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Abstract
Background Atrial fibrillation (AF) is the most common clinical arrhythmia, although the pathophysiology remains poorly understood. Population-based genetic studies have identified a susceptibility locus for AF at the gene ZFHX3.
Hypothesis We hypothesized that a cardiac-restricted knockout of the transcription factor ZFHX3 in a mammalian model organism would perturb normal cardiac development and function, and illuminate the role of ZFHX3 in AF.
Methods and results We generated a murine cardiac-restricted knockdown of ZFHX3 using cre-lox recombination. Knockdown was confirmed by organ specific genotyping. Among knockdown mice, increased inducibility of atrial arrhythmias was observed at in vivo electrophysiology testing at 3 months of age (% arrhythmia induction maneuvers 10 vs 0, p<0.001). Cardiac structure and function was normal in the knockdown mice at 3 months. Premature mortality was noted among the knockdown mice, with death occurring at 9–10 months of age (cox proportional HR sp<0.01) (figure 1). Cardiac magnetic resonance imaging revealed severe cardiomyopathy prior to death, with impaired left ventricular function and atrial enlargement. Histology of the affected hearts revealed severely dilated and fibrosed atria containing a large mass consistent with thrombus. Immunofluorescence of mouse hearts from early embryonic to adult stages confirmed cardiac expression of ZFHX3, with a marked atrial predominance. Optical mapping of Langendorff-perfused hearts demonstrated increased conduction velocity in knockdown atria (p<0.01) with a trend toward shorter action potential duration. Whole-RNA sequencing of atrial tissue revealed disruption of the left-right axis, with the left atrium transcript profile of knockdown animals more closely resembling that of the control right atrium, and vice versa. Several genes important for sidedness were differentially expressed, including SFRP5, an inhibitor of Wnt signaling. SFRP5 was downregulated in the knockdown left atria (FDR=1.6 × 10-11) and upregulated in the knockdown right atria (FDR=1.1 × 10-12). Functional analysis further implicated differential control of the hedgehog signaling pathway as the most significantly enriched pathway for differentially expressed genes common to both atria (BH value=7.9 × 10-6), with global upregulation in the left atrium and downregulation in the right atrium. Functional analysis of differentially expressed genes by atrium also revealed abnormalities in calcium ion binding and ion transmembrane transporter activity.
Structural and EP characteristics. A) and B) 2-D echocardiography and in vivo electrophysiology studies showing similar basic characteristics between control and knockdown animals; C) intracardiac recordings of atrial arrhythmias induced in knockdown mice; D) quantification of arrhythmia burden
A) Kaplan-Meier plot of increased mortality in knockdown mice; B) Masson-Trichrome stain for knockdown heart at 9 months showing atrial dilatation, fibrosis and mass; C) immunofluorescence showing atrial expression of ZFHX3; D) principle component analysis plot of RNA sequencing showing reduction in Euclidean distance between knockdown left atrium and control right atrium
Conclusion We have uncovered a role for ZFHX3 in the left-right patterning of cardiac atria. Disruption of this developmental process predisposes to atrial cardiomyopathy, affects atrial electrophysiology properties, changes which may increase the susceptibility to AF.