Fischer rats exhibit maladaptive structural and molecular right ventricular remodelling in severe pulmonary hypertension: a genetically prone model for right heart failure

Figures

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  Figure 1. (A) Kaplan Meier Survival analysis of Fischer (F) (n = 9) and SD (S) (n = 12) rats after SUHx (Log rank /Mantel-Cox analysis). Pulmonary hemodynamics. (B) Right ventricular systolic pressure (RVSP) was similar in both strains after SUHx. *P < 0.05 vs CTL (matched to strain). N = 4–6 per group. (C) Representative hematoxylin and eosin staining of paraffin-embedded lung sections from animals 4-weeks post-SU depicting Grade 0, Grade 1 or Grade 2 remodelling of pulmonary vessels in SUHx vs control lungs. Black arrows indicate vessels, scale bar = 200 μm. (D) Bar graph shows quantitative analysis of percentage of Grade 0, Grade 1 or Grade 2 vessels. Similar extent of pulmonary vascular remodelling was observed between both strains. Statistical analysis using One-way ANOVA with Tukey’s multiple comparisons test. N = 3–4 per group.

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  Figure 2. (A) Macroscopic images of Masson’s Trichrome stained heart sections and microscopic images of RV cross-sections illustrating cardiomyocyte fibre thickness. (B) Quantification of RV area relative to LV+S area. (C) Cardiomyocyte cross sectional area. Statistical analysis using One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 vs CTL (matched to strain). N = 3–6 per group.

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  Figure 3. Assessment of RV structure and function by echo at 4 weeks post SU. (A, B) RVID-d/LVID-d: end diastolic RV/LV internal diameter in M-mode echo. (C) RVFW: RV free wall thickness (M-mode echo). (D) Cardiac output. Data represented as mean ± SEM. Statistical analysis using One-way ANOVA with Bonferroni’s post-hoc test. *P < 0.05 vs CTL (matched to strain), #P < 0.05 vs S-SUHx. N = 5–14 per group.

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  Figure 4. Evaluation of RV function by MRI. Representative short-axis views of RV and LV in (A) end-diastole and (B) end-systole using MRI at 4 weeks post SU. (C) End-diastolic RV/LV volumes and (D) RV ejection fraction (%). Statistical analysis using One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 vs CTL. #P < 0.05 vs S-SUHx. N = 3–5 per group.

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  Figure 5. Microarray analysis of gene expression in the right ventricle at 4 weeks post SU. (A) Principal component analysis of RV gene expression changes in SD and Fischer rats treated with/without SUHx. (B) Heatmap of differentially expressed genes in SD and Fischer RVs in CTL and SUHx-induced PH. Genes with significant fold changes were filtered at a threshold of P < 0.05, >3-fold change. (C) Volcano plot of RV gene expression changes in SD and Fischer rats treated with SUHx relative to CTL. N = 3 per group.

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  Figure 6. Inadequate RV angiogenesis in response to SUHx-induced PH at 4 weeks following SU. (A) Volcano Plot graph of PCR-array of angiogenic genes in right ventricles. The graph shows Log 2 of the ratio of fold change in each gene’s expression in Fischer rats to SD rats. The fold change for each gene’s expression in SUHx group was calculated relative to CTL group. P-values were derived using student t-test. The dashed line indicates P < 0.05. N = 4 per group. (B) Immunohistochemistry staining for CD31 reveals a reduction in capillary density after SUHx in both strains and (D) quantification of capillary density. N = 3 per group. (C) Single slice confocal microscopy images of RV after fluorescence microangiography. (E) 3D capillary volume rendering from FMA stacks was expressed as capillary volume %. N = 3–4 per group. One-way ANOVA with Tukey’s multiple comparisons test where *P < 0.05 vs CTL; #P < 0.05 vs SD-SUHx. (F) Negative control for CD31 IHC. RV section was stained as described in the methods section in absence of primary antibody (CD31) and used as a negative control for CD31 IHC.

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  Figure 7. Natural killer (NK) cell infiltration in the RV. (A) Representative images (scale bar = 100 μm for 10X and 40X images) and (B) bar graph demonstrating NCR1 positive natural killer cell infiltration in the RV of Fischer CTL, SD CTL, SD SUHx and Fischer SUHx rats. Statistical analysis using One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 vs CTL; #P < 0.05 vs SD-SUHx. N = 3 per group.