The European respiratory journal · 2022
Single-cell RNA sequencing reveals that BMPR2 mutation regulates right ventricular function via ID genes.
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Du Mingxia, Jiang Haibin, Liu Hongxian, Zhao Xin, Zhou Yu, Zhou Fang, Piao Chunmei, Xu Guoqiang, Ma Feng, Wang Jianan, Perros Frederic, Morrell Nicholas W, Gu Hong, Yang Jun
Аннотация
BACKGROUND: Mutations in bone morphogenetic protein type II receptor (BMPR2) have been found in patients with congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH). Our study aimed to clarify whether deficient BMPR2 signalling acts through downstream effectors, inhibitors of DNA-binding proteins (IDs) during heart development to contribute to the progress of PAH in CHD patients.
METHODS: To confirm that IDs are downstream effectors of BMPR2 signalling in cardiac mesoderm progenitors (CMPs) and contribute to PAH, we generated cardiomyocyte-specific Id 1/3 knockout mice (Ids cDKO), and 12 out of 25 developed mild PAH with altered haemodynamic indices and pulmonary vascular remodelling. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) human embryonic stem cells that recapitulated the BMPR2 signalling deficiency of CHD-PAH induced pluripotent stem cells (iPSCs).
RESULTS: Cardiomyocytes differentiated from iPSCs derived from CHD-PAH patients with BMP receptor mutations exhibited dysfunctional cardiac differentiation and reduced calcium (Ca2+) transients, as evidenced by confocal microscopy experiments. Smad1/5 phosphorylation and ID1 and ID3 expression were reduced in CHD-PAH iPSCs and in Bmpr2 +/- rat right ventricles. Moreover, ultrasound revealed that 33% of Ids cDKO mice had detectable defects in their ventricular septum and pulmonary regurgitation. Cardiomyocytes isolated from mouse right ventricles also showed reduced Ca2+ transients and shortened sarcomeres. Single-cell RNA sequencing analysis revealed impaired differentiation of CMPs and downregulated USP9X expression in IDs KO cells compared with wild-type cells.
CONCLUSION: We found that BMPR2 signals through IDs and USP9X to regulate cardiac differentiation, and the loss of ID1 and ID3 expression contributes to cardiomyocyte dysfunction in CHD-PAH patients with BMPR2 mutations.
METHODS: To confirm that IDs are downstream effectors of BMPR2 signalling in cardiac mesoderm progenitors (CMPs) and contribute to PAH, we generated cardiomyocyte-specific Id 1/3 knockout mice (Ids cDKO), and 12 out of 25 developed mild PAH with altered haemodynamic indices and pulmonary vascular remodelling. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) human embryonic stem cells that recapitulated the BMPR2 signalling deficiency of CHD-PAH induced pluripotent stem cells (iPSCs).
RESULTS: Cardiomyocytes differentiated from iPSCs derived from CHD-PAH patients with BMP receptor mutations exhibited dysfunctional cardiac differentiation and reduced calcium (Ca2+) transients, as evidenced by confocal microscopy experiments. Smad1/5 phosphorylation and ID1 and ID3 expression were reduced in CHD-PAH iPSCs and in Bmpr2 +/- rat right ventricles. Moreover, ultrasound revealed that 33% of Ids cDKO mice had detectable defects in their ventricular septum and pulmonary regurgitation. Cardiomyocytes isolated from mouse right ventricles also showed reduced Ca2+ transients and shortened sarcomeres. Single-cell RNA sequencing analysis revealed impaired differentiation of CMPs and downregulated USP9X expression in IDs KO cells compared with wild-type cells.
CONCLUSION: We found that BMPR2 signals through IDs and USP9X to regulate cardiac differentiation, and the loss of ID1 and ID3 expression contributes to cardiomyocyte dysfunction in CHD-PAH patients with BMPR2 mutations.