Ectopic Pitx2 expression and sinoatrial node dysfunction in mice recapitulating a novel cardiac syndrome identified in unrelated families with deletions in a gene desert on chromosome 4q25

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Councin/NWO Introduction Several unrelated families presenting with a complex, heterogeneous cardiac syndrome including sinoatrial node dysfunction and atrial fibrillation were found to share overlapping deletions in a 1.5 Megabase pair gene desert on chromosome 4q25. These overlapping regions contain evolutionarily conserved CTCF binding sites that modulate the separation of topologically associating domains containing the transcription factor-encoding gene PITX2 and noncoding RNA genes. Aim To understand mechanistically how the deletion in the gene desert causes sinoatrial node dysfunction and atrial arrhythmias. Methods Mice lacking the orthologue of the minimally overlapping genomic region deleted in affected patients were generated. Electrophysiology, morphology, tissue composition, and transcriptomes were analysed in this mouse model. Results These mice recapitulated major morpho-functional features of the syndrome, including sinoatrial node dysfunction and atrial arrhythmogenesis . Out of the 15 genes flanking the gene desert, only Pitx2 expression was deregulated in the sinoatrial node. During development, cardiac Pitx2c expression is normally restricted to the left side where it drives left sinus venosus and left atrial morphogenesis and prevents the formation of a left-sided sinoatrial node. The PITX2 locus has also been associated with atrial fibrillation in GWAS. Immunohistochemistry together with 3D volume analysis showed that Pitx2c was ectopically expressed in half of the pacemaker cardiomyocytes of the developing and postnatal sinoatrial node while its expression in the left atrium was maintained. Key transcription factors that drive pacemaker cell differentiation, Tbx3, Isl1 and Shox2, were lost where Pitx2c was ectopically expressed. Moreover, a conserved long non-coding RNA (Playrr) previously linked to Pitx2 regulation in the developing dorsal mesentery, was downregulated in the sinoatrial node of homozygous mutants. The downregulation of the pacemaker gene program additionally coincided with the activation of the atrial myocardial gene program in half of the pacemaker cardiomyocytes. Conclusion We generated a mouse model of a novel human cardiac syndrome including sinoatrial node dysfunction associated with the deletion of critical CTCF binding sites in a gene desert on chromosome 4q25. This deletion induced ectopic Pitx2c expression in the sinoatrial node alongside a substantial loss of pacemaker cardiomyocyte identity and a gain in a working atrial myocardium-like phenotype in the sinoatrial node of affected mice.Loss of pacemaker cardiomyocyte identitySinoatrial node dysfunction in mutants