Modeling familial sinus node dysfunction with a large intergenic deletion between PITX2 and ANK2 using iPS cell-derived sinoatrial nodal-like cardiomyocytes

Abstract

Abstract Background Pituitary homeobox 2 (PITX2) encoded by PITX2, is one of the transcription factors and plays an important role in establishing the left-right axis during development. In the heart, PITX2 inhibits the development of the sinoatrial node (SAN) in the left atrium, whereas the absence of PITX2 allows the development of SAN in the right atrium. In a multicenter study, we recently identified a sizeable intergenic deletion between PITX2 and ANK2 on chromosome 4q25 in familial sinus node dysfunction (SND). The deletion contains a CTCF-binding motif (19 bps) which forms a topologically associating domain (TAD) involved in PITX2, and putatively disrupts the TAD. In order to investigate how the intergenic deletion causes SND, we analyzed iPS cells (iPSCs) by specifically differentiating them into SAN cardiomyocytes. Methods and results We identified an intergenic heterozygous deletion of 30k bps in a Japanese family with SND by whole genome sequencing. In the SND family, all 11 genetically affected patients developed SND since childhood, and seven of them underwent pacemaker implantation (the mean age was 26). To elucidate the pathology, we utilized 3 iPSC lines: an iPSC line from a patient, a homozygous CTCF-binding motif knockout line by gene editing, and a control line from a healthy volunteer. We modified the previously reported SAN differentiation protocol1 and generated SAN-like iPSC-derived cardiomyocytes (SANLCMs). SANLCMs showed a higher beating rate than ventricular-like cardiomyocytes (VLCMs), and exhibited SAN-like action potential morphology recorded using a patch-clamp technique. SANLCMs contained lesser NKX2-5 positive cells than VLCMs by immunostaining analysis and their gene expression patterns were compatible with SAN cardiomyocytes: higher expression levels of SHOX2, TBX18, TBX3, and HCN4, and lower expression levels of PITX2, NKX2-5, and MYL2 compared with VLCMs. Then, we analyzed the gene expression profiles during the SAN differentiation. SANLCMs from CTCF-binding motif knockout iPSC lines showed lower beating rates than controls. We also found that the levels of PITX2 expression in SANLCMs from patient-derived and CTCF-binding motif knockout iPSC lines were significantly higher compared to those from the control. Conclusion Our findings suggest that the lack of CTCF-binding motif involved in a large intergenic deletion on chromosome 4q25 might be associated with a hypoplasia of SAN via PITX2 overexpression during cardiac development.