Single-cell RNA sequencing reveals thatBMPR2mutation regulates right ventricular functionvia IDgenes

FIGURE 1. Bone morphogenetic protein type II receptor (BMPR2) mutation impairs the differentiation and calcium (Ca2+) transients of cardiomyocytes (CMs) derived from congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) induced pluripotent stem cells (iPSCs). a) Immunofluorescence of EOMES, TBX5, NKX2.5, MESP1, α-Actinin, cTnT and inhibitor of DNA-binding proteins (ID)1 as determined using 4′,6-diamidino-2-phenylindole (DAPI) as a counterstain on days 2, 4, 6 and 16 of CM differentiation. Scale bars=50 μm. b) mRNA levels of Brachyury (T), GATA4, TBX5 and cTnT were measured using quantitative PCR during CM differentiation from iPSCs, n=3. c) Fluorescence-activated cell sorting analyses (left) and quantification (right) of the percentages of cTnT+ cells on day 16 of CM differentiation from three control, three CHD-PAH without BMPR mutation (w/o) and three CHD-PAH with BMPR mutation (mut) iPS cell lines; 4–6 repeats for each cell line. Cell line 1–1 and 1–2 in CHD-PAH w/o group were obtained from the same patient. n=12–14. d–f) Representative Ca2+ imaging as determined by recording Fluo-4/AM traces in d) control iPSC-derived CMs, e) CHD-PAH w/o iPSC-derived CMs and f)…

FIGURE 2. Reduced inhibitors of DNA-binding proteins (ID)1/ID3 expression in congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) induced pluripotent stem cells (iPSCs) with bone morphogenetic protein type II receptor (BMPR2) mutations (mut) and the coordinative regulation between ID1 and ID3. a) Representative immunoblot analysis of the pSmad1/5, ID1 and ID3 levels in CHD-PAH mut and control iPSCs treated with or without 30 ng·mL−1 BMP4 for 2 h. α-TUBULIN was used as loading control. b) Representative immunoblotting of BMPRII, pSmad1/5, ID1 and ID3 in the right ventricles of wild-type (WT) and Bmpr2+/– rats. The results from three rats (marked as 1/2/3) are presented simultaneously. GAPDH was used as the loading control. c) Immunoblot analysis of human embryonic stem cells (hESCs) cultured with or without 30 ng·mL−1 BMP4 for 2 h. α-TUBULIN was used as the loading control. d) Immunoblot time-course analysis of pSmad1/5, Samd1, ID1 and ID3 expression in hESCs stimulated without (0 h) and with 30 ng·mL−1 BMP4 for 0.5–36 h. Smad1 and α-TUBULIN were used as loading controls. e) Immunoblot analysis of the ID1 and ID3 levels in hESCs stimulated with DMSO or LDN193189…

FIGURE 3. Id 1/3 knockout mice (Ids cDKO) mice are susceptible to pulmonary arterial hypertension (PAH) and pulmonary vascular remodelling. a–e) Assessment of a) right ventricular systolic pressure (RVSP) and total pulmonary vascular resistance index (tPVRI) (tPVRI=RVSP/cardiac index); b) Fulton index (right ventricular hypertrophy, RV/(LV+S)); c) tricuspid annular plane systolic excursion (TAPSE); d) cardiac output (CO) and cardiac index (CI) (cardiac output/body weight); and e) left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) in control mice (control) and Id cDKO mice without spontaneous arterial hypertension (non-SPAH) or with SPAH at age 6 months. Mice were defined as having SPAH when their RVSP was >25 mmHg. The numbers of mice that did or did not develop SPAH among the total mice in each group are shown on the x-axis. f) Representative immunofluorescence staining of α-actin smooth muscle (α-SMA, green) and the endothelial cell marker CD31 (red) in lung sections revealed thickened arterial walls in Id cDKO mice with SPAH at 6 months of age. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Scale bars=15 μm. g) Representative…

FIGURE 4. Ventricular defects and reduced calcium (Ca2+) transients in cardiomyocytes (CMs) compromised the right heart function in Id 1/3 knockout mice (Ids cDKO) mice. a) Types of blood reflux in the hearts of 2-month-old wild-type (control) (n=20), Id1−/–Id3f/w;Mesp1-cre (n=11) and Id1+/–Id3f/f;Mesp1-cre (n=16) mice as detected by colour Doppler echocardiography. b) Representative echocardiographic images (yellow arrow) and analysis of Ids cDKO and control mice, revealing regurgitation across the pulmonary valve during diastole in Id cDKO mice. c) Representative images of interventricular septum (IVS) defects (yellow arrow) in Ids cDKO mice based on colour Doppler echocardiography. d) Haematoxylin and eosin (H&E) and immunofluorescence staining of the CM markers wheat germ agglutinin (WGA) and MF20 (myosin heavy chain 1, MYH1) in longitudinal heart sections from 6-month-old mice. Scale bars=200 μm (black), 1000 μm (yellow) and 100 μm (white). e) Immunofluorescence staining of WGA and MF20 and f) quantification of the CM areas in the hearts of control and Ids cDKO mice, revealing decreased CM areas in the middle and outer myocardial walls of the right ventricles (RVs) from 6-month-old…

FIGURE 5. Impaired cardiac differentiation in inhibitors of DNA-binding proteins (ID) double-knockout (KO) human embryonic stem cell (hESC) lines. a) Immunoblotting analysis of ID1 and ID3 in three IDs KO hESC lines (ID KO-#1/#2/#3) and three wild-type (WT-#1/#2/#3) cell lines. α-TUBULIN was used as a loading control. The experiment was performed three times for each cell line; n=3. b) Fluorescence-activated cell sorting (FACS) analyses (left) and quantification (right) of the percentages of cTnT+ cells on day 16 of cardiomyocyte (CM) differentiation. n=3, three repeats for each cell line; n=3, three cell lines in each group. c) Immunostaining of α-Actinin (green) and cTnT (red) in cells differentiated from WT-#1 and ID KO-#1 cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used as a counterstain and is shown in blue. Scale bars=50 μm. d) Immunoblotting of EOMES/ID1/ID3 and α-TUBULIN (as a loading control) at different stages of CM differentiation in WT and IDs KO cell lines. e) The mRNA levels of Brachyury (T), MIXL1, TBX5, GATA4 and ISL1 during CM differentiation were measured by quantitative PCR. n=3, three technical replicates for one cell line. All results are presented as…

FIGURE 6. Single-cell (sc) RNA-sequencing (seq) analysis revealed the major affected populations of cardiac mesoderm progenitors in the inhibitors of DNA-binding proteins (IDs) knockout (KO) line. a) Schematic of the differentiation of cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs). The mRNA levels of EOMES, MESP1 and ID1 at different stages were measured by quantitative PCR and revealed that day 4 was the key point during differentiation. b) t-Distributed stochastic neighbour embedding with the R-Seurat package showing the different sources of cells analysed (right) and six clusters for wild-type (WT) and IDs KO lines (left) on day 4 of CM differentiation. c) The z-score-scaled average expression levels of differentially expressed genes for six clusters are shown as a heatmap (top), with the selected lineage-specific markers shown on the right. Representative Gene Ontology (GO) terms of biological processes are shown at the bottom. d) The expression level distributions of representative marker genes are shown as violin plots across clusters. Cell lines and clusters are represented by different colours. BMP: bone morphogenetic protein.

FIGURE 7. USP9X is a downstream effector gene of deficient inhibitors of DNA-binding proteins (IDs) in cardiac mesoderm progenitors (CMPs). a) t-Distributed stochastic neighbour embedding (t-SNE) visualisation of the key marker genes (EOMES, GATA4, ISL1, BMP4, MESP1, IRX3 and HAND1) in clusters 2 and 5. Cells expressing the representative marker genes are denoted in red. b) The z-score scaled average expression levels of differentially expressed genes (DEGs) between wild-type (WT) cells (red line) and IDs knockout (KO) cells (blue line) for six clusters are shown as heatmaps. c) The expression level distributions of USP9X are shown by t-SNE (upper). Cells expressing representative marker genes are denoted in red. The expression level distributions of USP9X are shown as violin plots in various clusters (lower). Cell lines and clusters are represented by different colours. d) Differential expression level of USP9X, HAND1 and MESP1 and other genes are shown in cluster 2 and USP9X, CKS1B and other genes in cluster 5 as a volcano plot. The size of the dot denotes the percentage of cells within a cluster, while the colour denotes the average expression level across all cells within a cluster.

FIGURE 8. Deficient bone morphogenetic protein type II receptor (BMPR2)/inhibitors of DNA-binding protein (ID) signalling downregulates USP9X via E47 during cardiomyocyte (CM) differentiation. a, b) Immunoblot analysis and quantification of a) USP9X, ID1 and ID3 in wild-type (WT) and IDs knockout (KO) cells at different stages of CM differentiation and b) in different WT and IDs KO cell lines (three cell lines in each group) on day 4 of CM differentiation. α-TUBULIN was used as the loading control. c) Immunoblot analysis of USP9X, pSmad1/5, ID1 and ID3 expression and quantification of USP9X in control and congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) mut induced pluripotent stem cells (iPSCs) on day 4 of CM differentiation. Three cell lines, marked as 1, 2 and 3, were analysed in each group. Total Smad1 and α-TUBULIN were used as loading controls. d) Representative fluorescence intensity of ID1 (red) and USP9X (green) (left) and the corresponding ImageJ-based quantifications (right) of the fluorescence intensity in the cells on day 4 of CM differentiation from control or CHD-PAH mut iPSCs. n=5, five random fields. Scale bars=50 μm. e) Immunoblot analysis of…

FIGURE 9. By using bone morphogenetic protein (BMP) receptor mutant congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) patient induced pluripotent stem cells (iPSCs) together with single-cell RNA sequencing (scRNA-seq) on inhibitors of DNA-binding proteins (IDs) knockout (KO) human embryonic stem cells (hESCs), we found that BMPR2 signals via IDs and USP9X to drive cardiac differentiation. In normal controls, BMPR2 signals via ID1, leading to the inhibition of E47 binding on the USP9X promoter, which regulates USP9X gene expression during cardiomyocyte differentiation. In CHD-PAH patients with BMP receptor mutations, the loss of ID1 and ID3 expression contributes to CM dysfunction.