Myocardial proteomic profile in pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a rare, fatal, and incurable disorder. Although advances in the understanding of the PAH pathobiology have been seen in recent years, molecular processes underlying heart remodelling over the course of PAH are still insufficiently understood. Therefore, the aim of this study was to investigate myocardial proteomic profile of rats at different stages of monocrotaline-induced PAH. Samples of left and right ventricle (LV and RV) free wall collected from 32 Wistar rats were subjected to proteomic analysis using an isobaric tag for relative quantitation method. Hemodynamic parameters indicated development of mild elevation of pulmonary artery pressure in the early PAH group (27.00 ± 4.93 mmHg) and severe elevation in the end-stage PAH group (50.50 ± 11.56 mmHg). In early PAH LV myocardium proteins that may be linked to an increase in inflammatory response, apoptosis, glycolytic process and decrease in myocardial structural proteins were differentially expressed compared to controls. During end-stage PAH an increase in proteins associated with apoptosis, fibrosis and cardiomyocyte Ca²⁺ currents as well as decrease in myocardial structural proteins were observed in LV. In RV during early PAH, especially proteins associated with myocardial structural components and fatty acid beta-oxidation pathway were upregulated. During end-stage PAH significant changes in RV proteins abundance related to the increased myocardial structural components, intensified fibrosis and glycolytic processes as well as decreased proteins related to cardiomyocyte Ca²⁺ currents were observed. At both PAH stages changes in RV proteins linked to apoptosis inhibition were observed. In conclusion, we identified changes of the levels of several proteins and thus of the metabolic pathways linked to the early and late remodelling of the left and right ventricle over the course of monocrotaline-induced PAH to delineate potential therapeutic targets for the treatment of this severe disease.

Figure 1

The Volcano Plot of proteins expression in (A) left ventricle myocardium of rats with early PAH and (B) end-stage PAH, as well as in (C) right ventricle myocardium of rats with early PAH and (D) end-stage PAH. The graph shows the log2 fold change of protein expression versus p-value. The dashed line indicates threshold 0.05 for p value (n = 4 per group).

Figure 2

Heat map presentation of a hierarchical cluster of significantly changed proteins in (A) left ventricle myocardium (p < 0.05; n = 4) and (B) right ventricle myocardium (selected with fold change > 1.20 and < −1.20; p < 0.05; n = 4) of rats with end-stage monocrotaline-induced PAH. The green and red colors represent low and high expression levels, respectively.

Figure 3

Enriched GO network related to KEGG pathways (https://www.kegg.jp/kegg/pathway.html) in (A) left ventricle myocardium of rats and (B) right ventricle myocardium of rats in end-stage monocrotaline-induced PAH. (C) KEGG pathways significantly enriched in right ventricle myocardium of rats with end-stage PAH depicted as a circle chart (p < 0.05). Biological processes and genes shared between pathways in left/right ventricle were visualized with ClueGO (kappa score ≥ 0.4) under the Cytoscape 3.3.0 environment as a functional grouped network. Each node represents a GO term or a gene. The enrichment significance of the GO terms is reflected by the size of the nodes. Edges represent connections between the nodes. (ClueGO under the Cytoscape 3.3.0 environment, https://apps.cytoscape.org/apps/cluego).

Figure 4

Histological cross-sections (hematoxylin and eosin staining) of left (A–C) and right (D–F) ventricle samples showing different stages of PAH development. A—left ventricle non-PAH control group, B—left ventricle early PAH, C—left ventricle end-stage PAH, D—right ventricle non-PAH control group, E—right ventricle early PAH, F—right ventricle end-stage PAH.

Figure 5

Histological cross-sections (Wheat Germ Agglutinin–Alexa Fluor 488 and DAPI [4,6-diamidino-2-phenylindole hydrochloride] staining) of left ventricle myocardium in non-PAH control group (A, C) and end-stage PAH animals (B, D).

Figure 6

Schematic summary of the results of the study.