sp1 binding sites served as negative control. Error bars denote standard error of mean. *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S4.

Figure 6

Figure 6. Identification of global COUP-TFII binding targets in atria

(A) Enriched functional annotation terms by DAVID Bioinformatics Sources from ChIP-seq identified genes that contain COUP-TFII binding sites in E13.5 atria. (B–G) Expression levels of denoted genes in E14.5 CKO atria compared with control. (H) The ChIP-seq result of COUP-TFII binding in the MLC2v (Myl2) locus. COUP-TFII binding motifs are shown in capital letters in boxes. (I) ChIP-qPCR results on pooled E13.5 atria using anti-COUP-TFII antibody (solid bar) or IgG (open bar). Bar graphs show enrichment of DNA fragments pulled down by antibodies. N indicates a region in the Hbb locus without COUP-TFII binding sites served as negative control. Error bars denote standard error of mean. *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S5 and Table S6.

Figure 7

Figure 7. Atrialized ventricular myocytes by COUP-TFII overexpression

Adjacent coronal sections of E17.5 control (A & B) and COUP-TFII overexpression (C & D) hearts stained for MLC2a (A & C) and MLC2v (B & D). (E & F) High power view of adjacent section corresponding to areas being bracketed in (C and D) that are double-stained for COUP-TFII (green) with MLC2a (E) or MLC2v (F) in red. Nuclei were stained in blue by DAPI. (G–I) Relative mRNA levels of Tbx5 (G), Hey2 (H) and Irx4 (I) from E17.5 CTRL (black bar) and COUP-TFII overexpression (OE, grey bar) ventricles measured by qRTPCR. Each groups consisted of 3 individual ventricles. Levels are shown as relative folds over control. Error bars denote standard error of mean. **, p<0.01; ***, p<0.001.

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