A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

Summary

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

Abstract

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at >50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

Introduction

Heart failure with preserved ejection fraction (HFpEF) accounts for more than half of heart failure cases and has become a worldwide public health issue¹. Clinical observations have indicated several critical features of HFpEF: (1) ventricular diastolic dysfunction, accompanied by increased systolic stiffness, (2) normal ejection fraction at rest with impaired exercise performance, and (3) cardiac remodeling². The proposed mechanisms include hormonal dysregulation, systemic microvascular inflammation, metabolic disorders, and abnormalities in sarcomeric and extracellular matrix proteins³. However, experimental studies have shown that heart failure with reduced ejection fraction (HFrEF) causes these alterations. Clinical studies have explored the therapeutic effects of angiotensin receptor inhibitors and drugs for treating HFrEF in HFpEF^4,5. However, unique therapeutic approaches for HFpEF are needed. Compared with understanding the clinical symptoms, the alterations in pathology, biochemistry, and molecular biology of HFpEF remain poorly defined.

Animal models of HFpEF have been developed to explore the mechanisms, diagnostic markers, and therapeutic approaches. Laboratory animals, including pigs, dogs, rats, and mice, can develop HFpEF, and diverse risk factors, including hypertension, diabetes mellitus, and aging, were selected as induction factors^6,7. For example, deoxycorticosterone acetate alone or combined with a high fat/sugar diet induces HFpEF in pigs^8,9. Ventricular pressure overload is another technique used to develop HFpEF in large and small animal models¹⁰. In addition, specific EF cut-off values to define HFpEF have been adopted across continents in recent years, as seen in the European Society of Cardiology guidelines, the American College of Cardiology Foundation/American Heart Association¹¹, the Japanese Circulation Society/the Japanese Heart Failure Society¹². Thus, many previously established models may become appropriate for HFpEF studies if the clinical criteria are adopted. For example, Youselfi et al. claimed that a genetically modified mouse strain, Col4a3-/-, was an effective HFpEF model. This strain developed typical HFpEF cardiac symptoms, such as diastolic dysfunction, mitochondrial dysfunction, and cardiac remodeling¹³. A previous study used a high-energy diet to induce cardiac remodeling with a mid-range of EF in aged monkeys¹⁴, characterized by a metabolic disorder, fibrosis, and reduced actomyosin MgATPase in the myocardium. Mouse transverse aortic constriction (TAC) is one of the most widely used models to mimic hypertension-induced ventricular cardiomyopathy. The left ventricle progresses from concentric hypertrophy with increased EF to dilated remodeling with reduced EF^15,16. The transitional phenotypes between these two typical stages suggest that the aortic constriction technique can be used to study HFpEF.

The pathological features, cellular signaling, and mRNA profiles of a porcine HFpEF model were previously published¹⁷. Here, a step-by-step protocol is presented to establish this model and the approaches to evaluate the phenotypes of this model. The procedure is illustrated in Figure 1. Briefly, the surgical plan was made jointly by the principal investigator, surgeons, laboratory technicians, and animal care staff. The minipigs underwent health examinations, including biochemical tests and echocardiography. Following surgery, anti-inflammatory and analgesic procedures were performed. Echocardiography, histological examination, and biomarkers were used to evaluate the phenotypes.

Protocol

All animal studies were approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute (approval no. IACUC2017009). All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals (8th Ed., 2011, The National Academies, USA). The animals were housed in an AAALAC-accredited facility at the Guangdong Laboratory Animals Monitoring Institute (license no. SYXK (YUE) 2016-0122, China). Six male Tibetan minipigs (n = 3 each for the sham group and DAC group, 25-30 kg in weight) were used to develop the HFpEF model.

1. Animal and instrument preparation

1. Acclimate the animals to the facility for 14 days before surgery.
2. Perform health examinations, including biochemical tests and echocardiography, before the surgery. Exclude the animals with cardiac abnormalities in structure (ventricular dilation or hypertrophy) and function (EF <50%) according to the T/CALAS85-2020 Laboratory animals - Guidelines for the health assessment of major organs, such as the heart, liver, kidney, and brain of large laboratory animals (Chinese Association for Laboratory Animal Sciences, China).
3. Fast the animals for more than 12 h before anesthesia by not feeding on the day of the surgery.
4. Prepare the surgical room and devices (Figure 2). Check aesthesia ventilator station, veterinary and patient monitors, veterinary ultrasound system, aspirator, and other surgical devices. Autoclave the scissors, forceps, retractors, scalpel handles, aspirator head, surgical needles, etc. (see Table of Materials).

2. Sedation, tracheal intubation, and vein cannulation

1. Weigh the animals and calculate the anesthetic drugs. Sedate the minipigs with 1 mg/kg of zoletil injection (tiletamine and zolazepam for injection) and 0.5 mg/kg of xylazine hydrochloride injection (see Table of Materials).
2. Restrain and place the minipigs in the right lateral recumbent position on the operating surgery table. Turn on the heating system to maintain the body temperature of the animals.
3. Perform the echocardiography (step 5) and collect 2 mL of blood samples.
4. Intubate the minipigs with an endotracheal tube connected to a veterinary anesthesia ventilator station (Figure 3A) (see Table of Materials).
5. Initiate the ventilation at an 8 mL/kg of tidal volume and 30 breaths/min. Maintain the animals with 1.5%-2.5% of isoflurane during the surgical procedure.
6. Establish intravenous cannulation using a peripheral intravenous catheter (26 G) (see Table of Materials) from an ear vein (usually the marginal ear vein, Figure 3B).
7. Connect the animal to a veterinary monitor.

3. Surgical procedure

1. Shave the left thoracic area. Apply 0.7% of iodine and 75% alcohol to aseptically prepare the skin from the scapula to the diaphragm (Figure 3C).
2. Place sterile drapes over the surgical area.
3. Administer propofol (5 mg/kg) (see Table of Materials) by intravenous injection to maintain general anesthesia.
4. Mark the incision (~15 cm long) along the 4th intercostal space prior to skin incision with electrocautery.
5. Open the chest using a combination of cautery and blunt dissection of the muscle and connective tissue. Use an aspirator to remove blood during the operation.
6. Use a rib retractor to spread the ribs (Figure 3D).
7. Locate the thoracic descending aorta segment and determine the constriction site (Figure 3E). Use two 3-0 surgical sutures to loop around the segment twice (Figure 3F). Place three layers of medical gauze between the suture and the aorta to avoid tissue damage by sutures.
8. Set up pressure measurement units to determine the degree of constriction (Figure 3F-H).
   NOTE: The unit includes a catheter that punctures the vessel wall, connection tube, pressure transducer, and a patient monitor.
9. Tighten the surgical suture surrounding the descending aorta segment gradually to achieve the desired constriction degree. Allow the pressure readings to stabilize for 20 min and permanently tighten the surgical knots.
10. Use a drainage chest tube to evacuate the air and excess fluids in the chest cavity.
11. Close the chest wall in layers, reapproximate the ribs and divided muscles with absorbable sutures.
12. Check for any bleeding and ensure good hemostasis.
13. Apply a bottle of benzylpenicillin (800,000 units) (see Table of Materials) to the operation area post-surgery.
14. Monitor the presence of eye blinking and limb movement of the animal. Disconnect the ventilator but leave the endotracheal tube. Monitor the presence of spontaneous breathing.
15. Return the animal to its housing room and leave it to wake up automatically.

4. Post-surgery care

1. Apply benzylpenicillin daily for 1 week (20,000 U/kg).
2. Apply 1 mg/kg of flunixin meglumine (see Table of Materials) daily for 1 week.
   NOTE: Opioid and NSAID analgesics should be administered intra- and post-operatively.

5. Transthoracic echocardiography

1. Sedate the animal with 1 mg/kg of zoletil.
2. Place the animal in a mobile restraint unit with a canvas cover.
   NOTE: The mobile restraint unit (see Table of Materials) has four apertures designed to extend the forelimbs and hind limbs of the animal.
3. Shave the left chest of the animal.
4. Place fingers on the left-center of the chest to feel the apical pulse. Apply the ultrasonic gel to the surrounding area.
5. Place the ultrasound system's phased array transducer (3-8 Hz) in the third intercostal space. Move the transducer toward an anterior or posterior direction and adjust the notch angle.
6. Identify the atria, ventricles, and aorta. Record the B-mode and M-mode parasternal long-axis images.
   NOTE: The B-mode image represents the cross-section of the left ventricle at the papillary muscle level, and the M-mode image shows the movement of the left ventricle over time.
7. Turn the transducer head 90° clockwise to obtain the parasternal short-axis view. Identify the left ventricle, right ventricle, and papillary muscle. Record the B-mode and M-mode images.
8. Use the workstation provided by the manufacturer of the ultrasound system to assess the cardiac structure and function.

Results

Echocardiography
Cardiac structure and function were evaluated at weeks 0, 2, 4, 6, 8, 10, and 12. The B-mode and M-mode recordings of the parasternal short-axis view are displayed in Figure 4A. The echocardiographic measurement included the ventricular septum thickness (VST), posterior wall thickness (PWT), and left ventricular internal dimension (LVID). The VST at end-diastole increased in the DAC hearts, whereas the PWT at end-diastole increased and then decreased d...

Discussion

This study used DAC techniques to develop an HFpEF model for Tibetan minipigs. A step-by-step animal and instrument preparation protocol is presented here, including sedation, tracheal intubation, vein cannulation, surgical procedure, and post-surgery care. The recording techniques for echocardiographic B-mode and M-mode heart images are also presented. After DAC, the heart underwent left ventricular hypertrophy during weeks 4 and 6 and dilation after week 8. LVEF was preserved during the 12-week period. Fibrosis and inf...

Materials

Name	Company	Catalog Number	Comments
Absorbable surgical suture	Putong Jinhua Medical Co. Ltd, China	4-0
Aesthesia ventilator station	Shenzhen Mindray Bio-Medical Electronics Co., Ltd, China	WATO EX-35vet
Aspirator	Shanghai Baojia Medical Apparatus Co., Ltd, China	YX930D
Benzylpenicillin	Sichuan Pharmaceutical. INC, China	H5021738
Disposal endotracheal tube with cuff	Shenzhen Verybio Co., Ltd, China	20 cm, ID 0.9
Disposal transducer	Guangdong Baihe Medical Technology Co., Ltd, China
Dissection blade	Shanghai Medical Instruments (Group) Co., Ltd, China
Electrocautery	Shanghai Hutong Medical Instruments (Group) Co., Ltd, China	GD350-B
Enzyme-linked immunosorbent assay ELISA kit	Cusabio Biotech Co., Ltd, China	CSB-E08594r
Eosin	Sigma-Aldrich Corp.	E4009
Flunixin meglumine	Shanghai Tongren Pharmaceutical Co., Ltd., China	Shouyaozi(2012)-090242103
Forceps	Shanghai Medical Instruments (Group) Co., Ltd.,China
Hematoxylin	Sigma-Aldrich Corp.	H3136
Isoflurane	RWD Life Science Co., Ltd, China	Veteasy for animals
Laryngoscope	Taixing Simeite Medical Apparatus and Instruments Limited Co., Ltd, China	For adults
LED surgical lights	Mingtai Medical Group, China	ZF700
Microplate reader	Thermo Fisher Scientific, USA	Multiskan FC
Microscope	Leica, Germany	DM2500
Mobile restraint unit	Customized	N/A	A mobile restraint unit, made by metal frame and wheels, with a canvas cover
Oxygen	Local suppliers, Guangzhou, China
Paraformaldehyde	Sigma-Aldrich Corp.	V900894
Patient monitor	Shenzhen Mindray Bio-Medical Electronics Company, China	Beneview T5
Peripheral Intravenous (IV) Catheter	Shenzhen Yima Pet Industry Development Co., Ltd., China	26G X 16 mm
Propofol	Guangdong Jiabo Phamaceutical Co., Ltd.	H20051842
Rib retractor	Shanghai Medical Instruments (Group) Co., Ltd.,China
Ruler	Deli Manufacturing Company, China
Scalpel handles	Shanghai Medical Instruments (Group) Co., Ltd.,China
Scissors (g)	Shanghai Medical Instruments (Group) Co., Ltd.,China
Suture	Medtronic-Coviden Corp.	3-0, 4-0
Ultrasonic gel	Tianjin Xiyuansi Production Institute, China	TM-100
Veterinary monitor	Shenzhen Mindray Bio-Medical Electronics Company, China	ePM12M Vet
Veterinary ultrasound system	Esatoe, Italy	MyLab30	Equiped with phased array transducer (3-8 Hz)
Xylazine hydrochloride injection	Shenda Animal Phamarceutical Co., Ltd., China	Shouyaozi(2016)-07003
Zoletil injection	Virbac, France	Zoletil 50	Tiletamine and zolazepam for injection