Pulmonary Hypertension Induced by Right Pulmonary Artery Occlusion: Hemodynamic Consequences of Bmpr2 Mutation

Abstract

Background: The primary genetic risk factor for heritable pulmonary arterial hypertension is the presence of monoallelic mutations in the BMPR2 gene. The incomplete penetrance of BMPR2 mutations implies that additional triggers are necessary for pulmonary arterial hypertension occurrence. Pulmonary artery stenosis directly raises pulmonary artery pressure, and the redirection of blood flow to unobstructed arteries leads to endothelial dysfunction and vascular remodeling. We hypothesized that right pulmonary artery occlusion (RPAO) triggers pulmonary hypertension (PH) in rats with Bmpr2 mutations.

Methods and results: Male and female rats with a 71 bp monoallelic deletion in exon 1 of Bmpr2 and their wild-type siblings underwent acute and chronic RPAO. They were subjected to full high-fidelity hemodynamic characterization. We also examined how chronic RPAO can mimic the pulmonary gene expression pattern associated with installed PH in unobstructed territories. RPAO induced precapillary PH in male and female rats, both acutely and chronically. Bmpr2 mutant and male rats manifested more severe PH compared with their counterparts. Although wild-type rats adapted to RPAO, Bmpr2 mutant rats experienced heightened mortality. RPAO induced a decline in cardiac contractility index, particularly pronounced in male Bmpr2 rats. Chronic RPAO resulted in elevated pulmonary IL-6 (interleukin-6) expression and decreased Gdf2 expression (corrected P value<0.05 and log2 fold change>1). In this context, male rats expressed higher pulmonary levels of endothelin-1 and IL-6 than females.

Conclusions: Our novel 2-hit rat model presents a promising avenue to explore the adaptation of the right ventricle and pulmonary vasculature to PH, shedding light on pertinent sex- and gene-related effects.

Keywords: bone morphogenetic protein receptor type 2; pulmonary arterial hypertension; pulmonary artery occlusion; pulmonary hypertension.

Figure 1. Surgical procedure.

A, Anatomical view of rats, both before and after the induction of right pulmonary artery occlusion, in an open‐chest setting. B, The process of conducting right heart catheterization in rats during the initial acute experiment. In (C), the same procedure is depicted, with the addition of 2 Millar catheters directly inserted into the left atrium and the main pulmonary artery.

Figure 2. Average expression stability values (M statistic) of remaining control genes estimated by the geNorm algorithm.

The lower the M factor, the more stable is the housekeeping gene indicated in the x axis.

Figure 3. Hemodynamic effect of acute RPAO.

The hemodynamic effects of acute RPAO in rats are presented in delta form (post RPAO–pre RPAO), in order to mitigate the effect of weight and size. A, Right ventricular systolic pressure. B, Cardiac output. C, Stroke volume. D, Index of contractility. E, RVSP/CO ratio. F, Left lung weight/right lung weight ratio. Descriptive statistics and modelizations are available in Table 2 and Tables S3 and S4. A–E: Female (F), WT N=8, Bmpr2 N=6; male (M), WT N=15, Bmpr2 N=17. F: Female (F), WT N=8, Bmpr2 N=6; male (M), WT N=7, Bmpr2 N=7. CO indicates cardiac output; IC, index of contractility; LLW/RLW, left lung weight/right lung weight ratio; RPAO, right pulmonary artery occlusion; RVSP, right ventricular systolic pressure; SV, stroke volume; and WT, wild type.

Figure 4. Acute assessment of the effect of RPAO in WT and Bmpr2 rats.

Delta of (A) PVR, (B) PP, (C) PA stiffness, and (D) mPAP. Descriptive statistics and modelizations are available in Table 3, Tables S3 and S4. Only male rats were used in this analysis. WT N=7, Bmpr2 N=7. mPAP indicates mean pulmonary arterial pressure; PA, pulmonary artery; PP, pulse pressure; PVR, pulmonary vascular resistance; RPAO, right pulmonary artery occlusion; and WT, wild type.

Figure 5. Representative pressure tracings.

From top to bottom, representative pressure tracings of RVP, PAP, and LAP (A–C). Representative pressure tracings of RVP, PAP, and LAP in WT rats after RPA occlusion (A), in Bmpr2 mutant rats after RPA occlusion (B), and in sham‐operated rats (C). LAP indicates left atrial pressure; PAP, pulmonary arterial pressure; RPAO, right pulmonary artery occlusion; RVP, right ventricular pressure; and WT, wild type.

Figure 6. Hemodynamic effect of chronic RPAO.

Absolute values of the parameters, in males and females, with WT or Bmpr2 ^+/− genetic background are presented. A, Right ventricular systolic pressure. B, Mean pulmonary artery pressure. C, Cardiac output. D, RVSP/CO ratio. E, Index of contractility. F, Stroke volume. G, Pulmonary vascular resistance. H, Pulse pressure. I, PA stiffness. J, Heart rate. Descriptive statistics and modelizations are available in Table 4, and Tables S5 and S6. Sham: Female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=4; RPAO: female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=5. CO indicates cardiac output; HR, heart rate; IC, index of contractility; mPAP, mean pulmonary artery pressure; PA, pulmonary artery; PP, pulse pressure; PVR, pulmonary vascular resistance RPAO, right pulmonary artery occlusion; RVSP, right ventricular systolic pressure; SV, stroke volume; and WT, wild type.

Figure 7. Echocardiographic assessment in WT, Bmpr2 rats with and without RPAO.

A, Pulmonary acceleration time. B, Tricuspid annular plane systolic excursion. Descriptive statistics and modelizations are available in Table 5, Tables S5 and S6. Sham: Female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=4; RPAO: female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=5. PAAT, pulmonary acceleration time; RPAO, right pulmonary artery occlusion; TAPSE, tricuspid annular plane systolic excursion; and WT, wild type.

Figure 8. Gene, sex, and RPAO effect on right ventricle and pulmonary vascular remodeling in the nonobstructed territories.

A, RV thickness. B, RV thickness normalized relative to the size of the animal. C, Fulton index. D, Medial wall thickness of pulmonary arteries with an external diameter <50μm. E, MWT of PA with an external diameter between 50 and 100 μm. F, Pictures of distal pulmonary microvessels of male rats, illustrating the increase in MWT caused by RPAO and the interaction between RPAO and Bmpr2 mutation. Scale bar: 50μm. Descriptive statistics and modelizations are available in Table 6, Tables S5 and S6. Sham: Female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=4; RPAO: Female (F), WT N=5, Bmpr2 N=5; male (M), WT N=5, Bmpr2 N=5. LV, left ventricular; MWT, medial wall thickness; PA, pulmonary artery; RPAO, right pulmonary artery occlusion; RV, right ventricular; and WT, wild type.

Figure 9. UMAP analyses.

UMAP visualization of individuals in 2‐dimensional reduction unveiling distinct clusters for male and female rats based on the gene signature, and a notable shift occurred post‐RPAO (A). Link between genes' cycle threshold values and UMAP's dimensions illustrated by vectors of which coordinates are based on correlations coefficients (B). RPAO, right pulmonary artery occlusion; UMAP Uniform Manifold Approximation and Projection for Dimension Reduction; and WT, wild type.

Figure 10. Volcano plot analyses.

Differential gene expression in the pulmonary unobstructed territories of rats with chronic RPAO compared with sham‐operated rats (A). Differential gene expression in the lungs of male rats compared with female rats (B). Upregulations are reported on the right and downregulations on the left. RPAO, right pulmonary artery occlusion;