A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Published: July 21, 2023

doi:

Gregor Gierlinger, Lavinia Rech, Sitaram M. Emani, Pedro J. del Nido, Ingeborg Friehs

¹Department of Cardiac Surgery, Boston Children’s Hospital,Harvard Medical School

Summary

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.

Abstract

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.

To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.

A neonatal rat heart is transplanted into an adolescent rat's abdomen and connected to the recipient's infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.

This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Introduction

Endocardial fibroelastosis (EFE), defined by the accumulation of collagen and elastic fibers in the subendocardial tissue, presents as a pearly or opaque thickened endocardium; EFE undergoes most active growth during the fetal period and early infancy¹. In an autopsy study, 70% of cases with hypoplastic left heart syndrome (HLHS) were associated with the presence of EFE².

Cells expressing markers for fibroblasts are the main cell population in EFE, but these cells also concomitantly express endocardial endothelial markers, which is an indication of the origin of these EFE cells. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts through endothelial-to-mesenchymal transition (EndMT)³. EndMT can be detected using immunohistochemical double-staining for endothelial markers such as cluster of differentiation (CD) 31 or vascular endothelial (VE)-cadherin (CD144) and fibroblast markers (e.g., alpha-smooth muscle actin, α-SMA). Furthermore, we also previously established the regulatory role of the TGF-ß pathway in this process with activation of the transcription factors SLUG, SNAIL, and TWIST³.

EndMT is a physiological process that occurs during embryonic cardiac development and leads to the formation of the septa and valves from endocardial cushions⁴, but it also causes organ fibrosis in heart failure, kidney fibrosis, or cancer and plays a key role in vascular atherosclerosis⁵^,⁶^,⁷^,⁸. EndMT in cardiac fibrosis is mainly regulated through the TGF-β pathway, as we and others have reported³^,⁹. Various stimuli have been described to induce EndMT: inflammation¹⁰, hypoxia¹¹, mechanical alterations¹², and flow disturbances, including alterations of the intracavitary blood flow¹³, and EndMT may also be a consequence of a genetic disease¹⁴.

This animal model was developed using the key components of cardiac EFE development, which are immaturity and alterations of the intracavitary blood flow, specifically flow stagnation. Immaturity was fulfilled by using neonatal rat hearts as donors, since neonatal rats are known to be developmentally immature immediately after birth. Heterotopic heart transplantation offered the provision of intracavitary flow restriction¹⁵.

From a clinical point of view, this animal model allows for better investigating the impact of EndMT on the growing left ventricle (LV). The growth restriction imposed on the fetal and neonatal heart through EndMT-induced EFE formation¹⁶ precludes patients with left ventricular outflow tract obstructions (LVOTO) such as congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair¹⁷. This animal model facilitates the study of the cellular mechanisms and regulation of tissue formation through EndMT and allows for the testing of pharmacological treatment options³^,¹⁸.

Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, EFE formation can be macroscopically observed within 3 days after transplantation. EFE tissue shows the same histopathological characteristics as human EFE tissue from patients with LVOTO.

Hence, this animal model, though developed for pediatric use in the spectrum of HLHS, can be applied when studying various diseases based on the molecular mechanism of EndMT.

Protocol

All the animal procedures were conducted in accordance with the National Research Council. 2011. Guide for the Care and Use of Laboratory Animals: Eighth Edition. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital. Prior to surgery, all the surgical instruments are steam-autoclaved, and modified Krebs-Henseleit buffer, with a final concentration of 22 mmol/L KCl, is prepared as a cardioplegic solution (Ta…

Representative Results

Graft viability and beating In this work, the graft viability was visually assessed after all the clamps had been removed, and an approximate reperfusion time of 10-15 min was allowed with an open abdomen for observation of the graft. The same scoring system to objectively verify graft viability was used for visual assessment at the end of surgery and for the echocardiography on POD 1, POD 7, and POD 14. 0 = no organ function; 1 = (rest) organ function, only minimal cont…

Discussion

This animal model of heterotopic transplantation of a neonatal donor rat heart into the recipient's abdomen creates the possibility to study EndMT-derived fibrosis through detailed histological tissue evaluation, identify regulatory signaling pathways, and test treatment options. Since EndMT is the underlying mechanism for fibrotic diseases of the heart, this model has great value in the field of pediatric cardiac surgery and beyond. In this model, many factors can negatively influence the outcome of the procedure. T…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was funded by Additional Ventures – Single Ventricle Research Fund (SVRF) and Single Ventricle Expansion Fund (to I.F.) and a Marietta Blau scholarship of the OeAD-GmbH from funds provided by the Austrian Federal Ministry of Education, Science and Research BMBWFC (to G.G.).

Materials

Advanced Ventilator System For Rodents, SAR-1000	CWE, Inc.	12-03100	small animal ventilator
aSMA	Sigma	A2547	Antibody for Immunohistochemistry
Axio observer Z1	Carl Zeiss		inverted microscope
Betadine Solution	Avrio Health L.P.	367618150092
CD31	Invitrogen	MA1-80069	Antibody for Immunohistochemistry
DAPI	Invitrogen	D1306	Antibody for Immunohistochemistry
DemeLON Nylon black 10-0	DemeTECH	NL76100065F0P	10-0 Nylon suture
ETFE IV Catheter, 18G x 2	TERUMO SURFLO	SR-OX1851CA	intubation cannula
Micro Clip 8mm	Roboz Surgical Instrument Co.	RS-6471	microvascular clamps
Nylon black monofilament 11-0	SURGICAL SPECIALTIES CORP	AA0130	11-0 Nylon
O.C.T. Compound	Tissue-Tek	4583	Embedding medium for frozen tissue specimen
p-SMAD2/3	Invitrogen	PA5-110155	Antibody for Immunohistochemistry
Rodent, Tilting WorkStand	Hallowell EMC.	000A3467	oblique shelf for intubation
Silk Sutures, Non-absorbable, 7-0	Braintree Scientific	NC9201231	Silk suture
Slug/Snail	Abcam	ab180714	Antibody for Immunohistochemistry
Undyed Coated Vicryl 5-0 P-3 18"	Ethicon	J493G	5-0 Vicryl
Undyed Coated Vicryl 6-0 P-3 18"	Ethicon	J492G	6-0 Vicryl
VE-Cadherin	Abcam	ab231227	Antibody for Immunohistochemistry
Zeiss OPMI 6-SFR	Zeiss		Surgical microscope
Zen, Blue Edition, 3.6	Zen		inverted microscope software

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Gierlinger, G., Rech, L., Emani, S. M., del Nido, P. J., Friehs, I. A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition. J. Vis. Exp. (197), e65426, doi:10.3791/65426 (2023).