Recently, mutations in BMPR2 have been found in patients with congenital heart disease associated PAH(CHD-PAH), especially in patients with postoperative PAH.
Our study aimed to identify the molecular mechanism by which BMPR2 downstream signaling predisposes patients to CHD-PAH.
Comparison of the expression of the Id genes, BMPR2 downstream effectors, between inducible pluripotent stem cell-cardiac mesoderm progenitors (iPSC-CMP) from healthy donors and CHD-PAHs with BMPR2 mutation, we showed a lower level of ID1 and ID3 total protein in CHD-PAHs, accompanied by a reduced cardiomyocytes (CMs) differentiation rate. Using human embryonic stem cell(hESC) lines with inducible overexpression(OE) of Id1 or Id3, we were able to show the reciprocal regulation between Id1 and Id3, and we unexpectedly found increased beating CMs formation during EC differentiation of the Id1 OE line. Double knockout(KO) of Id1 and Id3 in hESCs leads to decreased CMs differentiation.
With single-cell RNA-seq analysis, we revealed deviated differentiation from cardiac progenitor cells(CPCs) and identified downregulated USP expression in all clusters of KO lines compared with that in wild-type(WT) cells. Further analysis with the differentiated KO line revealed that CMs differentiation could be rescued by USP overexpression. In BMPR2 mutant CHD-PAH patients, we also found reduced USP expression.
By using single-cell sequencing facilitated development-based analysis, we conclude BMPR2/ID1 signals through USP to drive CPC lineage determination during heart development in CHD-PAH with BMPR2 mutation.
National Key Research and Development Program of China-stem cell and translational research (2016YFA0102300);
Nature Science Foundation of China (81870051, 81670054);
CAMS Innovation Fund for Medical Sciences (CIFMS)( 2016-I2M-4-003).