Mutations in bone morphogenetic proteintype II receptor (BMPR2) have been found in patients with congenital heartdisease-associated pulmonary arterial hypertension (CHD-PAH). Our study aimedto clarify whether deficient BMPR2 signalling acts through downstreameffectors, inhibitors of DNA-binding proteins (IDs), during heart developmentto contribute to the progress of PAH in CHD patients.

We found that Cardiomyocytes (CMs)-specific Id 1/3 knockout mice(Ids cDKO) developed mild PAH. Moreover, Ultrasound revealed that 33% of IdscDKO mice had detectable defects in their ventricular septum. CMsdifferentiated from induced pluripotent stem cells (iPSCs) derived from CHD-PAHpatients with BMPR mutations exhibited dysfunctional cardiac differentiationand reduced Ca²⁺ transients. Smad1/5 phosphorylation and ID1 and ID3expression were reduced in CHD-PAH iPSCs and in Bmpr2 ^+/- rat rightventricles. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) humanembryonic stem cells. Single-cell RNA(scRNA)-seq analysis revealed impaireddifferentiation of CMPs and downregulated USP9X expression in IDs KO cellscompared with wild-type (WT) cells.

We found that BMPR2 signals through IDs and USP9X to regulatecardiac differentiation, and the loss of ID1 and ID3 expression contributes toCM dysfunction in CHD-PAH patients with BMPR2 mutations.