Introduction

Pulmonary arterial hypertension (PAH) is characterized by a progressive increases in pulmonary vascular resistance (PVR) and pressure, which can lead to a deterioration in right ventricular function1,2. The major factors contributing to the elevated PVR include a sustained increase in pulmonary vascular contraction obliterative pulmonary vascular remodeling, thrombosis, intimal hyperplasia and fibrosis, etc3. Animal models have greatly contributed to the study of PAH associated with congenital heart disease (PAH-CHD), and suitable animal models are essential to understand the pathophysiology of PAH-CHD and for the development of new therapeutics.

There are many causes of PAH, and the pathogenesis of PAH from different causes is not exactly the same4,5. CHD is one of the most common causes of PAH. Currently, modeling approaches for PAH mainly include hypoxia, arteriovenous shunts, and monocrotaline6,7,8. However, none of these modeling methods can perfectly reproduce the hemodynamic characteristics of PAH-CHD. Inducing PAH via atrial septostomy is feasible with gradual development of heart and lung lesions. Therefore, atrial septostomy-induced PAH, rather than previously used sugen/hypoxia or monocrotaline, in nonhuman primates is the ideal experimental model for studying many cardiopulmonary diseases in humans. Although the PAH model of an arteriovenous shunt (abdominal aorta–inferior vena cava shunt, common carotid artery–jugular vein shunt) results in similar hemodynamic characteristics of PAH-CHD, this model does not fully reproduce the pathophysiological process of PAH-CHD. At present, arteriovenous shunts are used in some large animals (e.g., dogs and pigs).

In this study, we examined a miniaturized model of PAH-atrial septal defect (ASD) in rats that was constructed by thermal ablation with an atrial septal puncture. This model accurately reproduced the hemodynamic properties of PAH-ASD. The establishment of this model provides a solid preliminary basis for further research into the pathogenesis of PAH-ASD and the development of novel therapeutic measures.

Materials and methods

Animals

Adult male Sprague–Dawley (SD) rats (200–250 g) were purchased from Changsha Tianqin Biotechnology Limited Company (Certificate No. SCXK [Hunan] 2022–0011). All animal experimentation was approved by the Animal Protection and Use Committee of Zunyi Medical University and complied with Directive 2010/63/EU of the European Parliament (ethics review number: ZMU21-2301-034). The rats were provided with water and food ad libitum. All surgeries were performed with sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All methods were performed in accordance with the relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines (https://submission.springernature.com/submission/f36b1992-9396-42df-93cc-eff077a11641/file/d2a32db4-e884-4cab-9041-dbc16f29ea6c).

Experimental protocol

The experimental protocol was designed as shown in Fig. 1A. In this study, a total of 58 rats were used, out of which 22 rats died during the modeling process, leaving 36 rats for successful modeling and the mortality of this model is 38%. Thirty-six rats were randomly divided into three groups, n = 6/group, comprising the Sham, PAH-ASD 2 weeks, and PAH-ASD 4 weeks groups.

Figure 1
figure 1

Stereoscopic images of the heart with atrial septal puncture in a rat model of PAH-ASD. (A) Schematic representation of the experimental protocol to construct the PAH-ASD model using atrial septal puncture in rats. (B) Stereoscopic images of complete hearts. (C) Velocity of blood flow (Doppler image) of pulmonary artery after the atrial septostomy. (D) Statistical analysis of blood flow velocity in pulmonary arteries in rats. *P < 0.05 versus the sham group, n = 3/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks. (E) Survival rate of rats at 4 weeks after PAH-ASD modeling.

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Surgical operation

Healthy SPF grade SD rats aged 6–8 weeks were deprived of water and fasting for 12 h before surgery. The rats were then weighed and administered 1% pentobarbital sodium at the dose of 10 µL/g. The adequacy of anesthesia was measured by observing muscle twitching and pinching the tail. After successful anesthesia, hair was removed from a rat’s chest with a shaver. The rat was fixed on the operating table, and its tongue was pressed with toothless tweezers. Under the illumination of luminescent tweezers, oral secretions of the rat was cleaned with cotton swabs. An 18 G Y-type indwelling needle was then used for tracheal intubation from the oral cavity. The end of the indwelling needle was connected with an ear washing ball, and the ear washing ball was pressed to observe the fluctuations in the rat’s chest. The end of the indwelling needle was connected to the ventilator. The initial parameters of the ventilator were a respiratory rate of 70 times/min, inspiratory-expiratory ratio of 1:2, and tidal volume of 4 mL. The tidal volume was adjusted according to the observed state of the rats during the operation. A diameter 0.18-mm lacquer-coated nickel wire was passed through the inner diameter of a 0.5-mm stainless steel needle, and the needle was connected to a glass syringe. This setup was then connected to direct current-regulated power supply equipment.

The operation was performed in a relatively sterile environment. The thoracic cavities were opened horizontally along the left sternum of the rat between the third and fourth ribs, and the fourth rib was cut slightly along the parasternal line. After entering the chest and exposing the pericardium, the pericardium was opened and the left atrial appendage was exposed. The left atrial appendage was gently clamped with toothless tweezers, and then the atrial septum in the heart was punctured from the left atrial appendage to the right atrial appendage using a thermal ablation puncture needle. The DC-regulated power supply was turned on, and the puncture needle was heated for 45 s (5.2 V, 1 A). After the puncture was completed, the puncture needle was withdrawn, and a cotton ball and electric coagulation hemostatic device were used to stop any bleeding. Finally, the chest was closed and the skin was sutured.

Pulmonary arterial pressure measurements

The rats were anesthetized (the anesthesia was performed as described above) before pressure measurement. The hair of the rat’s chest was removed and the rats were placed on an operation table. After the tracheal intubation was connected to a small animal ventilator, the chest cavities were opened and a PE catheter (dimeter 2 mm) was inserted into the right ventricle. Connect the end of the PE catheter to the pressure sensor. The pressure of the right ventricle was tested using the Biomedical Signal Acquisition and Processing System (Tai Meng, Chengdou, China). The mean pulmonary arterial pressure (mPAP) was calculated from right ventricular systolic pressure (RVSP) using the following equation: mPAP = (0.61 × RVSP + 2) mmHg9. Meanwhile, the PVR was calculated from mPAP using the following equation:

$$ {\text{PVR}} = {\text{mPAP}}/{\text{right }}\;{\text{ventricular}}\;{\text{ cardiac}}\;{\text{output}}\; \, \left( {{\text{RVCO}}} \right) \times 16.65 \left( {{\text{Wood}}\;{\text{unit}}} \right) $$

Morphometric analysis

After the pressure measurements were completed, lung tissue was removed and the remaining blood was washed with ice normal saline. The left lung tissue of the rat was fixed with 4% paraformaldehyde, and then the lung tissue was dehydrated, transparent, impregnated with wax, and embedded. The lung tissue was cut into 3 μm thick lung sections.The sections were then dewaxed. Hematoxylin and eosin (H&E) (Servicebio, Wuhan, China) staining, elastin van Gieson (EVG) (Servicebio, Wuhan, China) staining, and α-smooth muscle actin (SMA) (Servicebio, Wuhan, China) immunofluorescence staining were performed on the sections.

H&E staining

Sections were stained with hematoxylin staining solution for 4 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned to blue with blue return solution and rinsed with running water. Sections were sequentially dehydrated in 85% and 95% gradient alcohol for 5 min each, and stained in eosin staining solution for 5 min. Sequentially, Sections were placed in anhydrous ethanol I, II and III for 5 min each, and dimethyl I and II for 5 min each for transparency, and then sealed with neutral gum.

Elastin van Gieson

Sequentially, the sections were put into environmental protection dewaxing solution I and II for 20 min each, anhydrous ethanol I and II for 5 min each, 75% alcohol for 5 min, and washed with tap water. EVG staining was performed, EVG dye solution A:B:C was mixed with 5:2:2 to form EVG dye solution, the sections were stained with EVG dye solution for 5 min, washed with tap water, EVG dye solution B was diluted twice and then slightly differentiated, washed with tap water, and so on. Then dye VG, EVG dye solution E:D mixed into VG dye solution according to 9:1, dyeing 2 min, rapid water washing, anhydrous ethanol three tanks of rapid dehydration. Two vats of clean xylene were transparent for 20 s and 5 min each, and the slices were wet-sealed with neutral gum.

Immunofluorescence

Take rat lung tissue to make paraffin sections, bake the tissue sections at 60 °C for 2 h, xylene deparaffinization for 2 h, placed in 100%, 95%, 90%, 80% of ethanol rehydration; followed by antigen repair, sealing, dropwise addition of rabbit monoclonal α-SMA antibody diluted with phosphate buffer solution (PBS) (dilution ratio of 1:100), put into a wet box, covered with a lid, placed in a 4 °C refrigerator overnight; on the second day of 37 °C incubation of sheep anti-rabbit secondary antibody 0.5 h, PBS rinse and add DAPI staining of cell nuclei, add anti-fluorescence quencher sealing, fluorescence inversion microscope on the 2nd day, incubate the sheep anti-rabbit secondary antibody at 37 °C for 0.5 h. After rinsing with PBS, add DAPI to stain the nuclei of the cells, add anti-fluorescence quencher to seal the film, and then read the film by fluorescence inverted microscope, and measure the average optical density by Image Pro Plus 6.0(Media Cybernetics) (https://mediacy.com).

Twenty to 40 pulmonary arteries with a diameter of 50–300 µm were randomly identified from each lung tissue section. Slideviewer software 2.6 (3DHISTECH Ltd.) (https://www.3dhistech.com) image analysis was used to draw a line on the inner wall of the lumen area and measure this area. A line was then drawn around the outer wall to measure the total vascular area. A point was randomly selected in the four quadrants of the blood vessel to calculate the thickness of the blood vessel wall. The long axis and short axis of blood vessels were measured, and the outer diameter of pulmonary vessels was calculated ([long axis of blood vessels + short axis of blood vessels]/2). The vascular wall thickness index was calculated as pulmonary vascular wall thickness/pulmonary vascular outer diameter. The vascular wall area was calculated as the total vascular area minus the lumen area. The vascular wall area index was calculated as follows: (total vascular area − lumen area)/total vascular area.

Determination of the right ventricular hypertrophy index

The heart was removed after pressure measurements were completed. The left and right atria and large blood vessels were cut off along the atrioventricular groove of the heart. We cut into the right ventricular free wall and the left ventricle with ventricular septum along the ventricular septum of the heart. The heart was dried with filter paper and weighed. The right ventricular hypertrophy index was calculated as follows: right ventricular weight/left ventricular + interventricular septum weight.

Pulmonary vascular obstruction score

Images of the lungs were captured by an orthogonal fluorescence microscope (Nikon, Japan) + scanner (3DHISTECH). EVG-stained sections were observed under a microscope at 20 × magnification. Ten to 15 high-power fields were randomly observed in each rat and images were recorded. Ten to 15 pulmonary arteries with a diameter of 100–200 μm were counted to observe formation of the neointima. The degree of vascular obstruction was scored as follows: a score of 0, no neointimal formation; a score of 1, lumen obstruction < 50%; and a score of 2, lumen obstruction > 50%10.

Echocardiography measurements

Conscious sedation was achieved with midazolam 0.2–0.3 mg subcutaneous injection and the anterior chest was shaved. Trans-thoracic echocardiograms were noted in conscious-sedated rats using a Vevo 770 High-Resolution echocardiograph (Visual Sonics, Toronto, ON, Canada)11.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). All statistical analyses were performed using the SPSS statistical software package version 29.0 (SPSS Inc., Chicago, Illinois, USA). Comparisons of multiple groups were analyzed with one-way analysis of variance followed by Tukey’s post-hoc test. P values < 0.05 were considered statistically significant.

Ethics approval and consent to participate

The study was carried out in accordanc e with ARRIVE guidelines.

Results

Observation of ASD in rats

A perfect animal model is important for studying these diseases, but current animal models of PAH-CHD are still relatively rare, especially in rodents. In this study, the PAH-ASD model was constructed using the thermal ablation atrial septal puncture technique. We observed artificial ASD in the heart of rats 2 weeks and 4 weeks after the operation (Fig. 1B). The whole heart was enlarged 2 weeks and 4 weeks after modeling. Meanwhile, ultrasound Doppler results showed that the blood flow velocities of pulmonary arteries in the PAH-ASD 4 weeks rats were higher than the sham group (P < 0.05) (Fig. 1C,D). In addition, survival rates of PAH-ASD rats were counted and the survival rate of the PAH-ASD rats within 4 weeks was approximately 62.0% (Fig. 1E).

Pulmonary vascular remodeling in PAH-ASD rats

H&E staining of rat lung tissues showed pulmonary vascular remodeling (Fig. 2A). Vascular thickness and the vascular thickness index in pulmomary arteries with an outer diameter of 50–300 μm in the PAH-ASD 2 and 4 weeks groups were significantly higher than those in the sham group (all P < 0.05) (Fig. 2B–G). The vascular area and vascular area index in pulmonary arteries with an outer diameter of 50–300 μm in the PAH-ASD 2 and 4 weeks groups were significantly higher than those in the sham group (all P < 0.05) (Fig. 2H–M).

Figure 2
figure 2

Pulmonary vascular remodeling in PAH-ASD rats. (A) H&E staining of cross-sections of the pulmonary artery with different vascular outer diameters. Scale bar = 50 μm. (B–G) Analysis of vascular thickness and the vascular thickness index in the pulmonary artery with different vascular outer diameters. (H–M) Analysis of the vascular area and vascular area index in the pulmonary artery with different outer diameters. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks.

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Semi-quantitative evaluation of medial thickness in pulmonary arteries with an outer diameter of 50–300 µm

Histological changes in PAH lesions are mainly neointimal formation and medial hypertrophy12. Pulmonary vascular remodeling in PAH occurs mainly in microscopic arteries with an outer diameter < 300 µm. Therefore, we semi-quantitatively evaluated changes in the tunica media in pulmonary arteries with an outer diameter of 50–300 µm. Immunofluorescence α-SMA staining showed that medial thickness and the medial thickness index in pulmonary arteries with an outer diameter of 50–100 µm at 2 and 4 weeks after modeling were significantly higher than those in the sham group (all P < 0.05) (Fig. 3A–C). The medial area and medial area index in pulmonary arteries with an outer diameter of 50–100 µm at 2 and 4 weeks after modeling were significantly higher than those in the sham group (all P < 0.05) (Fig. 3D,E). In addition, the medial thickness, medial thickness index, medial area, and medial area index in pulmonary arteries with an outer diameter of 101–200 µm and 201–300 µm at 2 and 4 weeks after modeling were significantly higher than those in the sham group (all P < 0.05) (Figs. 4A–E and 5A–E).

Figure 3
figure 3

Proliferation of pulmonary vascular media with an outer diameter of 50–100 µm. (A) Immunofluorescence α-SMA staining in pulmonary vascular media with an outer diameter of 50–100 µm. Scale bar = 20 μm. (B,C) Analysis of pulmonary vascular medial thickness and the pulmonary vascular medial thickness index with an outer diameter of 50–100 µm. (D,E) Analysis of the pulmonary vascular medial area and pulmonary vascular medial area index with an outer diameter of 50–100 µm. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks.

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Figure 4
figure 4

Proliferation of pulmonary vascular media with an outer diameter of 101–200 µm. (A) Immunofluorescence α-SMA staining in pulmonary vascular media with an outer diameter of 101–200 µm. Scale bar = 50 μm. (B,C) Analysis of pulmonary vascular medial thickness and the pulmonary vascular medial thickness index with an outer diameter of 101–200 µm. (D,E) Analysis of the pulmonary vascular medial area and pulmonary vascular media area index with an outer diameter of 101–200 µm. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks.

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Figure 5
figure 5

Proliferation of pulmonary vascular media with an outer diameter of 201–300 µm. (A) Immunofluorescence α-SMA staining in pulmonary vascular media with an outer diameter of 201–300 µm. Scale bar = 100 μm. (B,C) Analysis of pulmonary vascular media thickness and the pulmonary vascular media thickness index with an outer diameter of 201–300 µm. (D,E) Analysis of the pulmonary vascular medial area and pulmonary vascular medial area index with an outer diameter of 201–300 µm. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks.

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Semi-quantitative evaluation of muscularization in pulmonary arterioles

Muscularization of pulmonary arterioles is one of the main pathological changes of pulmonary vascular remodeling. Therefore, in this study, the rate of muscularization in pulmonary arterioles in PAH-ASD rats was determined. Immunofluorescence staining with α-SMA showed that muscularization of pulmonary arterioles in the PAH-ASD 2 and 4 weeks groups was significantly greater than that in the sham group (both P < 0.05) (Fig. 6A,B).

Figure 6
figure 6

Muscularization of the pulmonary artery with an outer diameter of < 50 µm. (A) Immunofluorescence α-SMA staining in the pulmonary artery with an outer diameter of < 50 µm. Scale bar = 200 μm; scale bar in the inset micrograph = 20 μm. (B) Muscularization of the pulmonary artery with an outer diameter of < 50 µm. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks.

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Hemodynamic studies and semi-quantitative evaluation of arterial luminal occlusion

The PE catheter that we used for right heart catheterization in rats had a straight catheter tip. We used right ventricular systolic pressure (RVSP) as a surrogate measure for pulmonary artery systolic pressure, and estimated measure pulmonary arterial pressure (mPAP), and estimated PVR from mPAP and RVCO (Fig. 7A–C). We found that mPAP in the PAH-ASD 2 weeks and 4 weeks groups was significantly higher than that in the sham group (both P < 0.05) (Fig. 7D,E). EVG staining showed severe luminal occlusion > 50% in the PAH-ASD model groups. The vascular obstruction score in the PAH-ASD 2 and 4 weeks groups was significantly higher than that in the sham group (both P < 0.05) (Fig. 7F,G). Increased PVR due to sustained pulmonary vasoconstriction and concentric pulmonary vascular remodeling in animals with experimental PAH increases the afterload burden of the right ventricle and results in right ventricular hypertrophy. Right ventricular hypertrophy is a secondary change in PAH and may reflect PAP to a certain extent. We found that the right ventricular hypertrophy index in the PAH-ASD 2 and 4 weeks groups was significantly higher than that in the sham group (both P < 0.05) (Fig. 7H). Meanwhile, the data of H&E staining showed that the cross-sectional area of the right ventricular myocardial cells in rats at 2 and 4 weeks post-modeling was significantly increased when compared to the Sham group (P < 0.05) (Fig. 7I,J). The increase in PVR serves as a crucial indicator of PAH. The results demonstrated that at both 2 weeks and 4 weeks after atrial septostomy in rats, the PVR was significantly higher compared to the sham group (P < 0.05) (Fig. 7K).

Figure 7
figure 7

Right heart catheterization in rats to measure pulmonary hemodynamics. (A) Diagram showing the position of the catheter in the right ventricle. (B,C) Images of the cardiac four-chamber view and a simplified protocol to measure pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR). (D) mPAP in PAH-ASD rats. (E) Statistical analysis of mPAP in PAH-ASD rats. (F) EVG staining of the pulmonary artery. Scale bar = 50 μm. (G) The pulmonary vascular obstruction score. (H) The right ventricular hypertrophy index. (I) H&E staining of cardiomyocytes in right ventricle. Scale bar = 50 μm. (J) Statistical analysis of the cross-sectional area of myocardial cells. (K) The PVR in PAH-ASD rats. *P < 0.05 versus the sham group, n = 6/group. ASD atrial septal defect; PAH pulmonary arterial hypertension; W weeks; WU wood units.

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Discussion

PAH is a rare and devastating disease in which a progressive increase in mPAP is due to intense pulmonary vascular remodeling. This increase in mPAP leads to right ventricular overload, hypertrophy, and ultimately right heart failure and death13. Although there have been great advances in medication for PAH-CHD in recent years, the efficacy and safety of these medications are unsatisfactory. Therefore, the development of a reliable CHD (ASD or ventricular septal defect)-induced PAH model is crucial to investigate the underlying mechanisms and pharmacological intervention of PAH-CHD.

Despite intensive investigations on multiple animal models (e.g., rats, monkeys, pigs, and transgenic mice) over the last few decades, the etiology of PAH-CHD has not been completely determined and there is no therapeutic strategy to date14,15. Therefore, well-established animal models are an important basis for the study of clinical diseases.

Rats were selected as the experimental animal to model PAH-ASD in this study after a comprehensive consideration of research costs, operability, animal tolerance, and other conditions. The specific advantages of the rat model are as follows. First, rats are easy to feed and moderately priced compared with other large animals. Second, experimental reproducibility is high. Third, compared with rabbits and dogs, rats have a genetic background closer to that of humans. Other small animals, such as mice, have a smaller body size and heart size than rats, and the operation is more difficult in the modeling process, and the success rate of constructing the model is lower and the mortality rate of modeling is higher.

This study showed that PAH and pulmonary vascular remodeling occurred at 2 weeks post-modeling in rats, and that artificial atrial septal ostomy was still clearly observed. These findings suggest that PAH is already present in rats 2 weeks after atrial septostomy. At 4 weeks post-modeling, the mPAP and PVR exhibited a statistically significant elevation compared to the sham group. Furthermore, pathological findings showed that pulmonary vascular remodeling was more pronounced at 4 weeks than at 2 weeks after modeling, and PAH was further increased. In this experiment, we also observed that even when ASD is fully healed, the remodeled pulmonary artery does not spontaneously resolve due to the absence of ASD, and the remodeling of the pulmonary artery continues to worsen. Therefore, in addition to surgical treatment of congenital heart disease, early prevention and treatment of PAH-CHD are important.

Pulmonary vascular remodeling is a process in which pathological changes in the tissue structure and function of pulmonary blood vessels are stimulated by various injuries, shear forces, hypoxia, and drugs16,17. Pulmonary vascular remodeling is an important pathophysiological basis for the occurrence and development of PAH-CHD18,19. However, the mechanism of pulmonary vascular remodeling is relatively complex and is still not completely understood. Previous methods of establishing a PAH model mainly focused on hypoxia, monocrotaline, and arteriovenous shunt. Unfortunately, none of these PAH models perfectly simulate PAH-CHD. The model developed in this study accurately reproduces the hemodynamic processes of PAH-ASD. Additionally, the successful construction of this model provides a good research tool to investigate potential drugs and targets for PAH-ASD in the future.

From a technical point of view, the traditional methods for constructing PAH mainly include perforation of the atrial septum with a percutaneous balloon catheter and surgical resection of the atrial septum in canines20,21. An arteriovenous shunt has also been successfully used for constructing PAH in some large animals (e.g., pigs and rabbits)22,23. The method used for creating an atrial septal defect involves making a left axillary incision and cutting off a rib to expose the left atrium. This allows application of electrode thermal ablation techniques for transatrial septal perforation by directly viewing the left atrium. Not only is the cost and technical difficulty of this method lower than conventional methods, but its trauma and complications are also less owing to its simplified operation and accurate localization. Conventional methods, which use a posterior lateral incision in the right chest, involve relatively large incisions and trauma, high complications and mortality, and difficult postoperative care of the thoracic drainage tube24,25,26. In the modeling method used in this study, complications such as pericardial tamponade and arrhythmia were not detected during follow-up, the puncture was easily performed without ultrasound guidance, the model reproducibility was good, and the modeling success rate was high.

In the PAH-ASD model, as the time increased and blood flow increased, there was a gradual increase in mPAP, pulmonary vascular smooth muscle hyperplasia, vascular intima thickening, and luminal narrowing. These findings indicated that the modeling of PAH-ASD was successful, and these pathological changes are consistent with the pathophysiological process of PAH-CHD. Additionally, the survival rate and success rate in our model are better than those in the certain existing ASD modeling methods. In summary, the modeling approach of this model should be generalized and applied.

However, our study has some limitations. We did not directly measure the pressure of the pulmonary arteries owing to the difficulty of pulmonary arterial cannulation. We measured the pressure of the right ventricle to convert mPAP, which may have had some deviation. Furthermore, clinically, ventricular septal defect is the most common cause of PAH-CHD, followed by ASD. An atrial septal stoma was chosen for this study because of technical difficulties with a ventricular septal stoma, which may have caused modeling to fail in some rats.