Heterozygous Tropomodulin 3 mice have improved lung vascularization after chronic hypoxia

Abstract

The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.

Figure 1

SNPs associated with No-HAPH in the selected interval constitutes lung specific sQTL of TMOD3 gene. (A) SNPs (top line marks) near TMOD3 in the selected interval that differs significantly between HAPH versus No-HAPH, i.e. allele frequency differences are >50%. The ‘alternative event’ track shows the predicted alternative promoter. These are further highlighted/aligned with SNP position. The blocks represent exons; lines indicate introns, drawn such that no exons overlap. The bar plots represent the alternate allele frequency (%) distribution in the HAPH, No-HAPH and NES (method) of sQTL. (B) A representative violin plots measuring normalize intron-excision ratio of one of the SNPs (rs2570233) that is overlapping an alternative promoter (alternative splicing) sites (NES = −0.42). In the lungs, the SNPs are associated with the excision level of an intron (position chr15:51916133-51 934 250, identified as cluster clu_21347, Supplementary Material, Fig. S12), where the level of intron-defining reads (in the RNA-seq data of GTEx lung sample) flanking base position 51 916 133 to 51 934 250 on chromosome 15 is high in A/A, medium in A/G and low in G/G genotype (P-value < 0.0001). The clusters are identified using LeafCutter that employs split-reads to uncover alternative choices of intron excision by finding introns that share splice sites (Ref. 38). A detailed explanation on how LeafCutter identifies sQTL is provided in the Methods section. (C) The expression of TMOD3 significantly increases in HPAEC when cultured under 5% O₂ environment. Error bar represents SEM; ^*, P-value < 0.05.

Figure 2

Hemodynamic changes in Tmod3^+/+ and Tmod3^−/+ after treatment with 4 weeks (chronic) hypoxia are comparable. (A) Representative graphs of RVP and (B) RV ± dP/dt. (C) RVSP; (D) mPAP and (E) RV ± dp/dt_max measurements depict significant increase under hypoxic, i.e. 10% O₂, condition in both Tmod3^+/+ and Tmod3^−/+ mice. (F) HR remain comparable in 21% and 10% O₂ environments. No differences were detected between the Tmod3^+/+ and Tmod3^−/+ mice when compared under respective O₂ environments. Normoxia, Tmod3^+/+ (n = 4) and Tmod3^−/+ mice (n = 4); hypoxia, Tmod3^+/+ (n = 6) and Tmod3^−/+ mice (n = 9). Error bar represent SEM.

Figure 3

Improved vascularization in the lungs of Tmod3^−/+ mice after 4 weeks of chronic hypoxia treatment. (A) Representative angiography images of the lungs of Tmod3^+/+ and Tmod3^−/+ mice under normoxia and after 4 weeks of 10% O₂ treatment. Image of whole lung at 8x and part of the lung at 30x are simultaneously depicted. (B) The total length of branches (mm/mm²) increase significantly in the Tmod3^−/+ mice at 4 weeks of 10% O₂ exposure. (C) Number of branches per mm² also depicts a significant increase in the Tmod3^−/+ mice under hypoxic environment. (D) Chronic hypoxia exposure also indicates an increasing trend in the number of junctions per mm². Normoxia, Tmod3^+/+ (n = 3) and Tmod3^−/+ mice (n = 4); hypoxia, Tmod3^+/+ (n = 3) and Tmod3^−/+ mice (n = 5). ^*, P-value < 0.05 (Mann–Whitney test).

Figure 4

Increase cell migration of Tmod3^−/+ endothelial cells. (A) Representative image of endothelial cell migration for cells isolated from the lungs of Tmod3^+/+ and Tmod3^−/+ mice. (B) Area covered by endothelial cells at 6, 12 and 18 h after scrape/injury showed that the cells isolated from Tmod3^−/+ mice have significantly increased restorative ability (migration) compared with the endothelial cells isolated from control mice, i.e. Tmod3^+/+. P-values, ^* = 0.0178 and ^** = 0.0078. Error bar represents SEM.