Development of RV failure in PAH rats

All animal studies were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Adult male Sprague Dawley rats (250–350g) received either a single subcutaneous injection of endothelial toxin Monocrotaline (MCT, 60mg/kg, MCT group, n=7) and were followed for ~30 days or VEGF-receptor antagonist Sugen (SU5416, 20mg/kg, SuHx group, n=7) and kept in hypoxia (10% oxygen) for 3-weeks followed by 2-weeks of normoxia. Phosphate buffered saline (PBS) treated rats served as controls (CTRL group, n=7). Transthoracic echocardiography was performed to monitor cardiopulmonary hemodynamics using a Vevo 2100 high-resolution image system. The right ventricular systolic pressure (RVSP) was measured directly by a catheter connected to a pressure transducer (ADInstruments) into the RV.

RNA Sequencing and functional analysis

Libraries for RNA-seq were prepared using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio) and sequenced using Hiseq 3000 (Illumina). Differential expression analysis was performed using the DESeq2 R package version 1.25.16. DEGs with false discovery rate (FDR) <0.05 based on Benjamini Hochberg algorithm were considered statistically significant. Gene set enrichment analysis was performed using the Bioconductor (release 3.1) fgsea¹⁰ and R (version 3.6.1) software package. Hallmark¹¹ and Reactome¹² gene sets were obtained from molecular signature database (MSigDB).¹³ Enriched pathways considered statistically significant were defined by adjusted p-value <0.05.

RNA Extraction and quantitative real-time PCR

Total RNA was isolated from rat RV and LV tissue using Trizol (Invitrogen). Quantitative real-time PCR was performed with iTaq Universal SYBR (Bio-Rad). GADPH was used as an internal reference control, and relative gene expression was normalized to PBS-treated group.

Western Blot Analysis

Protein lysates were prepared from rat RV and LV tissue using modified RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Sigma). Proteins were diluted in 4x Laemmli sample buffer, boiled, separated on 10% gels (Bio-Rad) by SDS page and subsequently transferred onto nitrocellulose membranes (Bio-Rad) using semi-dry blotting (TransBlot Turbo System, Bio-Rad). After transfer, membranes were blocked with 5% bovine serum albumin (Sigma) and incubated with antibodies directed against CTGF (Abcam #ab6992; 1:200) and Vinculin (Sigma #V9131; 1:5000). IR Dye-conjugated secondary antibodies (LI-COR) were used for detection and blots were scanned and quantified using the LI-COR Odyssey Infrared Imaging System and Image Studio Lite.

Histologic assessment and immunofluorescence staining

The RV wall, the left ventricular (LV) wall, and the interventricular septum (IVS) were dissected and weighed. The ratio of the RV to LV plus septal weight [RV/(LV + IVS)] was calculated as the Fulton index of RV hypertrophy. Rat heart OCT sections were stained with antibodies against anti-αSMA (Sigma #A2547; 1:400), anti-CD31 (Novus Biologicals #NB100–2284; 1:100), or anti-CTGF (CCN2, Abcam #ab6992; 1:500). Paraffin embedded human RV free wall sections were stained with Masson’s trichrome staining according to manufacturer’s protocol (Sigma) or with antibodies against anti-αSMA, anti-VWF, or anti-CTGF (CCN2). Percent RV tissue fibrosis was determined as previously described.¹⁴ VWF and αSMA positive vessels were counted manually at 10x objective from at least 20 images across the RV section. Mean fluorescence intensity was calculated by at least 15 images across the RV sections and then analyzed with ImageJ software (http://rsb.info.nih.gov/ij/). Sections were imaged with Nikon confocal microscope.

Human RV Tissue acquisition and characteristics

We acquired human RV sections (RV free wall) from non-heart failure donor hearts (control group; n=3) and right heart failure patients with PAH (RV Failure; n=5) from the Department of Pathology at University of California, Los Angeles (UCLA). De-identified clinical dataset were obtained for each patient. Right heart dysfunction due to PAH was evaluated by right heart catheterization (Table E6).

Similar transcriptome signature of RV failure in MCT and SuHx rats

After confirmation of severe RV dysfunction, RNA sequencing of RV tissue from MCT (n=4) and SuHx (n=4) rats revealed 7,842 and 3,146 differentially expressed genes (DEG), respectively, compared to control rats (n=4) based on FDR <0.05 (Figure 2, ​,3A,3A, Table E1 and E2). Hierarchical clustering of top DEGs (FDR <0.05) in the RV showed similar gene signatures between MCT and SuHx rats compared to control (Figure 2), with mild variability within the SuHx group. Both MCT- and SuHx-treated groups shared 2,693 DEGs in the RV that were either up- or down-regulated in a similar fashion (Figure 3A, ​,3C).3C). The top 20 up- and down-regulated transcripts according to log2fold change and p-value threshold of <0.05 in RV of MCT and SuHx-treated groups are shown in Figure 3B. SPP1 and AHRR were the most up-regulated DEG in MCT and SuHx-treated group, respectively compared to control. Myo16 and Proz were the most down-regulated DEGs in MCT and SuHx-treated group, respectively compared to control.

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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.

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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.

Multiple pathways are commonly altered in RV of MCT- and SuHx-induced RV failure

We performed a ranked-based directional enrichment analysis of RV transcriptome from MCT and SuHx rats compared to control. Since previous studies reported RV failure being associated with RV fibrosis, inflammation, and angiogenesis in MCT- and SuHx- models^15,16, we used a refined collection of gene sets (Hallmark) that includes these biological processes.¹¹ We identified 32 and 30 Hallmark pathways significantly enriched in MCT and SuHx rats, respectively with 25 pathways overlapping (Figure 4A, Table E3). The top up-regulated enriched pathways in RV of MCT and SuHx rats according to FDR <0.05 were EMT and tumor-necrosis factor-alpha (TNF-α) signaling via nuclear factor-kB (NFkB). The top down-regulated enriched pathways in both MCT and SuHx rats were oxidative phosphorylation and fatty acid metabolism (Figure 4B). Inflammatory response and angiogenesis were also significantly enriched in both MCT- and SuHx-treated groups.

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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.

To further explore specific biological processes, we used Reactome pathway enrichment analysis¹² of RV from MCT and SuHx rats. Extracellular matrix organization and interleukin-10 signaling were the top up-regulated enriched pathways in MCT and SuHx rats, respectively. The citric acid TCA cycle and respiratory electron transport were the top down-regulated enriched pathways in MCT and SuHx rats (Table E4).

qPCR validation of selected genes from enrichment pathway analysis

To validate the RNA sequencing results and gene expression changes, we selected DEGs from significantly enriched pathways that were identified by Hallmark analysis for qRT-PCR. PCR results of DEGs in the RV from EMT (SPP1, CCN2), TNF-α signaling via NFκB (IL7R), oxidative phosphorylation (ACAT1, COX7B), fatty acid metabolism (DECR1, HSP90A), inflammation (C5AR1) and angiogenesis (S100A4) were consistent with the RNA sequencing results (Figure 5A). In contrast, PCR results in the LV for SPP1, C5AR1, HSP90A, ACAT1, COX7B, and DECR1 showed no significant change in gene expression from SuHx and MCT rats compared to control. Though, we did observe significant up-regulation of S100A4 in SuHx (p=0.007) and MCT (p=0.003), CCN2 in SuHx (p<0.001), and IL7R in SuHx (p=0.006) compared to control (Figure 5B). No significant difference in gene expression levels between SuHx and MCT were noted.

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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.

Demonstration of EndMT/EMT in RV of MCT- and SuHx-induced RV failure

To evaluate the relevance of our RV transcriptome results in the context of RVF, we focused on EndMT/EMT, the top up-regulated enriched pathway in RV of MCT and SuHx rats. RV sections were stained with antibodies against α-SMA (myofibroblast marker) and CD31 (endothelial cell marker). Co-localization of α-SMA and CD31 is considered as evidence for EndMT (Figure 6).

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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.

To identify significant genes involved in EMT/EndMT, we created a list of genes based on Hallmark EMT gene set that were statistically significant (FDR <0.05) in both MCT and SuHx RV tissue (Table E5). Based on log2fold change, we identified SPP1 and CCN2 as the top up-regulated genes associated with EMT pathway. To validate their association with EMT and RV failure, we assessed CTGF (CCN2) immunolocalization in the RV which appears to be localized to vascular/perivascular areas and within the cardiomyocytes (Figure 7A). We also measured the expression of CTGF protein in MCT and SuHx rats using Western blot analysis. We found an increased RV expression of CTGF in SuHx (p=0.0362) and MCT rats (p=0.0016), whereas no significant differences were found in the LV (Figure 7B, ​,CC).

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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.

PAH animal models of RV failure

Traditionally, MCT rat model has been used to study RV failure, but off-target effects of MCT have greatly limited its translational relevance.^8,18 For example, myocarditis induced by MCT can affect both the left and right ventricle, which may complicate transcriptome results of RV hypertrophy and failure.¹⁹ A recent transcriptomic study showed the gene expression profile of RV failure using MCT rat model, and compared it with published microarray data from pulmonary arterial banding (PAB) mice model and patients with bone morphogenetic protein receptor type 2 mutations (BRMP2).²⁰ Potus et al. showed significant metabolic changes involving mitochondria and electron transport chain, as well as fibrosis, inflammation, and angiogenesis.²⁰ Our MCT-RV expression profile is also in agreement with their results and previous reported studies. However, we include for the first time, the expression profile of RV from SuHx animal model using RNA sequencing, which is considered a more relevant pre-clinical model due to its pulmonary neointimal lesions that closely resemble human plexiform lesions.^9,21

PAB model has been used alternatively to single injection of endothelial toxins to study RV remodeling since it is better tolerated and does not induce systemic or toxic effects. PAB is considered a pure RV pressure overload model without any pulmonary vascular injury. It typically results in far less RV decompensation and RV failure compared to MCT and SuHx animal models, and thus considered a better representation of RV adaptive remodeling.² A recent study comparing RV function of PAB with SuHx model showed that chronic progressive RV pressure overload alone does not cause RV failure, while RV failure from angioproliferative PAH results in exaggerated RVH, apoptosis, fibrosis, and capillary rarefaction.²² Clinically, RV function from adult patients with PAH can still deteriorate despite significant reduction in PVR²³, and survival has been shown to be significantly associated with changes in RV ejection fraction as opposed to PVR or CO.²⁴ Based on these observations, we find MCT and SuHx to be appropriate models for understanding the molecular fingerprints of maladaptive RVH as opposed to adaptive RVH. In this study, we defined maladaptive RVH and RV failure from MCT and SuHx rats by significant elevation of RVSP, decrease in PAT and PAT/PET ratio, RV dilation and fibrosis, which were confirmed prior to RNA sequencing. Thus, our results provide a comprehensive overview of genes involved in maladaptive RVH, but identifying key molecular determinants in the transition from adaptive to maladaptive RV remodeling are still needed.

Identification of top up-regulated genes in RV failure

Based on our differential expression analysis, the top up-regulated genes in the RV were SPP1 (Osteopontin, OPN) and AHRR (Aryl-Hydrocarbon Receptor Repressor) from MCT and SuHx rats, respectively. Previous reports have shown OPN, a matricellular cardiac fetal gene that is reactivated during cardiac stress, to be elevated in RV of MCT-induced RV failure rats, which can be reversed by estrogen.^14,25 OPN deletion has been shown to attenuate collagen deposition and deteriorate systolic function in the heart of angiotensin II-induced cardiac hypertrophy mouse model.²⁶ Also, RV remodeling and failure has been associated with extensive fibrosis, which is tightly linked to transformation growth factor beta (TGF-β) signaling, and changes in expression of extracellular matrix proteins and metalloproteinases.²⁷ We found that immunostaining of OPN in human RV tissue of patients with RV failure secondary to PAH localizes to interstitial and perivascular fibrotic areas (data not shown). Thus, these observations suggest a potential role of OPN in PAH-induced RV fibrosis.

Previous studies indicated that AHRR regulates AHR (aryl-hydrocarbon receptor), a basic helix-loop-helix transcription factor that acts as a receptor for endogenous metabolites and xenobiotics such as cigarette smoking.²⁸ AHRR mRNA is highly expressed in heart and brain²⁹, and plays a role in monocyte-to-macrophage differentiation³⁰, suppression of anti-inflammatory mediators³¹, and tumor biology.³² The role of AHRR in PAH and RV failure is unclear. Interestingly, studies have shown AHR and TGF-β signaling pathways to cross-regulate each other in a cell type-specific manner.^33,34 Given the role of TGF-β signaling in RV fibrosis, AHRR elevation may be involved in RV maladaptive remodeling in the setting of PAH.

EMT/EndMT activation in RV failure secondary to PAH

Based on our Hallmark enrichment analysis of RV from MCT and SuHx models, we identified up-regulated genes that were highly enriched in functions associated with EMT. EMT and EndMT play an integral role in early cardiovascular development, and are reactivated in numerous chronic cardiovascular disease states.^6,35 EMT/EndMT have been well characterized in myocardial fibrosis and diastolic dysfunction in pressure-overload rodent models^7,36, and play a critical role in tissue fibrosis and pulmonary vascular remodeling in PAH.³⁷ Given the evidence of EndMT in lungs of PAH patients³⁷ and cardiac fibrosis, and evidence of EndMT in RV of SuHx and MCT rats (Figure 6), we hypothesized that EndMT is most likely involved in RV failure of PAH patients. In this study, we identified five patients who met hemodynamic criteria for right-sided heart failure and either WHO group 1 (n=4) or group 3 (n=1) PH classification. MCT and SuHx models are considered more reflective of maladaptive RV hypertrophy in patients with WHO group 1 PAH.² We observed activation of EndMT by immunofluorescence using myofibroblast (α-SMA) and endothelial (VWF) markers. Further analysis of genes associated with EMT based on Hallmark gene set showed SPP1 and CCN2 as the common top up-regulated genes in RV failure from MCT and SuHx rats. CCN2, also known as connective tissue growth factor (CTGF), is a matricellular protein that is induced in the heart following cardiac injury^38,39, and has been associated with the development of tissue fibrosis.⁴⁰ We observed increased CTGF immunolabeling and protein expression in RV of SuHx and MCT rats (Figure 7). In human RV sections, we observed an increased expression of CTGF in perivascular areas. Our results are in agreement with a recent study showing up-regulation of CTGF in PAH-mediated RV failure samples compared to non-failing RV by immunoblot.⁴¹ Our findings using human RV tissue further validate the RV transcriptome from MCT and SuHx models, and highlight the importance of EMT/EndMT in RV failure secondary to PAH.

The role of inflammation in RV failure secondary to PAH

In our data, pro-inflammatory pathways were amongst some of the most up-regulated pathways in both models. Pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6 have been associated with the development and progression of RV failure in PAH animal models.^{15, 42–45} In MCT rats and PAB mice, expression of TNF-α in RV was increased⁴⁶ and contributed to adverse ventricular remodeling and dysfunction.⁴⁷ Decompensated RV failure from MCT model was also associated with increased TNF-α serum levels compared to compensated RV hypertrophy, suggesting a role of TNF-α as a biomarker for RV disease progression.^42,48 In addition, activation of NFkB has been associated with the development of cardiac hypertrophy and transition to heart failure.^49,50 A recent study showed an up-regulation of NFkB in RV of MCT model, which was modulated by exercise.⁴⁴ Attenuation of cardiovascular hemodynamics and RV hypertrophy in MCT-induced PAH by TNF-α antagonist suggests NFkB to be an important molecular mediator of TNF-α signaling.⁵¹

The role of metabolism in RV failure from PAH

We also identified multiple down-regulated genes in RV of MCT and SuHx rats that were involved with metabolism and fatty acid oxidation (FAO). During the process of RV adaptation and remodeling, there is a notable glycolytic shift in the RV of humans with PAH⁵² and animal models of PAH.⁵³ For instance, RV hypertrophy from MCT- and PAB-induced PAH causes an increase in glycolysis and Glut1 expression, and pyruvate dehydrogenase kinase (PDK)-mediated inhibition of pyruvate dehydrogenase (PDH).⁵³ This PDK-mediated metabolic switch is associated with impaired RV contractility and decreased cardiac output, and electrical remodeling.^53,54

FAO involvement in metabolic remodeling in RV failure remains unclear. A reciprocal relationship between FAO and glucose oxidation has been described (Randle’s Cycle) in RVH, where inhibiting FAO and thereby increasing glucose oxidation can limit cellular damage.^55,56 In fact, recent studies using FAO inhibitors, trimetazidine and ranolazine, showed improvement of RV function by increasing glucose oxidation in PAB⁵⁵ and MCT models.⁵⁷ Another study in MCT-induced PAH rats showed impaired mitochondrial function and ADP channeling within the RV, and decreased ratio of mitochondria per myofibrillar proteins⁵⁸, which supports the role of energy metabolism in contributing to contractile dysfunction in the RV.⁵⁹

NIH NHLBI Grant 1K08HL141995-01A1 (SU)