Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure

Abstract

Background: Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH.

Methods: We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5).

Results: We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV.

Conclusions: Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.

Keywords: fibrosis; monocrotaline; pulmonary hypertension; right ventricular failure; transcriptome.

Figure 1.

Development of RV failure in PAH rats. (A) Experimental protocol for MCT, SuHx, and PBS (Ctrl) treated rats. (B) Representative echocardiographic images of RVFAC from parasternal short-axis views at end-diastole. (C) Terminal hemodynamic assessment of RVSP, summary of echocardiographic assessment of RV function (RVID_d, RVFAC), and RV hypertrophy Fulton index (RV weight/LV weight + IVS weight). Data presented as mean ± SD. N= 6–7 rats per group.

Figure 2.

Heat map showing top differentially expressed genes in RV with FDR<0.05 of SuHx (blue) compared to MCT (red) and control (Ctrl; green) group. Gene expression scaled by row. N=4 rats per group.

Figure 3.

Common and unique DEGs in RV of MCT and SuHx rats. (A) Venn diagram representing total overlapping DEGs of MCT and SuHx rats that were considered statistically significant according to FDR <0.05 compared to control. (B) The top 20 up-regulated (left) and down-regulated (right) DEGs from MCT (red, top) and SuHx (blue, bottom) rats based on log2fold and FDR <0.05 compared to control. (C) Scatter plot of overlapping DEGs between MCT and SuHx with an R-squared of 0.911. N=4 per group.

Figure 4.

Hallmark pathways analysis of DEGs in RV of MCT and SuHx rats. (A) Venn diagram showing total number and overlapping pathways of MCT (red) vs SuHx (blue) with false discovery rate <0.05 compared to control. (B) Hallmark pathway enrichment analysis of overlapping pathways of MCT and SuHx rats. N=4 per group.

Figure 5.

qPCR validation of DEGs in RV (A) and LV (B) of Ctrl, SuHx, and MCT rats. Mean ± SD, n=6–7 per group for RV and n=3 per group for LV.

Figure 6.

Rat PH-mediated RV failure is associated with increased EndMT in RV. Representative images of immunofluorescence staining with antibodies against α-SMA (red) and CD31 (green) in rat RV sections from Control, SuHx and MCT groups. Arrows indicate vessels positive for both α-SMA and CD31. Arrowheads indicate co-localization (yellow). N=3 rats per group.

Figure 7.

Rat PH-mediated RV failure is associated with increased CTGF (CCN2) expression in RV. (A) Representative images of immunofluorescence staining with antibody against CCN2 (CTGF, red) in RV sections from Control, SuHx and MCT rats. Nuclei stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) in blue. (B, C) Quantification of CTGF (~38 KD) expression in RV and LV tissue of Control, SuHx and MCT rats. Vinculin (~117 KD) was used as a control. Mean ± SD, n=3 per group.

Figure 8.

Human PAH-mediated RV failure is associated with increased fibrosis, EndMT, and expression of CCN2 (CTGF). (A) Trichome staining of human RV cross-sections from PAH-mediated RV failure (RVF) and non-heart failure (CTRL) patients. Red and blue represents cardiomyocytes and collagen deposition (fibrosis), respectively. (B) Quantification of %RV fibrosis in RVF (n=5) and CTRL (n=3) patients. (C) Representative images of immunofluorescence staining with antibodies against α-SMA (red) and VWF (green) in human RV sections. Arrows indicate vessels positive for both α-SMA and VWF. Arrowhead indicates co-localization (yellow). (D) Quantification of major vessels positive for both α-SMA and VWF in RVF (n=5) and CTRL (n=3) patients by percentage of total VWF⁺ vessels and counts per high power field (hpf). (E) Representative images of immunofluorescence staining with antibody against CCN2 (CTGF, green) in RV sections from RVF and CTRL patients. Nuclei stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) in blue. (F) Quantification of CTGF expression in RV sections from RVF (n=5) and CTRL (n=3) patients by mean fluorescence intensity (MFI) per major vessel. Mean ± SD. Unpaired t-test.