Pitx2 impairs calcium handling in a dose-dependent manner by modulating Wnt signalling.

Atrial fibrillation (AF) is the most common type of arrhythmia in humans, yet the genetic cause of AF remains elusive. Genome-wide association studies (GWASs) have reported risk variants in four distinct genetic loci, and more recently, a meta-GWAS has further implicated six new loci in AF. However, the functional role of these AF GWAS-related genes in AF and their inter-relationship remain elusive. To get further insights into the molecular mechanisms driven by Pitx2, calcium handling and novel AF GWAS-associated gene expression were analysed in two distinct Pitx2 loss-of-function models with distinct basal electrophysiological defects; a novel Pitx2 conditional mouse line, Sox2CrePitx2, and our previously reported atrial-specific NppaCrePitx2 line. Molecular analyses of the left atrial appendage in NppaCrePitx2(+/-) and NppaCrePitx2(-/-) adult mice demonstrate that AF GWAS-associated genes such as Zfhx3, Kcnn3, and Wnt8a are severely impaired but not Cav1, Synpo2l, nor Prrx1. In addition, multiple calcium-handling genes such as Atp2a2, Casq2, and Plb are severely altered in atrial-specific NppaCrePitx2 mice in a dose-dependent manner. Functional assessment of calcium homeostasis further underscores these findings. In addition, multiple AF-related microRNAs are also impaired. In vitro over-expression of Wnt8, but not Zfhx3, impairs calcium handling and modulates microRNA expression signature identified in Pitx2 loss-of-function models. Our data demonstrate a dose-dependent relation between Pitx2 expression and the expression of AF susceptibility genes, calcium handling, and microRNAs and identify a complex regulatory network orchestrated by Pitx2 with large impact on atrial arrhythmogenesis susceptibility.