Differential gene expression of leaflet tissue in chronic ovine functional tricuspid regurgitation

Abstract

OBJECTIVES

Severe functional tricuspid regurgitation (FTR) is associated with subvalvular remodelling, but leaflet tissue alterations may also contribute. We set out to investigate molecular mechanisms driving leaflet remodelling in chronic ovine FTR.

METHODS

Thirteen adult sheep (55 ± 4 kg) underwent left thoracotomy, epicardial echocardiography and pulmonary artery banding to induce right heart failure and FTR. After 16 weeks, 13 banded (FTR) and 12 control animals underwent median sternotomy for epicardial echocardiography and were subsequently sacrificed with each tricuspid leaflet tissue harvested for RNA-seq and histology.

RESULTS

After 16 weeks, 7 animals developed severe, 2 moderate and 4 mild tricuspid regurgitation. Relative to control, FTR animals had increased pulmonary artery pressure, tricuspid regurgitation, tricuspid annular diameter and right atrial volume, while tricuspid annular plane systolic excursion and right ventricle fractional area change decreased. FTR leaflets exhibited altered constituents and an increase in cellularity. RNA-seq identified 85 significantly differentially expressed genes with 17, 53 and 127 within the anterior, posterior and septal leaflets, respectively. RRM2, PRG4 and CXCL8 (IL-8) were identified as differentially expressed genes across all leaflets and CXCL8 was differentially expressed between FTR severity grades. RRM2, PRG4 and CXCL8 significantly correlated with tricuspid annular plane systolic excursion, and this correlation was consistent regardless of the anatomical location of the leaflet.

CONCLUSIONS

Pulmonary artery banding in our ovine model resulted in right ventricle failure and FTR. Leaflet RNA-seq identified several differentially expressed genes, specifically RRM2, PRG4 and CXCL8, with known roles in tissue remodelling. These data, along with an overall increase in leaflet cellularity, suggest tricuspid leaflets actively remodel in FTR.

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INTRODUCTION

Secondary alterations in the valvular and subvalvular apparatus of the tricuspid valve are recognized as the patho-mechanisms of functional tricuspid regurgitation (FTR) [1]. Although FTR has drawn substantial interest in recent years and is clinically linked to high morbidity and mortality [2], durable surgical treatment of severe FTR remains challenging. Annular and ventricular remodelling have been implicated in the pathophysiology of FTR, yet the possible contribution of leaflet tissue alterations has not been extensively investigated. The role of increased pressure and mechanical stress in altering tricuspid valve molecular pathologies is poorly understood, though this role has been more thoroughly studied in aortic and mitral valves [3, 4]. Additionally, recent studies have highlighted the role of the immune system in the development and progression of valve disease, particularly through the actions of pro-inflammatory cytokines and immune cells [4, 5]. Mechanical stress on the aortic valve has been shown to increase the expression of the proinflammatory cytokines TGF-β and TNF-α that promote lymphocytic infiltration [3]. We set out to investigate the molecular alterations present in tricuspid valve (TV) leaflets in an ovine model of chronic functional tricuspid insufficiency. Due to similarities in both anatomy and physiology of the ovine heart to human’s, this model is currently considered a gold standard for chronic valve studies [6]. We hypothesized that the pressure overload induced by pulmonary artery banding (PAB) and subsequent FTR would stimulate alterations in TV leaflet molecular profile. Exploring the molecular pathways present in tricuspid leaflets with FTR will accelerate our understanding of the complex multilevel pathophysiology of this disease and facilitate improvements in diagnosis and treatment options for this burdened group of patients.

MATERIALS AND METHODS

All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research. The study protocol was approved by the Michigan State University Institutional Animal Care and Use Committee, protocol number PROTO202100064. Animals were housed and cared for at Michigan State University’s large animal facility.

Surgical preparation

The surgical protocol has been reported by our group in detail previously [7] and will be described here only briefly. Thirteen healthy adult male Dorset sheep (55 ± 4 kg) had an external right jugular intravenous catheter placed under local anaesthesia with 1% lidocaine injected subcutaneously. Animals were then anaesthetized with propofol (2–5 mg/kg IV), intubated and mechanically ventilated. General anaesthesia was maintained with inhalational isoflurane (1–2.5%) with fentanyl (5–20 mcg/kg/min) infused as additional maintenance anaesthesia. A sterile limited left thoracotomy was made through the 4th intercostal space, and epicardial echocardiography was performed to assess biventricular function and tricuspid and mitral valve competence. The main pulmonary artery was then encircled with an umbilical tape proximally to its bifurcation (Fig. 1). While monitoring systemic and pulmonary pressures, the umbilical tape was tightened down with progressive clip approximations to the brink of haemodynamic stability as described previously [8]. Subsequently, animals were monitored for 16 weeks with surveillance transthoracic echocardiography for evolving heart failure symptoms.

Terminal procedure

Sixteen weeks after completion of the PAB procedure, all experimental animals (FTR = 13) as well as 12 healthy control sheep (CTL = 12) were brought to the operating room and anaesthetized as described above. A median sternotomy was performed with subsequent epicardial echocardiography to assess biventricular function and valvular competence. At the conclusion of the experiment, the animals were euthanized through the administration of sodium pentothal (100 mg/kg IV). The heart was excised, and each tricuspid leaflet was immediately harvested with tissue divided for RNA-seq and histologic analysis.

Echocardiographic data acquisition

Epicardial echocardiography was performed in all animals and used to assess biventricular function, size and valvular competence. Images were acquired using the same equipment (2–4 MHz transducer, Vivid S6, GE Healthcare, USA). Right ventricle (RV) size and function were based on apical four-chamber and focused views. RV fractional area change was defined as (end-diastolic area – end-systolic area)/end-diastolic area. Tricuspid annular plane systolic excursion (TAPSE) was measured using M-mode from an apical four-chamber view. Tricuspid regurgitation (TR) grading included a comprehensive evaluation of colour flow and continuous-wave Doppler and was categorized by an experienced echo sonographer as none or trace (0), mild (+1), moderate (+2) or moderately severe (+3) or severe (+4).

RNA-seq

Excised tissue from tricuspid leaflets was stored in RNALater (Thermo Fisher) following surgery, incubated at 4°C overnight and subsequently frozen at –80°C. RNA extractions were performed on the tissue using a TRIzol extraction kit (Invitrogen) and RNeasy MinElute Cleanup kit (Qiagen). RNA integrity check and stranded total RNA library preparation with ribosomal reduction paired-end 50 bp sequencing was performed at Van Andel Research Institute Genomics Core (Grand Rapids, MI) on a Novaseq 6000 Illumina sequencer to an average depth of 100 M reads. Read quality was assessed with FastQC and mapped with Salmon (1.5.2) to sheep transcriptome (GCF_016772045.1_ARS-UI_Ramb_v2.0_rna.fna.gz). All FastQC and Salmon quality were assessed with MultiQC. Differentially expressed genes (DEGs) were identified by Limma-based differential analysis. Pathway analysis was performed with STRING functional tools for DEGs in TV leaflets between CTL and FTR animals using GO term enrichment for both sheep and humans.

Histology

Excised tissue was dissected using sterile technique and fixed in 10% formalin for 24–72 h. Subsequently, tissue was alcohol-dehydrated and infiltrated with paraffin using a Tissue-Tek VIP Processor. Medial leaflet tissue was then embedded in a paraffin block on 1 end and transversely sectioned (5 µM) by the Corewell Health (Grand Rapids, MI) histology core and stained with, haematoxylin and eosin, Ki67 and Movat’s pentachrome stains. Slides were scanned with a Leica Aperio AT2 Scanner (Leica Biosystems) interfaced and imaged with Aperio eSlide Manager through Aperio ImageScope (Leica Biosystems). Confocal images were obtained for slides stained for CD45, actin and 4',6-diamidino-2-phenylindole (DAPI) visualized using a Nikon A1 Laser Scanning Confocal Microscope. Images were analysed using ImageJ software (2.1.0/1.53c/Java1.8.0_172) to quantify structural constituents.

Data analysis

MedCalc software (version 20.114) was used for statistical comparisons. Histology data comparisons were made with a two-way analysis of variance, with all pairwise comparisons Bonferroni corrected. The surgical treatment group (FTR or CTL) as well as anatomical leaflet location were used as factors in the analysis. Data sets not meeting the assumptions of equal variance and normal variance were log-transformed, and in cases where data were unable to meet the assumptions of the test, non-parametric equivalent Kruskal–Wallis tests were used with a Conover test for pairwise comparisons. Echocardiographic data between CTL and FTR groups were compared using the Mann–Whitney U-test. Relationships between gene expression from RNA-seq data sets and echocardiographic measurements were analysed with linear regression with anatomical leaflet location treated as a subgroup. Comparisons of regression lines were made with an ANCOVA Bonferroni corrected for multiple comparisons. The level of significance was set at P < 0.05, and error bars represent the SEM.

RESULTS

Echocardiographic data

Immediately after PAB, peak pulmonary artery pressure increased from 19 ± 4 to 56 ± 9 mmHg (P < 0.001) in the FTR animals and, after 16 weeks, decreased to 40 ± 6 mmHg (P < 0.001), suggestive of RV failure. The mean TR grade (+0 to 4) was 0.5 ± 0.5 and 2.8 ± 1.3 for CTL and FTR groups, respectively (P < 0.001). Seven sheep developed severe, 2 moderate and 4 mild tricuspid insufficiency in the FTR group. Initial peak pulmonary artery pressure after banding did not differ between FTR animals, which subsequently developed severe FTR versus those with non-severe FTR (56 ± 8 vs 55 ± 10 mmHg, P = 0.8) indicative of heterogenous tricuspid valvular complex response to right ventricular pressure overload. Right ventricular fractional area contraction (56 ± 5 vs 34 ± 10%, P < 0.001) and tricuspid annular plane systolic excursion (TAPSE; 14 ± 8 vs 7 ± 3 mm, P < 0.001) were significantly lower in the FTR group, while tricuspid annulus diameter and RV end-diastolic volume increased from 26 ± 2 to 33 ± 4 mm (P < 0.001) and from 21 ± 6 to 34 ± 18 ml (P = 0.039), respectively. In summary, the FTR group presented with moderately severe TR, RV dysfunction and chamber remodelling and hypertrophy reflective of the clinical disease as summarized in Table 1.

TV leaflet histology

Our group has previously reported alterations in leaflet size due to PAB in an 8-week model of ovine FTR [9]. Changes in leaflet size and cellularity in our current chronic model of FTR (16 weeks) are reported in Fig. 2. Haematoxylin and eosin staining of CTL and FTR leaflets demonstrated a trend towards increased transverse area, estimated through our histological approach, in all 3 tricuspid leaflets (P = 0.078). To determine alterations in specific areas of the leaflets, we defined approximate regions as annulus, belly and free edge, roughly splitting the transverse section of the leaflets into thirds. Concomitant to the trend towards an increase in leaflet size, a significant increase in the number of nuclei was observed across all leaflets in the annular (P = 0.001) and belly (P = 0.012) regions in the FTR group, while this only trended towards significance in the free edge region (P = 0.056). The clear change in leaflet length and width is demonstrated by representative images of haematoxylin and eosin-stained transverse sections of septal (STL) leaflets in Fig 2A, B, E and F where the increase in cellularity can also be observed. This increase in cellularity was further investigated revealing a significant decrease in Ki67 proliferative index (P = 0.042) and concomitant significant increase in CD45 positivity (P = 0.005) in the STL leaflet (Supplementary Material, Fig. S1).

Movat’s pentachrome staining of transverse sections of leaflet tissue to identify collagen (yellow), elastin (black), glycosaminoglycans (GAGs) (blue), muscle (red/purple) and fibrin (red) is demonstrated in representative images in Fig. 3. Elastin was quantified in the annular (Fig. 3A), belly (Fig. 3B) and free edge (Fig. 3C) regions. A significant increase in elastin was observed in the FTR group only in the annular region (P = 0.031) across all 3 leaflets. In contrast, collagen (Fig. 3H–J) and GAGs (Fig. 3K–M) showed significant alterations in the FTR group across all regions and leaflets. Collagen content was significantly decreased (P = 0.001), while GAGs were significantly increased (P = 0.005) in all leaflet regions in the FTR group. These alterations in macromolecule content were observed across anterior, posterior and STL leaflets, with the most pronounced alterations in the STL.

Transcriptional analysis

Changes in gene expression in TV leaflets were determined by RNA sequencing of each TV leaflet from CTL and FTR animals. Three-dimensional principal component analysis of CTL versus FTR leaflets (Fig. 4A) revealed a definitive clustering dividing CTL and FTR leaflets. This analysis failed to clearly cluster anterior, posterior or STL leaflets (Fig. 4B), with all 3 anatomical leaflets being dispersed across the principal component analysis. Limma-based differential analysis identified 85 DEGs within each tricuspid leaflet from FTR animals compared to their respective CTL tissue (Fig. 4C). We identified 17, 53 and 127 DEGs within the anterior, posterior and STL leaflets, respectively. Pathway analysis of DEGs identified several differentially expressed pathways using sheep or human ontology annotations for genes (Supplementary Material, Fig. 2), including T cell extravasation (GO: 0072683, strength 1.44, –LOG10 FDR 1.62), response to cytokine (GO: 0034097, strength 0.31, –LOG10 FDR 1.47) and IL-3 signalling (WP286, strength 0.83, –LOG10 FDR 2.59).

Animals in the FTR group that underwent the PAB procedure displayed a range of responses in the severity of FTR, TV constitution and TV transcriptional alterations (Fig. 4). Due to this heterogeneous response in the setting of consistent degree of PAB, we sought to elucidate correlations between echocardiographic measurements and gene expression. CXCL8 (IL-8) was shown to have significant upregulation across all leaflets (Fig. 5A, P < 0.001), demonstrated a significant increase in expression between mild and severe TR (Fig. 5B, P = 0.048) and had a significant correlation with TAPSE (Fig. 5C, r = 0.412, P < 0.001). In addition, RRM2 (Fig. 6A, P < 0.001) and PRG4 (Fig. 6B, P < 0.001) were upregulated across all leaflets in FTR with similar significant correlations for RRM2 (Fig. 6B, r = 0.404, P < 0.001) and PRG4 (Fig. 6C, r = 0.195, P = 0.003) observed with TAPSE. Although other echocardiographic parameters were also found to be significantly different between the CTL and FTR groups (Table 1), no correlations with transcript levels for these DEGs were found using these metrics (data not shown).

DISCUSSION

The growing evidence of the deleterious long-term effects of untreated severe tricuspid insufficiency [1] and suboptimal outcomes of surgical treatment [2] was the impetus for this study to elucidate molecular pathways associated with leaflet remodelling in FTR. Tricuspid valve leaflets have been thought to be biologically quiescent tissue not actively participating in response to mechano-anatomical changes associated with the pathophysiology of FTR, though previous studies reporting tricuspid valve macromolecule analyses [10, 11] have demonstrated discrepancies in content, both between leaflets and regions within the leaflet. In contrast to this point of view, our study has corroborated alterations in leaflet ECM in response to mechanical stress [12], and recent publications have demonstrated the complexity of tricuspid leaflet tissue with numerous distinct cell types, including resident lymphoid and myeloid immune cells, valvular interstitial cells, valvular endothelial cells and myofibroblasts [13]. Studies such as these leveraging molecular techniques to identify unique cell types within this tissue suggest that understanding the molecular pathologies driving leaflet response in FTR may be a fruitful path to identifying novel mechanisms fuelling this valvular disease. Our study sought to identify molecular alterations in the tricuspid leaflets associated with right ventricular pressure overload and resultant FTR.

We have previously reported echocardiographic findings in sheep with FTR after 8 weeks of pulmonary banding [14]. In the current study, echocardiographic parameters of right ventricular dysfunction and annular and chamber remodelling continued their respective trends up to 16 weeks after PAB and reflect clinical characteristics observed in patients with significant FTR. FTR severity did not increase further in this more chronic model, which may be due to progressive RV dysfunction or valvular complex compensatory mechanisms.

Tricuspid valve leaflets in our PAB model of FTR enlarged under continued stress of ventricular pressure overload, with important histologic alterations observed in the leaflet tissue. While our prior observations at an earlier time point demonstrated a decrease in leaflet cellularity [9], currently a significant increase was observed at 16 weeks, which may signal a switch from a stress response to remodelling. All 3 leaflets in FTR animals had similar responses to PAB, demonstrating significant decrease in collagen content and a concurrent increase in GAGs from the annular hinge to the free edge. These alterations in leaflet tissue suggest myxomatous degeneration, the pathological condition characterized by the accumulation of GAGs leading to excessive tissue laxity and disruption of collagen and elastin fibres [15]. Such ultrastructural changes possibly promote mechanical weakening of the valve, which along with mechanical stresses due to tethering, may lead to stretching and elongation of the leaflets [16]. As the valvular complex expands, tissue laxity may be an active response, increasing the ability of the leaflet to stretch and compensate for the new environment. Trends towards increases in GAGs were observed in our prior study after 8 weeks of pulmonary banding, but significant alterations in neither GAGs, elastin nor collagen were seen [9]. By extending the duration of right ventricular pressure overload in this same model, we could demonstrate that these alterations progress, leading to significant increases in GAGs and decreases in collagen and elastin. Future studies are needed to determine if this myxomatous response is an adaptive response or a sign of degeneration. Myxomatous degeneration of the tricuspid valve is a relatively rare condition. However, there have been case reports of this pathology affecting the TV [17]. The observation of myxomatous TV in our PAB model of isolated right-sided heart failure with FTR indicates that this disruption of normal tissue architecture can occur in isolation and can be induced by pulmonary hypertension-like conditions.

Transcriptomic data from our study provide novel information on molecular signalling events taking place within the TV leaflets in the setting of significant FTR. RNA-seq is a powerful tool for hypothesis generation and allows many subsequent studies to validate these interesting findings. We identified several DEGs including pro-inflammatory cytokine IL-8, and pathway analysis showed the activation of cytokine response, T cell extravasation and the IL-3 signalling pathway. Interestingly, IL-8 has been shown to stimulate the expression of matrix metalloproteinases, which are known to lead to extracellular matrix degradation and valve remodelling similar to the TV leaflet alterations observed in our study [18]. Meng et al. found that inflammation caused by over-secretion of proinflammatory mediators, including IL-8, can contribute to the development of calcific aortic valve stenosis [19], and IL-8 has also been implicated in the recruitment and activation of immune cells in the valve leading to chronic inflammation and valve dysfunction [20]. These findings suggest that IL-8 may play a key role in the pathogenesis of valve disease as a possible master regulator of immune-mediated signalling in valve tissue. CXCL8 expression in our ovine study was also differential across FTR severity grades, which suggests a closer association with patho-mechanisms of the disease. The TV is a site of immune privilege [21], limiting its regenerative capacity but also providing a protective capacity against potentially damaging immune responses. It is likely that unique immune signalling cascades inherent to heart valves are responsible for the complex response to the mechanical stress induced by pressure overload, yet the potential therapeutic role of IL-8 and immune signalling in valve inflammation and remodelling remains unknown.

Proteoglycan 4, a mucin-like glycoprotein, was also found to be upregulated in FTR animals. Best known for its lubrication function in synovial joints, proteoglycan 4 participates in fibroblast adhesion and proliferation in the extracellular matrix of tissues [22]. In the aortic valve, it provides plasticity and resistance to compression [23]. Previously, a study demonstrated that PRG4 was upregulated in stenotic human aortic valves, a condition that is characterized by inflammation and thickening of valve tissue [24]. Our study corroborates an important role for PRG4 in valve disease and indicates that the expression of this glycoprotein is upregulated in TV leaflets following PAB with demonstrable increases in total GAGs. RNA-seq results also revealed increased expression of proliferation-related genes, including RRM2. RRM2 is a subunit of the enzyme ribonucleotide reductase, which is involved in DNA synthesis and repair as well as cell proliferation [25]. Previous studies demonstrated that cardiomyocytes exposed to stress conditions overexpressed RRM2 and showed higher proliferation rates [25]. In a rat model, it was shown that overexpressing RRM2 improved cardiac output and performance [26]. Increased proliferation-related RRM2 in the diseased tricuspid leaflets may indicate the valve’s attempt at regeneration and restoration of function, and the presence of these markers in a chronic model suggests an ongoing process. Interestingly, our attempt to identify alterations in proliferation indicated a significant decrease in Ki67 proliferative index and a significant increase in pan leucocyte marker CD45 positivity, indicating that the increase in cellularity either occurred via proliferation prior to the 16-week window of the study or is due instead to immune cell infiltration and other mechanisms. Future work using a time course to investigate the cell dynamics and the trajectory of leaflet remodelling would help to fully elucidate the leaflets response to PAB.

Particularly interesting, exploratory examination of the data set identified a negative correlation of several DEGs, including CLCX8 (IL-8), PRG4 and RRM2, with TAPSE, an echocardiographic measurement of right ventricular systolic function. While this observation is correlative in nature, we believe this may indicate a potential relationship between the expression of these markers and the disease state. TAPSE is a predictor of clinical deterioration in patients with pulmonary hypertension [27] and may also be useful for identifying early signs of TV disease. Non-invasive metrics identifying intervention points in TV disease may have notable clinical value, especially if future studies validate these alterations in molecular pathologies to be pharmacologically targetable. Our understanding of the molecular pathways playing a role in heart disease is expanding. For example, in patients with atrial fibrillation, higher levels of TNF-α, IL-6 and IL-8 were found to be predictors of successful AF ablation [28]. In a study of mitral leaflet response in ischaemic mitral regurgitation, Howsmon and colleagues identified 4 pathways with significant alterations. Core enrichment for these pathways consisted of a single gene, CXCL9, which is involved in immunoregulatory and inflammatory processes similar to IL-8 identified in our study [29]. Further work is needed to elucidate the cause-and-effect role of the DEGs as while some identified expression patterns and pathway activations may be indicative of active remodelling of leaflet tissue, others may simply be the result of the disease state. The transcriptional alterations identified in this study require proteomic validation; however, the identification of IL-8 as a possible mediator of TV disease is of particular interest due to the numerous compounds targeting IL-8 and its receptors [30] making it a potential pharmacologic target for the treatment of FTR.

Limitations

The results of this study must be viewed in the context of several important limitations. The primary limitation of our study is that the onset of FTR in our animal model was sudden rather than progressive, as in most clinical instances. Additionally, our model did not emulate left-sided heart disease, which is the usual culprit in the development of FTR. However, RV geometrical and functional alterations seen with functional tricuspid insufficiency clinically were replicated in our animals. Moreover, we captured only 1 time point at 16 weeks of disease progression; thereby, we were unable to characterize the temporal evolution of molecular pathways associated with leaflet remodelling, which would be technically demanding in large animal models. Finally, our study was conducted on sheep and may not fully resemble the human clinical condition, as the translation of pre-clinical data to clinical efficacy should be done with great caution.

CONCLUSIONS

Our study revealed alterations in tricuspid leaflet histology and gene expression in an ovine model of chronic FTR. Activated CXCL8 amongst numerous cytokines points to complex immunological and inflammatory mechanisms involved in leaflet alterations associated with right-sided pressure overload and RV remodelling. The correlation of TAPSE with multiple DEGs may indicate a more direct relationship between gene expression and disease severity that requires subsequent investigation. Elucidating further, the molecular pathways of leaflet remodelling may facilitate the identification of novel targets for diagnosis and treatment of patients with FTR.

SUPPLEMENTARY MATERIAL

Supplementary material is available at EJCTS online.

FUNDING

This study was funded by internal funds from the Michigan State University-Corewell Health Research Alliance and The National Institute of Health [R01HL165251 to M.K.R. and T.A.T.] and R21HL161832 to M.K.R. and T.A.T.].

Conflict of interest: Artur Iwasieczko was a Peter C. and Pat Cook Endowed Research Fellow in Cardiothoracic Surgery.

DATA AVAILABILITY

The data underlying this article will be shared on reasonable request to the corresponding author, and RNA-seq data will be deposited into a public repository.

Author contributions

Boguslaw Gaweda: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing—original draft. Austin Goodyke: Data curation; Formal analysis; Investigation; Visualization; Writing—original draft. Jeremy Prokop: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Writing—original draft. Sanjana Arora: Data curation; Investigation; Writing—original draft. Artur Iwasieczko: Conceptualization; Investigation; Methodology. Magda Piekarska: Methodology; Validation. Joseph Zagorski: Conceptualization; Methodology. Kazimierz Widenka: Conceptualization; Methodology. Manuel K. Rausch: Investigation; Methodology; Supervision. Aitor Aguirre: Investigation; Methodology; Supervision. Tomasz A. Timek: Conceptualization; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Validation; Writing—original draft.

Reviewer information

European Journal of Cardio-Thoracic Surgery thanks Leo Pölzl and the other anonymous reviewers for their contribution to the peer review process of this article.

REFERENCES

ABBREVIATIONS

ABBREVIATIONS
 
• CTL

  Control

 
• DEG

  Differentially expressed gene

 
• FTR

  Functional tricuspid regurgitation

 
• PAB

  Pulmonary artery banding

 
• RV

  Right ventricle

 
• STL

  Septal

 
• TR

  Tricuspid regurgitation

 
• TAPSE

  Tricuspid annular plane systolic excursion