IRAG1 Deficient Mice Develop PKG1β Dependent Pulmonary Hypertension

Figures

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  Figure 1. IRAG1 is downregulated in hypoxic mice lungs and isolated mice pulmonary arterial smooth muscle cells under hypoxic condition. (A,B) Western blots of protein lysates isolated from normoxic and hypoxic mice right ventricles (A) and lungs (B). Representative blot pictures are on the left and subsequent lognormal values of densitometry quantification are on the right. (C) Smooth muscle cells isolated from mice pulmonary arteries were cultured either in normoxia or hypoxia (1% O2) for 72 h. Representative western blot pictures of protein lysates from these cells are on the left and subsequent lognormal values of densitometry quantification are on the right. Densitometry was performed in the same blot pictures as presented here and n number corresponds to different animals in the same experiment. Number of animals used: (A) Right ventricles from five normoxic and five hypoxic WT mice; (B) lungs from four normoxic and four hypoxic WT mice; (C) mPASMCs isolated from five wildtype (WT) mice. Primary antibodies used in western blots were IRAG1, PKG1α, PKG1β, and IP3RI. Pan-actin was used as housekeeping gene. Fold change was calculated in relation to normoxic controls. All the western…

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  Figure 2. Disruption of IRAG1 gene in mice showed signs of right ventricular dysfunction in normoxic conditions. (A–H) WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O2) for five weeks and then evaluated for echocardiographic variables and went through right heart catheterization. Variables measured were right ventricular systolic pressure (RVSP, A), systemic arterial pressure (SAP, B), ratio of right ventricular weight to left ventricular wall plus septum weight [{RV/(LV + S)}, C], right ventricular internal diastolic diameter (RVID, D), cardiac output (CO, E), cardiac index (CI, F), Tricuspid annular plane systolic excursion (TAPSE, G), total pulmonary vascular resistance index (TPVRI, H). Number of animals used: (A) Normoxia—seven WT and seven IRAG1 KO, hypoxia—10 WT and 9 IRAG1 KO; (B) normoxia—seven WT and eight IRAG1 KO, hypoxia—10 WT and 9 IRAG1 KO; (C) normoxia—seven WT and nine IRAG1 KO, hypoxia—10 WT and 9 IRAG1 KO; (D) normoxia—seven WT and nine IRAG1 KO, hypoxia—seven WT and eight IRAG1 KO; (E) normoxia—seven WT and nine IRAG1 KO, hypoxia—eight WT and eight IRAG1 KO; (F) normoxia—seven WT and nine IRAG1 KO, hypoxia—eight WT and eight IRAG1 KO; (G) normoxia—seven…

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  Figure 3. Disruption of IRAG1 gene in mice showed pulmonary artery remodeling in normoxic conditions. (A) Formalin fixed paraffin embedded lung sections were immunostained for α-SMA (alpha-smooth muscle actin, violet) and vWF (von-Willebrand factor, brown). Bars indicate 20 μm. (B) Quantification of the relative percentage of fully, partially, and non-muscularized arteries were performed. Each group is represented as percentage of the total number of arteries counted. (C) Total muscularized arteries were quantified as the percentage of arteries that had any muscularization and represented as percentage of the total number of arteries counted. Number of animals used: (A–C) Normoxia—seven WT and eight IRAG1 KO, hypoxia—10 WT and 9 IRAG1 KO. All quantified data were represented as individual point and bar indicated the mean, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, comparison of multiple groups were performed by one-way ANOVA, followed by Tukey’s multiple comparisons test between the groups.

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  Figure 4. Disruption of IRAG1 gene in mice showed no obvious sign of mortality, increment in right ventricular (RV) fibrosis and area, and cardiomyocytes area. (A) Kaplan–Meier analysis was performed to analyze the difference of survival between the two groups. (B) Sirius red staining was performed to analyze fibrosis in mice heart and further quantified. Bars indicate 1000 µm. (C,D) H & E staining was performed with mice heart to analyze RV (C) and cardiomyocytes area (D) and further quantified. Bars indicate 1000 µm. Number of animals used: (A) 20 WT and 35 IRAG1 KO, (B) 6 WT and 4 IRAG1 KO, (C) 10 WT and 10 IRAG1 KO, (D) 10 WT and 10 IRAG1 KO. All quantified data were represented as individual point and bar indicated the mean, two-tailed unpaired t-Test.

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  Figure 5. IRAG1-deficient mice had endogenous dysregulation of PKGIβ in lung and RV (A,B) Western blots of protein lysates isolated from WT and IRAG1 KO mice lungs (A) and right ventricles (B). Representative blot pictures are on the left and subsequent lognormal values of densitometry quantifications are on the right. Densitometry was performed in the same blot pictures as presented here and n number corresponds to different animals in the same experiment. Number of animals used: (A) Lungs from four WT and five IRAG1 KO mice; lungs from four WT and four IRAG1 KO mice for PLN and Ppln (T17); (B) right ventricles from five WT and five IRAG1 KO mice. Primary antibodies used in western blots were IRAG1, PKG1α, PKG1β, IP3RI, Serca2a, Phospholamban (PLN), and phospho-Thr17 Phospholamban [pPLN (T17)]. Pan-actin was used as housekeeping gene. Fold change was calculated in relation to normoxic controls. All the western blot data were represented as individual point and bar indicates the mean, * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001, two-tailed unpaired t-Test.

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  Figure 6. IRAG1 and PKGIβ had stronger expression in lungs and isolated pulmonary artery smooth muscle cells (PASMCs) from end stage IPAH patients. (A,B) Western blots of protein lysates isolated from human lung homogenate (A) and human PASMCs (B). (A) 5 Donors and 6 IPAH patients lung samples were used. (B) 3 Donors and 3 IPAH patients PASMCs were used. Primary antibodies used in western blots were IRAG1, PKG1α, PKG1β, and IP3RI. Pan-actin was used as housekeeping gene.