Establishment of adult right ventricle failure in ovine using a graded, animal‐specific pulmonary artery constriction model

Abstract Background Right ventricle failure (RVF) is associated with serious cardiac and pulmonary diseases that contribute significantly to the morbidity and mortality of patients. Currently, the mechanisms of RVF are not fully understood and it is partly due to the lack of large animal models in adult RVF. In this study, we aim to establish a model of RVF in adult ovine and examine the structure and function relations in the RV. Methods RV pressure overload was induced in adult male sheep by revised pulmonary artery constriction (PAC). Briefly, an adjustable hydraulic occluder was placed around the main pulmonary artery trunk. Then, repeated saline injection was performed at weeks 0, 1, and 4, where the amount of saline was determined in an animal‐specific manner. Healthy, age‐matched male sheep were used as additional controls. Echocardiography was performed bi‐weekly and on week 11 post‐PAC, hemodynamic and biological measurements were obtained. Results This PAC methodology resulted in a marked increase in RV systolic pressure and decreases in stroke volume and tricuspid annular plane systolic excursion, indicating signs of RVF. Significant increases in RV chamber size, wall thickness, and Fulton's index were observed. Cardiomyocyte hypertrophy and collagen accumulation (particularly type III collagen) were evident, and these structural changes were correlated with RV dysfunction. Conclusion In summary, the animal‐specific, repeated PAC provided a robust approach to induce adult RVF, and this ovine model will offer a useful tool to study the progression and treatment of adult RVF that is translatable to human diseases.


| INTRODUC TI ON
Right ventricle failure (RVF) is associated with serious cardiac and pulmonary diseases that contribute significantly to the morbidity and mortality of patients. 1 The prevalence of RVF is significantly increased in the later stages of pulmonary hypertension (PH), congenital heart disease (CHD), and left heart failure with preserved ejection fraction (HFpEF). [2][3][4] Moreover, the mortality rate of these patients has not improved despite proposed therapeutic interventions. [3][4][5][6] The lack of effective treatment can be attributed to the incomplete understanding of the mechanisms of RVF and the lack of robust large animal models in adult RVF. [7][8][9] Preclinical (animal) models are powerful tools to investigate various human diseases including RVF. 7 Compared to small animal models, large animal models better mimic human physiology and pathophysiology [10][11][12][13] and thus are advantageous in studying both the pathogenesis and potential therapeutics that are more translatable to human patients. 14 To date, various methods have been used to establish RV pressure overload, the most common etiology of RVF. These methodologies include pulmonary artery (PA) ( Table 1) or pulmonary vein banding, 13,15 thromboembolic induction, 11,16,17 chronic hypoxia, 18 monocrotaline, 19,20 and the combination of sugen and chronic hypoxia. [21][22][23] However, some of these models are essentially the models of PH, which mainly focus on the pulmonary vascular disease and do not necessarily involve the establishment of RVF.
(Reviews of the PH models are cited here. [24][25][26][27] Compared to other RV pressure overload models, the PA banding/constriction (PAB or PAC) model is a model of RV adaptation or dysfunction alone with no pulmonary vascular diseases. While this model has been critiqued less realistic than the other PH models, it is unique and advantageous since the changes in the RV are the sole effect of the hemodynamic insult, that is, the pressure overload.
Such a model provides us an opportunity to investigate the biomechanical mechanism of RV failure without other confounding factors such as altered systemic inflammation from pulmonary vascular diseases. 28 PAB/PAC has been used in different animal species and with a mix of ages (from newborn to young adult) for RV adaptation or RVF studies (Table 1). To our knowledge, the only large animal study of chronic, adult RVF was performed in canine in the early 1990s, while the clinical standard of RVF was absent at the time. 29 Moreover, both adult and non-adult large animals have been used in the literature, with mixed goals of studying pediatric or adult RV diseases, as well as using PAC as a treatment option or means to induce RV dysfunction. For instance, lambs were commonly used and the response of the RV was associated with CHD. [30][31][32][33][34] In adult ovine studies, it was the acute changes in the RV that were examined and the chronic remodeling and outcomes were not studied. 10,35 Therefore, despite the "apparently" widely used PAC model in large animals, to date, no chronic RVF has been established in adult ovine.
The goal of the present study is to adopt an animal-specific, graded pressure overload method to establish chronic RVF in adult ovine and to investigate the structural and functional changes with RVF development. Ovine were chosen due to the widely reported similarities between human and ovine cardiovascular anatomy, function, and physiology. 14,16,25  Vet Products, IL) was placed around the main pulmonary artery and secured with two 2-0 polybutester sutures. Next, under pressure monitoring, saline was injected to the occluder acutely until the RV systolic pressure (RVSP) reached an equivalent number of its left ventricle (LV) systolic pressure as described previously. [30][31][32]34 The amount of saline was recorded and the saline was withdrawn from the occluder to allow the animal to recover from the surgical procedure for 2 days. This minimized the "surgical insult" to the RV and thus the response was mainly a result of hemodynamic overload induced via saline injections starting 2 days post-surgery. Besides the baseline measurements in the PAC sheep, age-matched, healthy intact sheep (n = 3) were used as additional controls (CTL).
Animal-specific, graded filling of the occluder with saline was induced in awake animals at weeks 0, 1, and 4 post-surgery ( Figure 1).
The amount of saline injections was determined by the procedure described above as well as the RV morphology and function from biweekly echocardiography. If we observed signs of heart failure (eg, difficulty in breathing, anorexia, grinding teeth, etc), continuous RV dilation and RV hypertrophy (eg, increased RV area or RV wall thickness), or function decline (eg, decreased flow velocity across the PA valve and decreased TAPSE), we reduced the injection volume or did not inject any saline further ( Table 2).

| Echocardiography
Transthoracic two-dimensional echocardiography was performed bi-weekly (weeks 0, 2, 4, 6, 8, 11) using a 2.5 MHz transducer on a GE Vivid 7 (GE Healthcare) ultrasound machine. Briefly, parasternal images were obtained in the awake sheep in lateral recumbency, using American Society of Echocardiography guidelines with minor imaging plane modifications in the sheep. 36 Ventricular dimensions (such as RV area, septum diameter, and RV or LV inner diameter; RVID/ LVID), tricuspid annular plane systolic excursion (TAPSE), and flow dynamics were measured.
TA B L E 1 Review of prior small and large animal models of pulmonary arterial (PA) banding/constriction. All studies adopted the same degree of constriction (ie, with a fixed diameter, area reduction, or pressure level) except for Leeuwenburgh et al, [30][31][32]34 Ramos et al, 58 Gold et al, 59 Gaynor et al, 60 and Verbelen et al, 35 which used the same criteria as our study to elevate the RV pressure to the individual's systemic pressure

| Hemodynamic measurements and terminal procedure
Prior to euthanasia, the CTL and PAC animals were anesthetized and RV catheterization was performed to obtain hemodynamic measure-

| Statistical and correlation analysis
One

| Hemodynamic and functional changes in the RV with PAC
Eleven weeks post-PAC, there was a significant increase in RVSP  Figure 2C, P < .05). Lastly, we found that TAPSE was significantly decreased at week 11 compared to the baseline ( Figure 2D, P < .05). These results indicate that pressure elevation was successful, and RVF was evident in these animals.

| Morphological changes in the RV with PAC
With the chronic pressure overload, there were significant increases in diastolic RV area and RVID as measured by echocardiography (Figure 3), suggesting a progressive dilatation of the chamber. Some global changes in the hearts were examined after tissue harvest (Table 3). Both the RV weight/body weight (P < .05) and wall thickness (P < .05) were larger in the PAC group, and the Fulton index as a routine RV hypertrophy index was significantly increased as well (P < .05).

| Structural changes in the RV with PAC
From the H&E staining, we quantified RV cardiomyocyte width and found that there was a significant increase in the cell width with PAC (Table 3, P < .05). From the Picro Sirius Red staining, we examined RV collagen content and fiber orientation in the CTL and PAC groups using polarized microscope images. PAC tended to lead to more collagen accumulation (P = .065), especially type III collagen accumulation in the RV (Table 3, P = .05). There was no difference in collagen fiber orientation between the CTL and PAC groups ( Table 3).

| Correlation analyses of the structure and function in the RV
We first examined the relations of RV pressure (RVSP) and the structures. As shown in Figure 4A,B, we found a significant correlation between RVSP and Fulton index (P < .05), which has been used to indicate RV hypertrophy at the tissue level 23 ; there was also a trend of correlation between the RVSP and the width of cardiomyocytes.
These correlations indicated that the degree of RV pressure overload was associated with RV hypertrophy at both cellular and tissue levels. As we found that both Fulton index and width of the cardiomyocyte were correlated with the RVSP, we further investigated the relations between these two hypertrophy indices. It was not surprising to see that there was a significant correlation between the cardiomyocyte width and Fulton index in the RVs of the experimental groups, suggesting that the enlarged cardiomyocytes would contribute to increased RV mass (P < .05, Figure 4C).

F I G U R E 2 Decline in RV
Interestingly, we also found a strong correlation between type III collagen and Fulton index (P = .05, r = .80, Figure 4D), and this relation was absent between the type I collagen and Fulton index (data not shown). These results indicated that RV fibrosis, especially the accumulation in type III collagen, was correlated with the RV hypertrophy.
Next, we examined the relations between the RV hypertrophy or fibrosis and its function indices. As shown in Figure 4E,F, we found a significant correlation between the Fulton index (hypertrophy) and RV ejection time (ET) and a significant correlation between the Fulton index and the stroke volume, respectively (P < .05). Furthermore, we found a significant correlation between type III collagen content and ejection time and a trend of moderate correlation between type III collagen content and stroke volume ( Figure 4G,H). However, there were no correlations between type I collagen content and the RV function (data not shown).

| D ISCUSS I ON
In this study, we described a revised PAC ovine model of adult RVF secondary to pressure overload. This model allowed for a customizable constriction between individual animals and at multiple time points. RV hypertrophy and fibrosis were evident in the pressure overloaded sheep. Surprisingly, the increase in type III collagen was more pronounced than the increase in type I collagen.

| The revised PAC ovine model of adult RV failure
To date, this study is the first report of an ovine model of adult, chronic RVF. Historically, lambs have been used in the study of RV dysfunction or therapeutics in pediatric patients (Table 1). Since ovine reach sexual maturity at 6-8 months old, 41  ( Table 1). If the PAC induction is too mild, RV adaptation rather than RVF will occur; on the other hand, if the PAC induction is too severe, animal deaths often occur prior to data collection. 42 In this study, we adopted the same criteria as used previously [30][31][32]34 to induce a similar degree of hemodynamic insult in these sheep and then examined the remodeling of the RVs. As a result, different (customized) degrees of PAC were induced ( Table 2) to ensure that the proper degree of pressure overload was achieved for RVF establishment in different individuals. Therefore, even with a small number of animals (n = 3 per group), we were able to confirm significant structural changes (ie, RV hypertrophy and fibrosis) and functional changes (ie, reduction in systolic function and ejection hemodynamics) in the RVs, from which RVF was evident. This pilot study had 0% of mortality in the PAC animals, which is rare in the similarly reported studies since the model is known for its drawbacks in surgical mortality, especially when the goal is to induce RVF. 42  There are some advantages of the animal-specific, graded PAC methodology. First, this method allows for a PA constriction that results in identical hemodynamic insult between animals. As shown in Table 1, many preclinical studies used a fixed degree of constriction (ie, increase to certain pressure value, reduce to certain PA diameter, etc) to induce pressure overload. However, each animal responds uniquely to PAC and thus a fixed constriction may lead to varied degrees of RV dysfunction (from adaptation to failure), which may complicate the assessment and diagnoses of RVF among animals. In addition, various degrees of constriction were reported (see Table 1) and there is a lack of guidance on the induction of PAC. Since RV

| New insights of RV failure from the study
In addition to the clearly adverse functional changes, we have observed morphological and structural changes in the RVs in the PAC group that are characteristic of RVF. 10,11,13 Firstly, RV dilatation and hypertrophy were evident and the RVID/LVID was gradually increased F I G U R E 4 Correlations between the structure and function in the RVs of CTL and PAC groups. A and B, correlations between the RVSP and Fulton index or width of the cardiomyocyte, respectively. C and D, correlations between the width of the cardiomyocyte or the type III collagen content and Fulton index, respectively. E and F, Correlations between the Fulton index and ejection time or stroke volume, respectively. G and H, Correlations between the collagen type III content and ejection time or stroke volume, respectively during the progression of RVF ( Figure 3C). In a recent study, the ratio of end-diastolic volumes (EDVs) of the RV to LV (RVEDV/LVEDV) was found to increase with increased RV free wall stiffness in PH patients, and this new index was strongly and inversely correlated with RV peak contractility. 46 Thus, we speculate that the increased RVID/LVID may indicate a gradual reduction in RV contractility and explain the impaired systolic function (SV) observed in the PAC sheep.
Moreover, RV fibrosis was revealed in the PAC sheep (Table 3).
This is not surprising because collagen deposition is universally reported in clinical and preclinical studies, large and small animals, as well as from early to late stage of RVF. 13,23,42,47 However, it was the collagen type III, not type I, that was more markedly increased in the PAC RVs. This is unexpected because type I collagen is the major isoform of collagen fibers in the RV, 48,49 and provides more mechanical strength than type III collagen. 50 We do not know why RVF led to a more significant increase in type III collagen, which will be examined in future investigations. Even in LVs, there is no consensus on whether type I or type III collagen plays a more significant role in its pathogenesis. 37,51 Future studies should also delineate the role of different subtypes of collagen in RVF.
Finally, we found some interesting correlations among the healthy and failing RVs. RV hypertrophy and fibrosis were strongly correlated with RV function ( Figure 4F,H). This indicated that the severity of RV hypertrophy or fibrosis was linearly linked to the adaptation of the RV and could be used as diagnostic parameters indicative of RVF. Indeed, both ventricular mass and collagen deposition have been used in preclinical and clinical settings and were found to correlate with the severity of ventricular dysfunction. 52,53 These data also confirm that our ovine model recapitulates the behavior and pathogenesis of human RVF. Furthermore, the amount of type III collagen was strongly correlated with the Fulton index ( Figure 4D), indicating that certain molecular mechanisms in type III collagen metabolism are linked with RV hypertrophy. To date, the proof of a mechanistic link between fibrosis and RV dysfunction is insufficient. 47,54 Despite the evidence that increased collagen accumulation is found in severe RVF, the treatment that reversed the collagen deposition in the RV failed to improve the RV function. 54,55 Here, we observed that type III collagen content was strongly correlated with ejection time and SV ( Figure 4G,H). We suspect that the different roles of type I and type III collagen in RV dysfunction may explain the discrepancy in the literature.

| Limitations
There are a few limitations to this study. Firstly, we did not have 3D measurements of the RV volume or strain, which are useful indices of RVF. 11,13,56 Cardiac magnetic resonance imaging or pressure-volume relations are the gold standard and should be used to investigate adult ovine RVF in the future. 44,57 Secondly, even though we observed significant functional impairment, other clinical signs such as peripheral edema or body weight loss were absent. 3,13 These signs are typically seen in the late stage of RVF and the RVF observed in this study may be in an early rather than a late stage.

| CON CLUS ION
In this study, we reported a revised animal-specific, graded pulmonary artery constriction model in adult ovine. The model led to successful right ventricle failure development with significant structural and functional changes as well as some correlations between right ventricle hypertrophy or fibrosis and functional decline. The model is robust and safe to induce various degrees of pressure overload and at multiple time points, which enables the flexibility to adapt to different protocols to answer various research questions related to the progression or treatment of right ventricle failure.

ACK N OWLED G EM ENTS
We thank Elisabeth Gray and Courtney Doherty for assistance with histology analysis.

CO N FLI C T O F I NTE R E S T
None.