Atrial fibrillation risk loci interact to modulate Ca2+-dependent atrial rhythm homeostasis
Brigitte Laforest, … , Christopher R. Weber, Ivan P. Moskowitz
Brigitte Laforest, … , Christopher R. Weber, Ivan P. Moskowitz
Published October 14, 2019
Citation Information: J Clin Invest. 2019;129(11):4937-4950. https://doi.org/10.1172/JCI124231.
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Research Article Cardiology Genetics

Atrial fibrillation risk loci interact to modulate Ca2+-dependent atrial rhythm homeostasis

Abstract

Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.

Authors

Brigitte Laforest, Wenli Dai, Leonid Tyan, Sonja Lazarevic, Kaitlyn M. Shen, Margaret Gadek, Michael T. Broman, Christopher R. Weber, Ivan P. Moskowitz

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Figure 3

Antagonistic interactions between TBX5 and GATA4 on Atp2a2 and Ryr2 expression and on an Ryr2 enhancer.

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Antagonistic interactions between TBX5 and GATA4 on Atp2a2 and Ryr2 expr...
(A) Relative gene expression by qPCR of known AF calcium genes and calcium-interacting proteins from left atrium of R26CreERT2 (n = 6–10) Tbx5fl/+;R26CreERT2 (n = 5–7), Gata4fl/+;R26CreERT2 (n = 7–10), and Gata4fl/+;Tbx5fl/+;R26CreERT2 (n = 9–13) mice 2 weeks after TM treatment. Tbx5 heterozygotes showed 20% decrease in Atp2a2 and Ryr2 gene expression, which was normalized in Gata4/Tbx5 compound heterozygotes. Data are normalized to GAPDH and relative to R26CreERT2. P values were determined by 1-way ANOVA followed by Tukey post-hoc test. (B) Relative transcript expression of Pitx2 by qPCR in the left atrium of Tbx5 heterozygotes, Gata4 heterozygotes, and Gata4/Tbx5 compound heterozygotes 2 weeks after TM treatment. Data are represented as means ± SEM normalized to GAPDH and relative to R26CreERT2 mice (set as 1) (n = 6 R26CreERT2, n = 5 Tbx5fl/+;R26CreERT2, n = 5 Gata4fl/+;R26CreERT2, and n = 5 Gata4fl/+;Tbx5fl/+;R26CreERT2). Experiments were performed in technical duplicates. P value was determined by 1-way ANOVA followed by post-hoc Tukey test. (C and D) Atp2a2 and Ryr2 genomic locus (Mm9) aligned with published ATAC-seq dataset from HL-1 cardiomyocytes and ChIP-seq dataset for TBX5 and GATA4. Green rectangle denotes the cis-regulatory regions with overlapping open chromatin as well as TBX5- and GATA4-binding motifs. (E–G) In vitro luciferase response assay of Atp2a2 and Ryr2 candidate enhancers in HEK293T cells cotransfected with TBX5 and/or GATA4 or HL-1 atrial cardiomyocytes and corresponding GATA mutant enhancer. Data are means ± SEM, normalized to scrambled vector. Experiments were performed in technical triplicates (n = 5 for Atp2a2 enhancer; n = 4 for Ryr2 in HEK293T cells and n = 5 for Ryr2 in HL-1 cardiomyocytes). P values were determined by 1-way ANOVA followed by Tukey post-hoc test. *P < 0.05; **P < 0.01.