Loss of the Atrial Fibrillation-Related Gene, Zfhx3 , Results in Atrial Dilation and Arrhythmias

Abstract

Background: ZFHX3 , a gene that encodes a large transcription factor, is at the second-most significantly associated locus with atrial fibrillation (AF), but its function in the heart is unknown. This study aims to identify causative genetic variation related to AF at the ZFHX3 locus and examine the impact of Zfhx3 loss on cardiac function in mice. Methods: CRISPR-Cas9 genome editing, chromatin immunoprecipitation, and luciferase assays in pluripotent stem cell–derived cardiomyocytes were used to identify causative genetic variation related to AF at the ZFHX3 locus. Cardiac function was assessed by echocardiography, magnetic resonance imaging, electrophysiology studies, calcium imaging, and RNA sequencing in mice with heterozygous and homozygous cardiomyocyte-restricted Zfhx3 deletion ( Zfhx3 Het and knockout, respectively). Human cardiac single-nucleus ATAC-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by ZFHX3 . Results: We found SNP rs12931021 modulates an enhancer regulating ZFHX3 expression, and the AF risk allele is associated with decreased ZFHX3 transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than Zfhx3 Het and wild-type mice, with alterations in conduction velocity, atrial action potential duration, calcium handling and the development of atrial enlargement and thrombus, and dilated cardiomyopathy. Zfhx3 loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF. Conclusions: Our findings implicate ZFHX3 as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac Zfhx3 are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for Zfhx3 in the control of cardiac genes and signaling pathways essential for normal atrial function.

Role of Genetic Variation in Transcriptional Regulatory Elements in Heart Rhythm

“…The locus harboring ZFHX3 is associated with AF [ 22 , 57 , 58 ]. Recently, loss-of-function experiments in mice revealed that Zfhx3 regulates a large number of atrial genes and signaling pathways required to maintain normal atrial function [ 35 ]. Loss of Zfhx3 resulted in atrial dilatation and arrhythmia.…”

“…Which of the associated variants causes the AF risk remained elusive. However, rs12931021, in an intron of ZFHX3 , was observed to influence activity of a putative RE and influenced the epigenetic state of that RE in hiPSC-cardiomyocytes [ 35 ]. Moreover, when the RE was deleted in hiPSC-cardiomyocytes, ZFHX3 expression was reduced.…”

Connecting the Dots From GWAS to Function in Atrial Fibrillation for ZFHX3

“…Functional genomics, cellular, and mouse studies are all required to go from the GWAS locus to the responsible gene and variant, and the disease mechanism, and Jameson et al 6 have elegantly accomplished these tasks. Their compelling findings demonstrate that Zfhx3 is a causal gene for AF in mice, providing a rational explanation for the association between genetic variants near ZFHX3 and AF in humans.…”

“…In this issue of Circulation Research, Jameson et al 6 have performed just these tasks, focusing on the second most significant locus associated with AF, on chromosome 16q22 (Figure). The lead AF-associated SNP at this locus, rs2106261, resides in ZFHX3 gene's intron, but that is insufficient to conclude ZFHX3 as the causal gene.…”

“…Nevertheless, now that Jameson et al, 6 identified the responsible gene and variant, they needed to identify the mechanism by which reduced expression of ZFHX3 leads to AF susceptibility. This required an animal model, and the easiest one to create and study is the mouse; thus, heart-specific Zhfx3 heterozygous and homozygous knockout mice were created by breeding Zfhx3 floxed mice with mice carrying a cardiomyocyte expressed Cre transgene.…”