Right Ventricle Remodeling Metabolic Signature in Experimental Pulmonary Hypertension Models of Chronic Hypoxia and Monocrotaline Exposure

Abstract

Introduction: Over time and despite optimal medical management of patients with pulmonary hypertension (PH), the right ventricle (RV) function deteriorates from an adaptive to maladaptive phenotype, leading to RV failure (RVF). Although RV function is well recognized as a prognostic factor of PH, no predictive factor of RVF episodes has been elucidated so far. We hypothesized that determining RV metabolic alterations could help to understand the mechanism link to the deterioration of RV function as well as help to identify new biomarkers of RV failure.

Methods: In the current study, we aimed to characterize the metabolic reprogramming associated with the RV remodeling phenotype during experimental PH induced by chronic-hypoxia-(CH) exposure or monocrotaline-(MCT) exposure in rats. Three weeks after PH initiation, we hemodynamically characterized PH (echocardiography and RV catheterization), and then we used an untargeted metabolomics approach based on liquid chromatography coupled to high-resolution mass spectrometry to analyze RV and LV tissues in addition to plasma samples from MCT-PH and CH-PH rat models.

Results: CH exposure induced adaptive RV phenotype as opposed to MCT exposure which induced maladaptive RV phenotype. We found that predominant alterations of arginine, pyrimidine, purine, and tryptophan metabolic pathways were detected on the heart (LV+RV) and plasma samples regardless of the PH model. Acetylspermidine, putrescine, guanidinoacetate RV biopsy levels, and cytosine, deoxycytidine, deoxyuridine, and plasmatic thymidine levels were correlated to RV function in the CH-PH model. It was less likely correlated in the MCT model. These pathways are well described to regulate cell proliferation, cell hypertrophy, and cardioprotection. These findings open novel research perspectives to find biomarkers for early detection of RV failure in PH.

Keywords: MCT; RV dysfunction; arginine; chronic-hypoxia; purine; tryptophan.

Figure 1

Characterization of pulmonary hypertension by echocardiography and right heart catheterization of CH-PH and MCT-PH rats and their respective controls. (A). Experimental protocol. Preparation and analysis of CH-PH rats, MCT-PH rats, Normoxia rats (as control for CH-PH rats), and Vehicle rats (as controls for MCT-PH). (B). Principal Component Analysis (PCA) projection from all echocardiography and catheterization parameters. (C). Representation of the record of right ventricular pressure (RVP) expressed in mmHg. (D–I). Bar graph of top-six altered parameters in normoxia (Nx), chronic hypoxia (CH), vehicle and monocrotaline (MCT) rats: (D). Right ventricular systolic pressure (RVS.P). (E). Pulmonary vascular resistance (PVR, evaluated by RVS.P/Cardiac output ratio). (F). RV hypertrophy by measuring the Fulton index. (G). RV free wall thickness index. (H). Cardiac output. (I). RV contractility index. t-tests were used after verification of normal distribution of values (Shapiro–Wilk normality test). p-values are shown in Supplementary Table S1. Significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2

Characterization of pulmonary vascular remodeling and RV fibrosis in CH-PH and MCT-PH rats and their respective controls. (A). Representative hematein–eosin–safran (HES) staining of the paraffin-embedded lung sections from the control, MCT, Vehicle, Chronic hypoxia (CH), and normoxia (Nx) groups. Scale bar = 100 μm. (B). Pulponary vessel occlusion (%) in the control, the control, MCT, Vehicle, CH and Nx groups (n = 4 different rats per condition). (C). Interstitial fibrosis was identified with trichrome Masson staining in the RV compartments of control, the control, MCT, Vehicle, CH, and Nx groups. Scale bar = 100 μm (D). Quantification of the percentage of fibrosis in RV tissues from control and MCT-exposed rats (n = 20 images per rat from 4 rats). t-tests were used after verification of normal distribution of values (Shapiro–Wilk normality test). Significance: ** p ≤ 0.01; *** p ≤ 0.001.

Figure 3

Top5 altered metabolites in PH rat models. Bar graph of metabolites discriminating chronic-hypoxia rats (CH) models from normoxia rats (Nx) and monocrotaline (MCT) rats from vehicle rats (Vhcl) (Mann–Whitney test). (A). In plasma. (B). In right ventricle. (C). In left ventricle. Significance versus the respective control group (Mann–Whitney test): * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001. Reader’s note: due to specific matrix effects, intensities in plasma and heart tissues are not comparable.

Figure 4

Metabolomic pathways mainly altered on CH-PH and MCT-PH rats. IDO: Indoleamine 2,3-Dioxygenase. TPH: Tryptophan hydroxylase. NOS: Nitric oxide synthase. Main impacted pathways in: (A) hypoxia rat model (A1: Arginine metabolic pathway, A2: Pyrimidine metabolic pathway, A3: Tryptophan metabolic pathway and A4: Purine metabolic pathway) and (B) MCT rat model (B1: Arginine metabolic pathway, B2: Pyrimidine metabolic pathway, B3: Tryptophan metabolic pathway and B4: Purine metabolic pathway). Green color: metabolites altered in both heart and plasma samples. Red color: metabolites altered only in heart samples. Blue color: metabolites altered only in plasma samples.

Figure 5

Interpretation of metabolic alterations detected in plasma and heart samples of rat models of PH. (green = increased; red = decreased).