Loss of the Atrial Fibrillation-Related Gene, <i>Zfhx3</i>, Results in Atrial Dilation and Arrhythmias.

Abstract

<i>ZFHX3</i> (zinc finger homeobox 3), 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 <i>ZFHX3</i> locus and examine the impact of <i>Zfhx3</i> loss on cardiac function in mice. 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 <i>ZFHX3</i> 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 <i>Zfhx3</i> loss (<i>Zfhx3</i> Het and knockout, respectively). Human cardiac single-nucleus ATAC (assay for transposase-accessible chromatin)-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by <i>ZFHX3</i>. We found single-nucleotide polymorphism (SNP) rs12931021 modulates an enhancer regulating <i>ZFHX3</i> expression, and the AF risk allele is associated with decreased <i>ZFHX3</i> transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than <i>Zfhx3</i> 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. <i>Zfhx3</i> loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF. Our findings implicate <i>ZFHX3</i> as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac <i>Zfhx3</i> are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for <i>Zfhx3</i> in the control of cardiac genes and signaling pathways essential for normal atrial function.