Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Summary

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Abstract

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

Introduction

Cardiovascular diseases, coronary artery disease (CAD) in particular, remain the major cause of morbidity and mortality in Hong Kong and worldwide¹. In Hong Kong, a 26% increase from 2012 to 2017 of the number of CAD patients treated under the Hospital Authority was projected². Among all CADs, acute myocardial infarction (MI) is a leading cause of death and subsequent complications, such as heart failure (HF). These contribute to significant medical, social, and financial burdens. In patients with MI, thrombolytic therapy or primary percutaneous coronary intervention (PCI) is an effective therapy in preserving life, but these therapies can only reduce cardiomyocyte (CM) loss during MI. The treatments available are unable to replenish the permanent loss of CMs, which leads to cardiac fibrosis, myocardial remodeling, cardiac arrhythmia, and eventually heart failure. The mortality rate at 1-year post-MI is around 7% with more than 20% patients developing HF³. In end-stage HF patients, heart transplantation is the only available effective therapy, but it is limited by a shortage of available organs. Novel therapies are necessary to reverse the development of post-MI HF. As a result, cell-based therapy is considered an attractive approach to repair the impaired CMs and ameliorate left ventricular (LV) function in HF following MI. Our previous studies found stem cell transplantation to be beneficial for heart function improvement after direct intramyocardial transplantation in small animal models of MI^4,5. Standardized preclinical large animal HF protocols are thus needed to further test the efficacy and safety of stem cell transplantation before clinical use.

Recent decades have witnessed the widespread use of pigs in cardiovascular research for stem cell therapy. HF pigs are a promising model of translational research due to their similarity to humans in terms of cardiac size, weight, rhythm, function, and coronary artery anatomy. Moreover, porcine HF models can mimic post-MI HF patients in terms of CM metabolism, electrophysiological properties, and neuroendocrine changes under ischemic conditions⁶. The protocol presented here uses such a standardized pig HF model, employing a closed-chest coronary balloon occlusion of the left circumflex artery (LCX) followed by rapid pacing induced by pacemaker implantation. The study also optimizes the route of intramyocardial administration of stem cells for the treatment of post-MI HF. The purpose is to produce a porcine animal model of chronic myocardial infarction that can be used to develop treatments that are clinically relevant for patients with severe CAD.

Protocol

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and regulations of the University of Hong Kong, and the protocol was approved by the Committee on the Use of Live Animals in Teaching and Research (CULTAR) at the University of Hong Kong.

NOTE: Female farm pigs weighing 35-40 kg (9-12 months old) were used for this study. The flowchart of this experiment is shown in Figure 1.

1. Surgical procedures

1. Anesthesia and preparation of the animal
   1. Fast the animals for 12 h and subject to water deprivation for 4 h before the experiment.
   2. Anesthetize the pigs through an intramuscular injection of tiletamine+zolezepam (2-7 mg/kg) and xylazine (0.5-1 mg/kg) prepared in 20 mL of normal saline. Monitor the animal's palpebral reflexes until they are absent.
   3. Remove the pig's hair and sterilize the skin at the neck and the groin for sections 1.3-1.5. Disinfect the operation area 3x with 70% ethanol and betadine.
   4. Place a 7 mm endotracheal tube into the porcine trachea and place a 22 G venous indwelling needle into the ear vena.
   5. Move the pig onto the operating table and place in a supine position. Connect the endotracheal tube to the respirator and mechanically ventilate (inspiratory/expiratory time ratio 1:2) the animal with isoflurane (1.5%-2.0% inhalation) and oxygen (0.5-1.5 L/min inhalation).
   6. Monitor the surface electrocardiogram and blood pressure, and continuously monitor the heart rate, heart rhythm, and arterial blood pressure via electrophysiology recording systems.
2. Echocardiography
   1. Move the pig to the left lateral decubitus position and fix on the table.
   2. Put the probe on the pericardial region and perform serial echocardiography, including 2D and M-mode imaging, using a high-resolution echocardiographic system and a 3-9 MHz transducer at the baseline, before cell transplantation and 8 weeks after cell transplantation (Supplemental Figure 1).
   3. Analyze all the obtained images using commercial software. Calculate the LV end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), LV ejection fraction (LVEF), and wall thickness after standard echocardiographic images are obtained from the parasternal long-axis view.
      NOTE: All the off-line analyses were conducted by another independent operator using a computer workstation. The variability of the measurements between different observers was 4% based on 20 repeated random images. All the echocardiographic measurements were performed in accordance with the American Society of Echocardiography recommendations.
3. Pacemaker implantation
   1. Move the pig to the supine position and fix the limbs of the pig on the table with straps.
   2. Locate the right carotid artery and jugular vein in the carotid triangle (behind the sternocleidomastoid and surrounded by the stylohyoid, the digastric muscle, and the omohyoid) and isolate the right carotid artery and jugular vein with hemostatic forceps under sterile conditions (Supplemental Figure 2). Ligate the distal end of the right carotid artery and jugular vein. Sew the two muscles with 2-0 Vicryl.
   3. Cannulate the right jugular vein with an angiocath and insert a pacemaker lead to the right ventricle under X-ray guidance (Figure 2).
   4. Isolate the sternocleidomastoid and the anterior scalene muscle using forceps. Implant a pacemaker between the two muscles and sew the two muscles with 2-0 silk. Connect the pacemaker to the lead.
   5. Reprogram the pacemaker to backup VVI mode (35 bpm) by a pacemaker generator after the transplantation.
   6. Apply rapid ventricular pacing (150 beats/min) to induce HF by a pacemaker generator 4 weeks after MI induction. Then set the pacemaker back to backup VVI mode at 8 weeks.
4. Invasive pressure volume loop analysis
   NOTE: Perform invasive hemodynamic assessment at baseline, before cell transplantation and 8 weeks after cell transplantation to assess changes in LV function.
   1. Isolate the right femoral artery and femoral vein in the femoral triangle (surrounded by the inguinal ligament, sartorius muscle, and adductor longus muscle) (Supplemental Figure 2).
   2. Cannulate the right femoral artery with an angiocath and place a guidewire into the artery via the angiocath. Remove the angiocath and cannulate a 9F sheath into the artery under the guidance of the guidewire. Remove the guidewire.
   3. Cannulate the right femoral vein with a 12F sheath as described in step 1.4.2. Insert a balloon catheter from the placed 12F sheath into the inferior vena cava (IVC) under X-ray guidance.
   4. Calibrate a 7 Fr pressure-volume (PV) catheter in isotonic saline with a PV signal processor.
   5. Insert the PV catheter into the LV apex from the placed 9F sheath under X-ray guidance. Suspend ventilation and measure the left ventricular maximal positive pressure derivative (+dP/dt), end-systolic pressure (ESP), and end-diastolic pressures (EDP) with the PV signal processor.
   6. Measure the end systolic pressure-volume relationship (ESPVR) by the PV signal processor during the occlusion of the IVC.
   7. Restart ventilation when the procedure is finished.
5. Induction of MI
   1. Intravenously administer amiodarone (5 mg/kg intravenously over 1 h) and lidocaine (1.5 mg/kg intravenous bolus) to the animal before induction of MI to prevent ventricular arrhythmias.
   2. Cannulate the right carotid artery with an 8F sheath as mentioned in step 1.4.3.
   3. Perform the coronary angiography through a 6F JR4 over-the-wire guiding catheter via the placed sheath guided by standard C arm fluoroscopy equipment.
   4. Occlude the left circumflex coronary artery (LCX) distal to the first obtuse marginal branch with percutaneous transluminal coronary angioplasty (PTCA) dilatation balloon catheter inflation under X-ray guidance (Figure 2).
   5. Inject 1 mL of 700 µm sponge microspheres mixed with 3 mL of saline prepared in a 10 mL syringe through the balloon catheter to block the LCX, then deflate the balloon and perform an angiogram to confirm the occlusion.
   6. Repeat the injection procedure to achieve successful complete blockage.
   7. Monitor the animal heart rate and rhythm to detect cardiac arrhythmias. If ventricular fibrillation happened, use an external, biphasic defibrillator to reestablish a sinus rhythm using 150-300 J shocks.
6. Stem cell injection
   1. Randomly assign all the animals with notable impairment of heart function (LVEF < 40% at 8 weeks after induction of MI) to two different groups: one that will receive intramyocardial administration of 2 x 10⁸ human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs), and the other that will not receive hiPSC-MSCs.
   2. Prepare the hiPSC-MSCs in 2 mL of normal saline for intramyocardial transplantation. Before intramyocardial hiPSC-MSCs transplantation, repeat the anesthesia and animal preparation steps mentioned in section 1.1, this time sterilizing 10 cm around the apex beat area. Perform left thoracotomy at the 4-5 intercostal space with a retractor. Perform pericardiotomy to expose the infarcted lateral wall.
      NOTE: The length of the incision was 10-12 cm.
   3. Use 5-8 intramyocardial injections (~0.3 mL per injection) around the infarcted area to administer culture medium (Table of Materials) to one group of animals or 2 x 108 hiPSC-MSCs to the other group (Figure 3). Carefully avoid any damage to coronary arteries to reduce the risk of hemorrhage.
   4. Close the intercostal space with iron wire and close the muscle layer with 2-0 silk. Sew the subcutaneous tissue and skin with 2-0 vicryl.
7. Intracardiac programmed electrical stimulation
   1. Perform programmed electrical stimulation using a programmable stimulator to assess the inducibility of ventricular tachyarrhythmia (VT) after the cell transplantation therapy.
   2. Insert a 6F electrophysiological catheter into the right ventricular apex via the femoral vein before sacrificing all the animals.
   3. Display the intracardiac recordings with the surface electrocardiogram leads I, II, and III on the electrophysiological recording system at a speed of 200 mm/s. Deliver a 2 ms pulse width at 2x the diastolic threshold using a stimulator.
   4. Deliver a pacing train of eight stimuli (S1) at two drive cycle lengths (200 ms and 300 ms), followed by one (S2) or two (S2 and S3) premature extra stimuli.
   5. Sequentially shorten the coupling intervals until a ventricular effective refractory period or arrhythmia is induced. Note the presence of inducible sustained VT (>10 s).

2. Postoperative protocol

1. Postoperative medicine
   1. Perform conventional pharmacological therapies for HF. In brief, orally administer metoprolol succinate (25 mg) and ramipril (2.5 mg) to all animals daily.
   2. Intramuscularly administer enrofloxacin (5 mg/kg) and buprenorphine (0.01 mg/kg) to all animals daily for 1 week after surgery to prevent infection and relieve pain.
   3. To minimize immunological rejection, orally administer a steroid (40 mg/day orally) and cyclosporine (200 mg/day orally) to all animals from 3 days prior to cell transplantation to 8 weeks after.
2. Infarct size assessment
   1. Euthanize the animals by an overdose of dorminal (pentobarbital sodium, 100 mg/kg, IV) at the end of the experiment.
   2. Open the chest and collect the heart. Rinse the heart in 0.9% saline.
   3. Serially section LV tissue samples with a scalpel at 1 cm thicknesses in the LV transverse direction.
   4. Select portions of the slices that contain the infarcted myocardium to measure the wall thickness and the infarct area.
   5. Capture the image of these slices and quantitatively analyze the wall thickness and the infarct area using commercial image analysis software.
   6. Fix the tissue in 10% formalin at 4 °C for a month. Embed the tissue within, adjacent and remote to the infarct sites (~1 cm² pieces) in paraffin. Section into 5 µm slices using a microtome for histological examination.
3. Cell survival
   1. Detect the engraftment of the transplanted cells by immunohistochemical staining with anti-human nuclear antigen (HNA) according to the protocol provided by the manufacturer.
   2. Capture the image in three different sections at five random fields in each animal and quantitatively analyze the positive cells in the peri-infarct zone.
      NOTE: The image capturing system and image analysis software were used to capture and analyze the images of the heart sections.

Results

Mortality
A total of 24 pigs were used in this study. Three of them died during MI induction because of sustained VT. One animal died in the open-heart surgery for cell injection because of wound bleeding. Two animals died because of severe infection. Two animals were excluded because of slight EF reduction (LVEF reduction > 40% of baseline). As a result, 16 animals completed the whole study protocol.

...

Discussion

Standard animal models are of paramount importance to understand the pathophysiology and mechanisms of diseases and test novel therapeutics. Our protocol establishes a porcine model of HF induced by left circumflex artery blockage and rapid pacing. Eight weeks after the induction of MI, the animals developed significant impairment of LVEF, LVEDD, LVESD, +dP/dt, and ESPVR. This protocol also tests the administration method of stem cell therapy for heart regeneration by intramyocardial injection. The infarct size, and card...

Materials

Name	Company	Catalog Number	Comments
Amiodarone	Mylan	-	-
Anaesthetic machines and respirator	Drager	Fabius plus XL	-
Angiocath	Becton Dickinson	381147	-
Anti-human nuclear antigen	abcam	ab19118	-
Axio Plus image capturing system	Zeiss	Axioskop 2 PLUS	Axioskop 2 plus
AxioVision Rel. 4.5 software	Zeiss	-	-
Baytril	Bayer	-	enrofloxacin
Betadine	Mundipharma	-	-
CardioLab Electrophysiology Recording Systems	GE Healthcare	G220f	-
Culture media	MesenCult	05420	-
Cyclosporine	Novartis	-	-
Defibrillator	GE Healthcare	CardioServ	-
Dorminal	TEVA	-	-
Echocardiographic system	GE Vingmed	Vivid i	-
EchoPac software	GE Vingmed	-	-
Electrophysiological catheter	Cordis Corp	-	-
Embozene Microsphere	Boston Scientific	17020-S1	700 μm
Endotracheal tube	Vet Care	VCPET70PCW	Size 7
Ethanol	VWR chemicals	20821.33	-
Formalin	Sigma	HT501320	10%
IVC balloon Dilatation Catheter	Boston Scientific	3917112041	Mustang
JR4 guiding catheter	Cordis Corp	67208200	6F
Lidocaine	Quala	-	-
Mersilk	Ethicon	W584	2-0
Metoprolol succinate	Wockhardt	-	-
Microtome	Leica	RM2125RT	-
Mobile C arm fluoroscopy equipment	GE Healthcare	OEC 9900 Elite	-
Pacemaker	St Jude Medical	PM1272	Assurity MRI pacemaker
Pacemaker generator	St Jude Medical	Merlln model 3330	-
Pressure-volume catheter	CD Leycom	CA-71103-PL	7F
Pressure–volume signal processor	CD Leycom	SIGMA-M	-
Programmable Stimulator	Medtronic Inc	5328	-
PTCA Dilatation balloon Catheter	Boston Scientific	H7493919120250	MAVERICK over the wire
Ramipril	TEVA	-	-
Sheath introducer	Cordis Corp	504608X	8F, 9F, 12F
Steroid	Versus Arthritis	-	-
Temgesic	Nindivior	-	buprenorphine
Venous indwelling needle	TERUMO	SR+OX2225C	22G
Vicryl	Ethicon	VCP320H	2-0
Xylazine	Alfasan International B.V.	-	-
Zoletil	Virbac New Zealand Limited	-	tiletamine+zolezepam