Different cardiovascular and pulmonary phenotypes for single- and double-knock-out mice deficient in BMP9 and BMP10

Abstract

Aims: BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension, but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10.

Methods and results: Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-knock-out (KO) mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After 3 weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single- and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay.

Conclusions: Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodelling, whereas BMP10 plays a role in hypoxia-induced cardiac remodelling in mice.

Keywords: Bone morphogenetic proteins; High-output heart failure; Pulmonary hypertension; Pulmonary vascular remodelling; Vascular anomalies.

Graphical Abstract

Figure 1

Combined loss of Bmp9 and Bmp10 leads to cardiomegaly, splenomegaly, and pulmonary hemosiderosis in DKO mice. Heart weight (A) and spleen weight (B) normalized to body weight and representative photographs of hearts (C) and spleens (D) (n = 11–12/group, male mice, scale bar = 4 mm). Representative photomicrographs of H&E-stained transverse heart sections (E) and quantitative analysis of the left ventricle + septum (LV+S), right ventricle (RV), and total cardiac tissue area (F) (n = 4/group, female mice, scale bar = 2 mm). Representative photomicrographs of fresh lungs (scale bar = 2 mm) (G) and lung sections stained with Prussian blue (scale bar = 200 µM) (H). All mice were injected with tamoxifen at the age of 2 months and euthanized at the age of 5 months. Data are presented as the means ± SEM and were analysed using Kruskal–Wallis tests followed by Dunn’s tests. *P < 0.05, ****P < 0.0001 vs. WT; ^#P < 0.05, ^##P < 0.01 vs. Bmp9-KO; ^$P < 0.05, ^$$P < 0.01 vs. Bmp10-cKO.

Figure 2

Combined loss of Bmp9 and Bmp10 leads to high cardiac output and cardiac hypertrophy in DKO mice. (A–H) Echocardiographic analysis of the left ventricle of 4- and 11-month-old WT and DKO female mice (n = 7–13/group). End-systolic (s) and end-diastolic (d) measurements of the interventricular septum (IV S), left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW) thickness, and the left ventricular internal diameter (LVID) (A), calculation of the left ventricular volume (LV Vol) (B), left ventricular mass/body weight (LV mass/BW) (C), heart rate (D), stroke volume (E), cardiac output (F), ejection fraction (G), and fractional shortening (H). All mice were injected with tamoxifen at the age of 2 months. Data are presented as the mean ± SEM and were analysed using Mann–Whitney tests to compare DKO vs. WT or 4-month-old vs. 11-month-old mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (I–L) Plasma analysis and immunofluorescence. ANP (atrial natriuretic peptide) (I) and BNP (brain natriuretic peptide) (J) plasma levels (n = 4–7 male mice/group). Quantitative analysis of cardiomyocyte size (K) and representative photomicrographs (L) of transverse heart sections stained with FITC-conjugated wheatgerm agglutinin to outline individual cardiomyocytes (n = 3–4 female mice/group, 100 cardiomyocytes/mouse). Mice were injected with tamoxifen at the age of 2 months and analysed at the age of 4–5 months. Data are presented as the means ± SEM and were analysed using Kruskal–Wallis tests followed by Dunn’s tests. *P < 0.05 vs. WT; ^#P < 0.05 vs. Bmp9-KO.

Figure 3

Combined loss of Bmp9 and Bmp10 leads to a reduction in arterial blood pressure (BP), vascular anomalies, and alveolar capillary dilatation in DKO mice. Non-invasive measurements of systolic (A), diastolic (B), and mean (C) BP in conscious adult mice (n = 9–16 male mice/group). Data are presented as the mean ± SEM and were analysed using Kruskal–Wallis tests followed by Dunn’s tests. **P < 0.01, ***P < 0.001 vs. WT. ^##P < 0.01 vs. Bmp9-KO. ^$P < 0.05, ^$$P < 0.01 vs. Bmp10-cKO. Representative photomicrographs of brain and lungs of mice that received an injection of 45-µm fluorescent beads in the left cardiac ventricle (n = 5 female and 5 male mice/group, scale bar = 2 mm) (D) and of mice that received an intravenous injection of 15-µm fluorescent beads (n = 4 female and 3 male mice/group) (E). Representative photomicrographs of the intestines of latex-blue injected mice (F) (n = 3–5 male and 3–5 female mice/group, scale bar = 1 mm). Representative photomicrographs (G) and quantitative analysis (H) of the size of capillaries from 500-nm thick lung sections stained with epoxy tissue stain. The red dotted lines outline several capillaries (scale bar = 50 µm). Data represent the mean capillary area of n = 16–20 independent lung sections from three different female mice/group. Data are presented as the means ± SEM and were analysed using Mann–Whitney tests. ****P < 0.0001 vs. WT. Mice were injected with tamoxifen at the age of 2 months and analysed at the age of 5 months.

Figure 4

RNAseq analysis of WT and DKO mouse lung tissue reveals modulation of the transcription of genes involved in inflammation, angiogenesis, blood pressure, cilium organization, and cardiac development. Five WT and seven DKO male mice were injected with tamoxifen at the age of 2 months and analysed at the age of 5 months. (A) A bi-clustering heatmap was used to visualize the expression profile of the top 40 differentially expressed genes (DEGs) between the WT and DKO conditions, with the lowest adjusted P-value by plotting their log2 transformed expression values (colour scale) for each sample. (B) Visualization of the comparison of the global transcriptional change across groups was visualized by volcano plots. Each data point in the scatter plot represents a gene. The log2 fold change of each gene is represented on the x-axis and the -log10 of its adjusted P-value on the y-axis. Genes that were not significantly differentially expressed (NS) are represented in grey, DEGs in blue, and a selection of DEG involved in specific biological processes in red (gene ontologies used for these volcano plots are listed in the Supplementary material online, Table S3). (C) Gene-set enrichment analysis (GSEA) performed using the functional database gene ontology/biological process non-redundant. The top 10 positive (blue) and negative (red) categories are presented. Each bar represents a gene ontology, the normalized enrichment score is represented on the x-axis, and the colour of the bar represents the FDR P-value. Enrichment plots for the top positive and negative terms are shown on the right..

Figure 5

Bmp9-KO, Bmp10-cKO, and DKO mice do not develop spontaneous PH under normoxic conditions and exhibit different susceptibility to the development of chronic hypoxia induced PH and cardiac remodelling. (A, D, G) Values of right ventricular systolic pressure (RVSP), (B, E, H) right ventricular hypertrophy, expressed by the Fulton Index RV/(LV+S), (C, F, I), and of heart weight of WT vs. Bmp9-KO mice (A–C), WT vs. BMP10-cKO mice (D–F), and WT vs. DKO mice (G–I). Data are presented as the means ± SEM of n = 5–10 male mice per group and were analysed using by ANOVA followed by Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. WT under normoxia; ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001 vs. WT under chronic hypoxia. AU, arbitrary unit; LV, left ventricle; ns, non-significant; RV, right ventricle; S, septum.

Figure 6

Loss of Bmp9 but not Bmp10 decreases the susceptibility of developing of chronic hypoxia-induced PH, as it protects against pulmonary arterial remodelling. Representative images of haematoxylin-eosin (H&E) staining and α-smooth muscle (SM)-actin and quantification of the percentage of muscularized and wall thickness of distal pulmonary arteries in the lungs of WT vs. Bmp9-KO mice (A), WT vs. Bmp10-cKO mice (B), and WT vs. DKO mice (C). Scale bar = 50 µm for all sections. Data are presented as the means ± SEM of n = 5–9 male mice per group and were analysed by ANOVA followed by Tukey’s test. *P < 0.05, ****P < 0.0001 vs. WT under normoxia; ^###P < 0.001, ^####P < 0.0001 vs. WT under chronic hypoxia.

Figure 7

Loss of Bmp9 in mice is associated with reduced endothelin-1 (ET-1) abundance and ET-1 plays a crucial role in BMP9-induced vasoconstriction in chick chorioallantoic membrane. ET-1 plasma (A) levels in WT, Bmp9-KO, Bmp10-cKO, and DKO mice (n = 8–10 male mice/group) and lung mRNA (B) in WT, Bmp10-cKO, and DKO mice (n = 5–13 male mice/group). Mice were injected with tamoxifen at the age of 2 months and analysed at the age of 5 months. Data are presented as the mean ± SEM and were analysed using Kruskal–Wallis tests followed by Dunn’s tests. *P < 0.05, **P < 0.01 vs. WT. Quantitative analysis (C) and representative photomicrographs (D) of FITC-Dextran-injected blood vessels from chick chorioallantoic membranes treated with bosentan (0, 50, and 500 µM), in combination with BMP9 (20 nM) or not for 24 h (scale bar = 250 um). Data are presented as the means ± SEM of n = 3–6 eggs/group and were analysed using Kruskal–Wallis tests followed by Dunn’s tests. *P < 0.05 vs. vehicle treated control and ^##P < 0.01 vs. the 20 nM BMP9 condition.