Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

A genetic and developmental biological approach for a family with complex congenital heart diseases-evidence of digenic inheritance.

Objective: Congenital heart disease (CHD) is caused by cardiovascular developmental defects and has a global prevalence of ∼1%. The etiology of CHD is multifactorial and remains generally unknown, despite advances in analytical techniques based on next-generation sequencing (NGS). The aim of our study was to elucidate the multi-genetic origin and pathogenesis of an intriguing familial case with complex CHD. Methods: We performed an original trio-based gene panel analysis using NGS of the family, including two siblings with CHD of single ventricular phenotype, and their unaffected parents. The pathogenicity of the detected rare variants was investigated in silico, and the functional effects of the variants were confirmed in vitro using luciferase assays. The combinatorial effect of gene alterations of the putative responsible genes was tested in vivo using genetically engineered mutant mice. Results: NGS-based gene panel analyses revealed two heterozygous rare variants in NODAL and in TBX20 common to the siblings and to just one of parents. Both variants were suspected pathogenic in silico, and decreased transcriptional activities of downstream signaling pathways were observed in vitro. The analyses of Nodal and Tbx20 double mutant mice demonstrated that Nodal+/-Tbx20-/- embryos showed more severe defects than Nodal+/+Tbx20-/- embryos during early heart development. The expression of Pitx2, a known downstream target of Nodal, was downregulated in Tbx20-/- mutants. Conclusions: Two rare variants on NODAL and TBX20 genes detected in this family were considered to be loss-of-function mutations. Our results suggest that NODAL and TBX20 may be complementary for the cardiac development, and a combinatorial loss-of-function of NODAL and TBX20 could be implicated in digenic inherence as the etiology of complex CHD associated with single ventricle defects in this family.