Research Article

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility

Department of Medical Biology, Amsterdam Cardiovascular Sciences, Amsterdam Reproduction and Development, Amsterdam University Medical Centers, University of Amsterdam, Netherlands
Department of Experimental Cardiology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, Netherlands
Department of Cardiology, Severance Hospital, College of Medicine, Yonsei University, Republic of Korea
Department of Physiology, University of Maastricht, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Netherlands

Jan 30, 2023

https://doi.org/10.7554/eLife.80317

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Version of Record published: February 14, 2023 (This version)
Accepted Manuscript published: January 30, 2023 (Go to version)
Accepted: January 29, 2023
Received: May 27, 2022
Preprint posted: May 14, 2022 (Go to version)

Figures
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Additional files

6 figures, 1 table and 9 additional files

Figures

Figure 1 with 1 supplement

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AF-associated noncoding variants are found in the *TBX5* locus.

(A) Hi-C heatmap from human lymphoblastoid line GM12878 shows AF-associated variants are found in the regulatory domain of *TBX5*. Zoom-in of the AF-associated region overlaid with promoter capture Hi-C (red arcs), regions of open chromatin in whole left atria (ATACseq LA), H3K27ac and H3K4me1 ChIPseq signatures in cardiomyocytes. (B) Mouse orthologue of the human region including ATACseq from left atrial and ventricular CMs, H3K27ac ChIPseq from whole juvenile hearts, EMERGE, and conservation tracks. CRISPR/Cas9-generated deletions in light gray (REint), and dark gray (REdown).

Figure 1—figure supplement 1

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AF-associated variants near *TBX5* are grouped into distinct haplotypes.

Linkage disequilibrium (LD) matrix plot of SNPs located at the *TBX5* locus associated with AF. The SNPs found in the last intron and downstream of the gene are clustered into two distinct haplotypes based on minimum linkage disequilibrium r2 of 0.1. The plot was generated using the LD matrix tool al ldlink.nci.nih.gov. Scale bar depicted above and below gene.

Figure 2 with 3 supplements

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Deletion of AF-associated regions results in increased *Tbx5* in adult atrial tissue.

(A) *Tbx5* expression in atria, ventricles, and lungs from P21 control, *Tbx5^RE(int)KO*, and *Tbx5^RE(down)KO* mice determined by RT-qPCR. (**B–C**) *Tbx5* expression levels in adult control and *Tbx5^RE(int)KO* (B) or *Tbx5^RE(down)KO* (C) left and right atria. (D) UCSC browser view of the human intronic region (gray) and tested fragments containing AF-associated SNPs (blue) overlaid with chromatin conformation, EMERGE, and conservation tracks. (E) Luciferase assay (n=6) shows enhancer activity differences between non-risk (dark gray) or risk (gray) alleles (Kruskal-Wallis p=0.0019). Statistical significance within each tissue type was determined with ANOVA followed by pairwise comparisons using Dunnett’s T3 multiple comparison test in A, unpaired t-tests in B, C, and Kruskal-Wallis tests followed by pairwise unpaired t-tests (p values shown in figure) in E.

Figure 2—figure supplement 1

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No difference in transcript levels of genes adjacent to *Tbx5* in deletion mutants.

(A) Fetal (E14.5) expression levels of *Tbx5* were normalized to controls within each litter. (B) Reference gene-corrected expression levels of *Rbm19*, *Tbx3*, and *Med13l* in juvenile whole atria (left) and ventricles (right) in WT (n=5), *Tbx5^RE(int)KO* (n=6), and *Tbx5^RE(down)KO* (n=6). Statistical significance within each gene and tissue type was determined with Kruskal-Wallis tests.

Figure 2—figure supplement 2

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Tbx5 is selectively expressed in cardiomyocyte nuclei in atria of WT and *Tbx5^RE(int)KO* mice.

(A) Immunoflourescence staining on paraffin sections of adult left atria for Tbx5 (red), PCM-1 which marks cardiomyocytes nuclei (green), and DAPI (blue). Red arrowheads point to non-cardiomyocyte (Tbx5-negative, PCM-1-negative) nuclei, and yellow arrowheads point to Tbx5 +cardiomyocytes. Scale bar is 50 µm.

Figure 2—figure supplement 3

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*Tbx5* splicing is not affected in deletion mutants.

(A) UCSC browser view shows exon read coverage including the ones flanking the intronic deletion is similar across genotypes. (B) RTq-PCR of WT and *Tbx5^RE(int)KO* (HOM) left and right atria using primers spanning exons 6–8 and 8–9. (C) Resulting amplicon sizes are not different in homozygous mice.

Figure 2—figure supplement 3—source data 1 Raw gel for Figure 2—figure supplement 3.: https://cdn.elifesciences.org/articles/80317/elife-80317-fig2-figsupp3-data1-v2.zip
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Figure 3 with 1 supplement

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Increased *Tbx5* in adult atria results in altered in vivo electrophysiology.

(**A–C**) Graphs show individual and average ECG measurements for heart rate (RR) (A), heart rate variation (SDNN) (B), and PR-interval (C) of adult *wild-type*, *Tbx5^RE(int)KO*, and *Tbx5^RE(down)KO* mice. (**D, E**) Graphs show changes in heart rate-corrected sinus node recovery times at 120ms (cSNRT₁₂₀) (D), and Wenckebach cycle length (WBCL) (E). (F) Bar graph depicts the total time each mouse spent in an atrial arrhythmia (AA) episode after two pacing passes, with the number of mice in which at least one episode lasting >1 s was observed below each bar. (G) Representative traces from *wild-type* (top) and two *Tbx5^RE(int)KO* (bottom) individuals showing disappearance of p waves or the start of atrial arrhythmia with variability in ventricular response after pacing stimulus. Significance for in vivo parameters was determined with Kruskal-Wallis test followed by Dunn’s multiple comparison tests in A, B, D, and AA duration in F (top graph), and one-way ANOVA followed by Tukey’s multiple comparisons test in C and E. AA inducibility significance was determined with pairwise Fisher’s exact test (F bottom of graph).

Figure 3—figure supplement 1

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Additional in vivo electrophysiology parameters.

(A) Lead 2 traces of WT, *Tbx5^RE(int)KO*, and *Tbx5^RE(down)KO* mice showing slow and variable RR, a sinus pause (arrowhead), or abnormal P waves (asterisk). (B) Graph shows no significant change in QRS duration in either mutant. Statistical significance was determined in B with Kruskal-Wallis test.

Figure 4

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Atrial function abnormalities in *Tbx5^RE(int)KO* mice.

(A) Typical action potentials (APs) and upstroke velocity measured at 6 Hz in single left atrial cardiomyocytes using the amphotericin-perforated patch clamp technique. (B) Average resting membrane potential (RMP), AP amplitude (APA), and AP upstroke velocity remain unchanged in mutants. (C) AP duration at 20, 50, and 90% of repolarization (APD₂₀, APD₅₀, APD₉₀, respectively) is increased in mutants. (D) APD₉₀ was prolonged at all tested frequencies in mutant left atrial cardiomyocytes. Error bars are SD. (E) Diastolic and systolic intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were not changed in *Tbx5^RE(int)KO* compared to controls. Statistical significance in B was determined with unpaired t-tests with Welch’s correction, and unpaired t-tests were used in B. Experimental groups were compared using two-way repeated measures ANOVA (**D, E**), followed by pairwise unpaired t-tests (C). Error bars are SEM.

Figure 5 with 4 supplements

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Transcriptomic analysis of *Tbx5^RE(int)KO* left atria.

(**A, B**) Volcano plot showing differentially expressed transcripts in *Tbx5^RE(int)KO* (n=3)(A) and *Tbx5^RE(down)KO* (n=3) (B) from *wild-type* (n=4) left atria. Dark red and light red dots indicate significantly upregulated genes by raw p-value and p-adjusted for multiple testing, respectively. Correspondingly, dark blue and light blue dots indicate downregulated genes. p Values were adjusted for multiple testing using the false discovery rate (FDR) method of Benjamini-Hochberg. (C) X-Y plot of all transcripts in *Tbx5^RE(int)KO* (x axis) and *Tbx5^RE(down)KO* (y axis), with deregulated genes common to both deletion mutants in purple, *Tbx5^RE(int)KO* deregulated genes in blue, and *Tbx5^RE(down)KO* deregulated genes in red. (D) Gene ontology (GO) analysis of upregulated and downregulated genes in *Tbx5^RE(int)KO* and *Tbx5^RE(down)KO* mutants. (F) Graph depicts fold change expression in control and mutant samples of genes known to affect action potential duration. * denotes significantly deregulated in one or both mutant lines.

Figure 5—figure supplement 1

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Ion channel expression in left atria of deletion mutants.

(A) Graph depicts differential ion channel expression (fold change) in control and mutant left atria. RPBPL; Reads per (transcript) base pair length.

Figure 5—figure supplement 2

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Comparison of genes differentially expressed between left atria of *Tbx5^RE(int)KO* and control mice, *Tbx5^G125R/+* and control mice and *Tbx5 iKO* and control mice.

(**A–B**) Scatterplots of Log₂(Fold Change) of transcripts in *Tbx5^RE(int)KO* (x axis) and (A) *Tbx5^G125R/+* or (B) *Tbx5 iKO* (y axis), with deregulated genes (Padj <0.05) common to both genotypes in purple, *Tbx5^RE(int)KO* deregulated genes in blue and *Tbx5^G125R/+* or *Tbx5 iKO* deregulated genes in red.

Figure 5—figure supplement 3

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*Pitx2* expression is not affected in *Tbx5^RE(int)KO* atria.

(A) RT-qPCR on left and right atria of controls and deletion mutants for *Pitx2*. Statistical significance was determined by unpaired t-tests (no significant differences detected).

Figure 5—figure supplement 4

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Modest *Tbx5* upregulation minimally alters genome-wide chromatin accessibility.

(A) Heatmaps depicting vertically sorted up or downregulated accessible regions spanning 1500 bp bins (Padj <0.05) in *Tbx5^RE(int)KO* (n=4) vs WT (n=4) cardiomyocytes. (B) UCSC browser view of the *Tbx5* locus showing differential accessibility profiles in mutants.

Figure 6 with 1 supplement

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A genetic interaction between *Tbx5* and *Prrx1*.

(**A, B**) Graph shows reference gene-corrected expression of *Tbx5* (A), and *Prrx1* (B) in PCM1-positive and -negative nuclei fractions of *wild-type*, *Tbx5^RE(int)KO*, *Prrx1^(enh)KO* and *double* mutant whole hearts determined by RT-qPCR. (C) Principal component analysis of transcriptomes of *wild-type*, *Tbx5^RE(int)KO* and *Double*KO left atrial samples. (**D–H**) Graphs show individual and average ECG measurements for RR (D), heart rate variation (SDNN) (E), PR interval (F), cSNRT at 120ms pacing (G), and WBCL (H) of *wild-type*, *Tbx5^RE(int)KO*, *Prrx1^(enh)KO* and *Double*KO mice. (I) Bar graph depicts the total time each mouse spent in an AA episode after two pacing passes, with the number of mice in which at least one episode lasting >1 s was observed below each bar. Statistical significance was determined using one-way ANOVA followed by Tukey’s test for pairwise comparisons in A, B, F, H, Kruskal-Wallis test followed by Dunn’s test for pairwise comparisons in D, E, G, and AA duration in I (top of graph), Fisher’s exact test for pairwise comparisons of AA inducibility in I (bottom of graph).

Figure 6—figure supplement 1

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Comparison of genes differentially expressed between left atria of *Prrx1^(enh)KO* and control mice, *Tbx5^RE(int)KO* and control mice, and *Double homozygous* mutants and control mice.

(**A–B**) X-Y plot of all transcripts in *Prrx1^(enh)KO* (A) or *Tbx5^RE(int)KO* (B) (x axis) and *Double homozygous* (y axis), with deregulated genes (Padj <0.05) common to both genotypes in purple, *Prrx1^(enh)KO,* or *Tbx5^RE(int)KO* deregulated genes in red and *Double homozygous* deregulated genes in blue. (C) Plot depicts comparison between *Tbx5^RE(int)KO* (x axis) and *Prrx1^(enh)KO* (y axis). (D) Box plots showing differences in Log₂(Fold Change) distribution of *Prrx1^(enh)KO, Tbx5^RE(int)KO*, and *Double homozygous* left atria.

Tables

Table 1

Gene ontology (GO) analysis of upregulated and downregulated genes in Tbx5^RE(int)KO, Prrx1^(enh)KO, and Double homozygous adult left atria.

	Prrx1(enh)KO				Tbx5RE(int)KO				Double
GO biological process	UP/DOWN	Fold enrich	P-value	FDR	UP/DOWN	Fold enrich	P-value	FDR	UP/DOWN	Fold enrich	P-value	FDR
Regulation of biological quality (GO:0065008)	DOWN	1.64	1.7E-08	3E-06	UP	1.55	1.5E-10	8E-07	UP	1.56	3.4E-08	1.5E-05
Localization (GO:0051179)	DOWN	2.11	1.5E-14	1.6E-11	UP	1.45	1.7E-09	6.5E-06	UP	1.44	2.9E-07	8E-05
Developmental process (GO:0032502)	UP	2.33	6.1E-20	1.6E-16	UP	1.39	6.8E-09	2.1E-05	UP	1.61	1.8E-14	2.9E-10
Cellular component organization (GO:0016043)	DOWN	1.85	4.3E-08	6.9E-06	UP	1.41	1.4E-08	3.1E-05	UP	1.54	1.3E-10	2.2E-07
Regulation of ion transport (GO:0043269)	DOWN	2.68	8.7E-08	1.3E-05	UP	2.22	1.7E-07	0.00018	UP	2.36	7.6E-07	0.00018
Regulation of cell communication (GO:0010646)	DOWN	2.18	2.8E-18	1.1E-14	UP	1.48	3.3E-07	0.00029	UP	1.57	3.4E-07	8.9E-05
Positive regulation of cell projection organization (GO:0031346)	DOWN	2.07	0.00033	0.0158	UP	2.44	1.5E-06	0.001	UP	2.08	3.9E-07	0.0001
Cardiac conduction (GO:0061337)	DOWN	7.1	0.00011	0.00597	UP	6.57	3.6E-06	0.00203	UP	7.36	1E-05	0.0015
Cell differentiation (GO:0030154)	UP	2.25	2.8E-11	8.2E-09	UP	1.39	7.9E-06	0.00276	UP	1.65	4.8E-10	5.8E-07
Metal ion transport (GO:0030001)	DOWN	3.43	4.5E-08	7.3E-06	UP	2.18	1E-05	0.00342	UP	2.25	0.00069	0.049
Regulation of cell motility (GO:2000145)	UP	2.96	2.1E-14	1.2E-11	DOWN	3.35	2.3E-38	9.1E-35	DOWN	2.28	2.5E-05	0.00564
Extracellular matrix organization (GO:0030198)	UP	7.57	2E-24	3.1E-20	DOWN	6.12	3.5E-34	4.2E-31	DOWN	7.87	3.2E-17	5.1E-13
Circulatory system development (GO:0072359)	UP	3.3	2.8E-17	3.7E-14	DOWN	3.2	3.3E-32	2.5E-29	DOWN	3.14	2.3E-10	9.1E-07
Vasculature development (GO:0001944)	UP	4.11	4.6E-17	4.8E-14	DOWN	3.97	1.6E-31	1E-28	DOWN	3.23	3.2E-07	0.00017
Regulation of response to stimulus (GO:0048583)	UP	1.91	1.8E-17	2.6E-14	DOWN	1.79	1.3E-28	5.8E-26	DOWN	1.61	2.2E-06	0.00072
Actin cytoskeleton organization (GO:0030036)	DOWN	3.17	4.6E-08	7.3E-06	DOWN	3.91	4.9E-28	1.9E-25	DOWN	3.26	7.9E-07	0.00034
Cell adhesion (GO:0007155)	UP	2.92	3.9E-12	1.5E-09	DOWN	3.1	1.8E-27	6.6E-25	DOWN	2.99	1.1E-08	1.9E-05
Biological adhesion (GO:0022610)	UP	2.94	2E-12	8.2E-10	DOWN	3.06	4.9E-27	1.6E-24	DOWN	2.95	1.4E-08	2E-05
Angiogenesis (GO:0001525)	UP	4.5	4.9E-12	1.8E-09	DOWN	4.87	4.9E-27	1.6E-24	DOWN	4.17	3.7E-07	0.00019
Animal organ development (GO:0048513)	UP	2.03	8.4E-17	7.4E-14	DOWN	1.84	1.3E-23	3.3E-21	DOWN	1.78	3.4E-07	0.00018

Additional files

Supplementary file 1 rs7312625 motif analysis +- 30 bp. Changed motifs in red.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp1-v2.xlsx
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Supplementary file 2 DESeq2 result WT vs Tbx5^RE(int)KO adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp2-v2.xlsx
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Supplementary file 3 DESeq2 result WT vs Tbx5^RE(down)KO adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp3-v2.xlsx
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Supplementary file 4 RNAseq comparison of Tbx5^RE(down)KO and Tbx5^G125R/+ adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp4-v2.xlsx
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Supplementary file 5 RNAseq comparison of Tbx5^RE(down)KO and Tbx5iKO adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp5-v2.xlsx
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Supplementary file 6 DESeq2 result WT vs Double adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp6-v2.xlsx
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Supplementary file 7 DESeq2 result WT v Prrx1^(enh)KO adult left atria.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp7-v2.xlsx
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Supplementary file 8 Deletion coordinates in mm10.: https://cdn.elifesciences.org/articles/80317/elife-80317-supp8-v2.xlsx
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MDAR checklist: https://cdn.elifesciences.org/articles/80317/elife-80317-mdarchecklist1-v2.docx
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Fernanda M Bosada
Karel van Duijvenboden
Alexandra E Giovou
Mathilde R Rivaud
Jae-Sun Uhm
Arie O Verkerk
Bastiaan J Boukens
Vincent M Christoffels

(2023)

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility

eLife 12:e80317.

https://doi.org/10.7554/eLife.80317

Figures

AF-associated noncoding variants are found in the TBX5 locus.

AF-associated variants near TBX5 are grouped into distinct haplotypes.

Deletion of AF-associated regions results in increased Tbx5 in adult atrial tissue.

No difference in transcript levels of genes adjacent to Tbx5 in deletion mutants.

Tbx5 is selectively expressed in cardiomyocyte nuclei in atria of WT and Tbx5^RE(int)KO mice.

Tbx5 splicing is not affected in deletion mutants.

Figure 2—figure supplement 3—source data 1

Increased Tbx5 in adult atria results in altered in vivo electrophysiology.

Additional in vivo electrophysiology parameters.

Atrial function abnormalities in Tbx5^RE(int)KO mice.

Transcriptomic analysis of Tbx5^RE(int)KO left atria.

Ion channel expression in left atria of deletion mutants.

Comparison of genes differentially expressed between left atria of Tbx5^RE(int)KO and control mice, Tbx5^G125R/+ and control mice and Tbx5 iKO and control mice.

Pitx2 expression is not affected in Tbx5^RE(int)KO atria.

Modest Tbx5 upregulation minimally alters genome-wide chromatin accessibility.

A genetic interaction between Tbx5 and Prrx1.

Comparison of genes differentially expressed between left atria of Prrx1^(enh)KO and control mice, Tbx5^RE(int)KO and control mice, and Double homozygous mutants and control mice.

Tables

Gene ontology (GO) analysis of upregulated and downregulated genes in Tbx5^RE(int)KO, Prrx1^(enh)KO, and Double homozygous adult left atria.

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

Supplementary file 8

MDAR checklist

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AF-associated noncoding variants are found in the TBX5 locus.

AF-associated variants near TBX5 are grouped into distinct haplotypes.

Deletion of AF-associated regions results in increased Tbx5 in adult atrial tissue.

No difference in transcript levels of genes adjacent to Tbx5 in deletion mutants.

Tbx5 is selectively expressed in cardiomyocyte nuclei in atria of WT and Tbx5RE(int)KO mice.

Tbx5 splicing is not affected in deletion mutants.

Figure 2—figure supplement 3—source data 1

Increased Tbx5 in adult atria results in altered in vivo electrophysiology.

Additional in vivo electrophysiology parameters.

Atrial function abnormalities in Tbx5RE(int)KO mice.

Transcriptomic analysis of Tbx5RE(int)KO left atria.

Ion channel expression in left atria of deletion mutants.

Comparison of genes differentially expressed between left atria of Tbx5RE(int)KO and control mice, Tbx5G125R/+ and control mice and Tbx5 iKO and control mice.

Pitx2 expression is not affected in Tbx5RE(int)KO atria.

Modest Tbx5 upregulation minimally alters genome-wide chromatin accessibility.

A genetic interaction between Tbx5 and Prrx1.

Comparison of genes differentially expressed between left atria of Prrx1(enh)KO and control mice, Tbx5RE(int)KO and control mice, and Double homozygous mutants and control mice.

Gene ontology (GO) analysis of upregulated and downregulated genes in Tbx5RE(int)KO, Prrx1(enh)KO, and Double homozygous adult left atria.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

Supplementary file 8

MDAR checklist

Downloads (link to download the article as PDF)

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Tbx5 is selectively expressed in cardiomyocyte nuclei in atria of WT and Tbx5^RE(int)KO mice.

Atrial function abnormalities in Tbx5^RE(int)KO mice.

Transcriptomic analysis of Tbx5^RE(int)KO left atria.

Comparison of genes differentially expressed between left atria of Tbx5^RE(int)KO and control mice, Tbx5^G125R/+ and control mice and Tbx5 iKO and control mice.

Pitx2 expression is not affected in Tbx5^RE(int)KO atria.

Comparison of genes differentially expressed between left atria of Prrx1^(enh)KO and control mice, Tbx5^RE(int)KO and control mice, and Double homozygous mutants and control mice.

Gene ontology (GO) analysis of upregulated and downregulated genes in Tbx5^RE(int)KO, Prrx1^(enh)KO, and Double homozygous adult left atria.