A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition
July 21st, 2023
This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.
Endocardial fibroelastosis hinders left ventricular growth and prevents biventricular surgical repair for patients with congenital critical aortic stenosis and hypoplastic left heart syndrome. While surgical resection is currently the only available treatment option, EFE frequently recurs. Therefore, we aim to investigate the mechanisms behind EFE and develop new therapeutic strategies using an animal model.
The main developments in the field of NDMT research are restricted to cell culture models. Which have inherent limitations, and research has been hampered due to a lack of in vivo models. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts.
A process called endothelial-to-mesenchymal transition. This animal model allows for a more generalized examination of NDMT-induced fibrosis in the heart. Whereas other models are designed to induce NDMT through genetic modifications, hypertension, or dietary restrictions.
As EFE often recurs after primary resection, sometimes with an even more infiltrative growth pattern, we are especially interested in the molecular interaction with the underlying myocardium. Begin harvesting the neonatal donor heart by anesthetizing and sterilizing the rat. And making a horizontal incision at the xiphoid with a 15 blade scalpel under a 12.5X surgical microscope.
Next, use scissors to create vertical incisions laterally up to the axillae on both sides. Remove the anterior thoracic wall with another horizontal incision beneath the neck. Dissect the inferior vena cava, or IVC, right and left superior vena cavi, or SVC, and pulmonary vessels with scissors.
Encircle and ligate all vessels using a 7-0 silk suture. Slightly push the diaphragm down with forceps. And administer three milliliters of ice cold high potassium modified Krebs-Henseleit solution to the right atrium by puncturing the IVC with a 30 gauge needle.
Cut the IVC, SVC, and pulmonary vessels using scissors. Ensure proper length by transecting the pulmonary arteries as far as possible. And the aorta distal to the brachiocephalic trunk using a number 11 blade scalpel.
Flush the heart with the ice cold cardioplegic solution using a three milliliter syringe. Place the anesthetized rat on an oblique shelf. Secure its front teeth with a string, and position the head towards the surgeon.
Place the light on the outside of the neck onto the area of the vocal cords. Grasp the tongue with two fingers, and gently push it upwards and to the left for optimal intubation visibility. Now use an 18 gauge two inch cannula for a 100 to 150 gram rat, and secure the intratracheal tube with tape.
Connect the intubation cannula to the small animal ventilator. And adjust the settings based on the animal size, following the manufacturer's instructions. After intubating the rat, perform a midline laparotomy using a 15 blade scalpel to make the skin incision.
Open the anterior abdominal wall with scissors. And mobilize the intestines toward the right upper quadrant. Next, expose the retroperitoneal abdominal aorta and IVC using cotton tip applicators.
Cover the intestines with warm saline-soaked gauze. And use retractors to maintain optimal exposure of the IVC and abdominal aorta. Now perform blunt dissection of the infrarenal IVC and abdominal aorta up to the bifurcation.
Ligate all infrarenal branching arteries and veins with a 10-0 nylon suture. Transport the harvested donor's heart in a sterile surgical basin containing krebs-henseleit buffer to the surgical field. Ensure to irrigate the donor heart intermittently with ice cold cardioplegic solution.
Next, separate the pulmonary trunk and descending aorta using micro scissors. And irrigate the donor heart with ice cold cardioplegic solution. Next, apply four atraumatic vascular clamps to the distal and proximal segments of the infrarenal aorta and IVC.
Perform an aortotomy using micro scissors to make two small horizontal cuts. Then, place the donor heart on the left side of the aorta. Secure the recipient's infrarenal aorta and the donor's ascending aorta end to side at the 12 and six o'clock positions of the aortotomy with sutures.
Proceed with the third and fourth sutures at the three and nine o'clock positions. Gently flipping the heart to the left side of the aorta after the third suture. Finalize the arterial anastomosis by adding one to two sutures to each interspace.
Now, rotate the rat counterclockwise so the head faces the surgeon's left hand. Shift the donor's heart to the left side of the abdominal aorta for an optimal view of the IVC. Perform a venotomy on the IVC slightly proximal to the aortic anastomosis.
Using an 11 blade for puncture, and micro scissors to adjust the size according to the donor's pulmonary trunk diameter. Flush the intracaval lumen with heparinised saline. Next, perform the venous anastomosis between the recipient's IVC and the donor's pulmonary trunk by placing interrupted 11-0 nylon sutures on the back wall of the vessel, starting at the 12 and six o'clock positions.
Place a continuous 11-0 nylon suture on the front wall. Place absorbable gelatin sponge strips over the anastomosis. And remove the microvascular clamps beginning with the distal ones.
Ensure that the coronary vessels fill. And that the donor's heart begins beating shortly upon release of the proximal clamps. Apply light pressure using a cotton tip applicator on the sponges to achieve optimal hemostasis.
Carefully place the intestines back into the abdomen, being cautious not to distort the arterial and venous anastomosis. Close the abdominal wall and allow the animal to recover. A horizontal slice of the donor graft resected at the mid ventricular level of the right and left ventricles is shown.
Histological analysis indicated that the endocardial fibroelastosis tissue had high amounts of organized collagen and elastin fibers. Endocardial fibroelastosis tissue double stained for VE-Cadherin and Alpha-SMA. CD-31, phospho-SMAD2, and SMAD3.
CD-31 and SLUG or SNAIL indicated endothelial-to-mesenchymal transition.
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Overview
This study investigates endocardial fibroelastosis (EFE) in congenital heart defects, focusing on the transition of endothelial cells to fibroblasts. An animal model is developed to explore the mechanisms of EFE and potential therapeutic strategies.
Key Study Components
Area of Science
- Cardiology
- Neuroscience
- Pathophysiology
Background
- Endocardial fibroelastosis is a condition that impedes left ventricular growth.
- Current treatment options are limited and often ineffective.
- Research has been primarily conducted in cell culture models.
- In vivo models are necessary for a better understanding of EFE.
Purpose of Study
- To investigate the mechanisms underlying EFE formation.
- To develop new therapeutic strategies for treating EFE.
- To utilize an animal model for detailed histological evaluation.
Methods Used
- Development of an animal model of EFE.
- Histological tissue evaluation.
- Identification of regulatory signaling pathways.
- Testing of potential treatment options.
Main Results
- Establishment of a model that mimics EFE in congenital heart defects.
- Identification of key signaling pathways involved in endothelial-to-mesenchymal transition.
- Evaluation of treatment options that may reduce EFE recurrence.
- Insights into the phenotypical changes of endothelial cells to fibroblasts.
Conclusions
- The animal model provides a valuable tool for studying EFE.
- Understanding the mechanisms of EFE can lead to better treatment strategies.
- Future research should focus on translating findings into clinical applications.
Frequently Asked Questions
Executive Industry Relevance
Modeling endothelial-to-mesenchymal transition (EndMT) in a neonatal heterotopic rat heart transplantation system addresses a critical gap in preclinical cardiac fibrosis research. This in vivo platform enables mechanistic de-risking of fibrotic pathways relevant to congenital heart disease, supporting predictive confidence for early-stage therapeutic hypothesis testing. The model's translational alignment with human endocardial fibroelastosis (EFE) pathology enhances its value for portfolio triage and target validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of EndMT-driven fibrotic mechanisms in a disease-relevant cardiac context.
- Supports functional target validation for anti-fibrotic interventions in congenital heart disease.
- Facilitates mechanistic de-risking by recapitulating human EFE histopathology in vivo.
- Provides a platform for evaluating molecular interactions between endocardium and myocardium.
Screening & Assay Development
- Prepares validated in vivo systems for downstream efficacy testing of candidate therapeutics.
- Enables reproducible induction and quantification of EFE tissue for comparative studies.
- Supports standardization of histological and molecular readouts for screening workflows.
- Allows for scalable assessment of intervention impact on EndMT and fibrosis progression.
Translational & Preclinical Research
- Aligns preclinical model outputs with human EFE tissue characteristics for translational continuity.
- Enables risk-adjusted advancement of anti-fibrotic strategies targeting EndMT pathways.
- Provides a bridge from mechanistic discovery to preclinical validation in congenital cardiac fibrosis.
- Supports biomarker development for monitoring EndMT and fibrotic progression in vivo.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum for cardiac fibrosis, enabling hypothesis testing, target validation, and translational assessment of anti-EndMT interventions.
- Discovery Biology: Supports mechanistic studies of EndMT and its role in EFE formation.
- Screening: Provides quantitative histological and molecular outputs for candidate evaluation.
- Analytics: Enables measurement of collagen, elastin, and EndMT markers for condition comparison.
- Translational Research: Aligns animal model pathology with human disease for preclinical relevance.
- Enterprise Reuse: Offers a reusable in vivo platform for fibrosis and EndMT research across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cardiac fibrosis research.
- Operational Value: Standardizes in vivo modeling of EndMT and EFE for reproducibility and scalability.
- Strategic Value: Improves go/no-go decisions for anti-fibrotic targets and interventions.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates targeting EndMT-driven fibrosis.
Implementation Considerations
- Requires expertise in microsurgical transplantation and in vivo cardiac modeling.
- Demands access to advanced histological and molecular analysis infrastructure.
- Necessitates cross-team standardization of tissue harvesting and readout protocols.
- Adaptation may be needed for different developmental stages or cardiac disease models.
- Model is limited to mechanistic and preclinical research; not validated for clinical translation.
