The authors acknowledge Alfreda and Kung Tak Chung for their excellent technical support during the animal experiments.

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
<ol>
	<li>Anesthesia and preparation of the animal
	<ol>
		<li>Fast the animals for 12 h and subject to water deprivation for 4 h before the experiment.</li>
		<li>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&#39;s palpebral reflexes until they are absent.</li>
		<li>Remove the pig&#39;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.</li>
		<li>Place a 7 mm endotracheal tube into the porcine trachea and place a 22 G venous indwelling needle into the ear vena.</li>
		<li>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).</li>
		<li>Monitor the surface electrocardiogram and blood pressure, and continuously monitor the heart rate, heart rhythm, and arterial blood pressure via electrophysiology recording systems.</li>
	</ol>
	</li>
	<li>Echocardiography
	<ol>
		<li>Move the pig to the left lateral decubitus position and fix on the table.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Pacemaker implantation
	<ol>
		<li>Move the pig to the supine position and fix the limbs of the pig on the table with straps.</li>
		<li>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.&#160;Sew the two muscles with 2-0 Vicryl. </li>
		<li>Cannulate the right jugular vein with an angiocath and insert a pacemaker lead to the right ventricle under X-ray guidance (Figure 2).</li>
		<li>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.</li>
		<li>Reprogram the pacemaker to backup VVI mode (35 bpm) by a pacemaker generator after the transplantation.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Calibrate a 7 Fr pressure-volume (PV) catheter in isotonic saline with a PV signal processor.</li>
		<li>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.</li>
		<li>Measure the end systolic pressure-volume relationship (ESPVR) by the PV signal processor during the occlusion of the IVC.</li>
		<li>Restart ventilation when the procedure is finished.</li>
	</ol>
	</li>
	<li>Induction of MI
	<ol>
		<li>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.</li>
		<li>Cannulate the right carotid artery with an 8F sheath as mentioned in step 1.4.3.</li>
		<li>Perform the coronary angiography through a 6F JR4 over-the-wire guiding catheter via the placed sheath guided by standard C arm fluoroscopy equipment.</li>
		<li>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).</li>
		<li>Inject 1 mL of 700 &#181;m sponge microspheres mixed with 3&#160;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.</li>
		<li>Repeat the injection procedure to achieve successful complete blockage.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Stem cell injection
	<ol>
		<li>Randomly assign all the animals with notable impairment of heart function (LVEF &#60; 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.</li>
		<li>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.</li>
		<li>Use 5-8 intramyocardial injections (~0.3 mL per injection) around the infarcted area to administer culture medium (Table of Materials)&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Intracardiac programmed electrical stimulation
	<ol>
		<li>Perform programmed electrical stimulation using a programmable stimulator to assess the inducibility of ventricular tachyarrhythmia (VT) after the cell transplantation therapy.</li>
		<li>Insert a 6F electrophysiological catheter into the right ventricular apex via the femoral vein before sacrificing all the animals.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Sequentially shorten the coupling intervals until a ventricular effective refractory period or arrhythmia is induced. Note the presence of inducible sustained VT (&#62;10 s).</li>
	</ol>
	</li>
</ol>
2. Postoperative protocol
<ol>
	<li>Postoperative medicine
	<ol>
		<li>Perform conventional pharmacological therapies for HF. In brief, orally administer metoprolol succinate (25 mg) and ramipril (2.5 mg) to all animals daily.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Infarct size assessment
	<ol>
		<li>Euthanize the animals by an overdose of dorminal (pentobarbital sodium, 100 mg/kg, IV) at the end of the experiment.</li>
		<li>Open the chest and collect the heart. Rinse the heart in 0.9% saline.</li>
		<li>Serially section LV tissue samples with a scalpel at 1 cm thicknesses in the LV transverse direction.</li>
		<li>Select portions of the slices that contain the infarcted myocardium to measure the wall thickness and the infarct area.</li>
		<li>Capture the image of these slices and quantitatively analyze the wall thickness and the infarct area using commercial image analysis software.</li>
		<li>Fix the tissue in 10% formalin at 4 &#176;C for a month. Embed the tissue within, adjacent and remote to the infarct sites (~1 cm2 pieces) in paraffin. Section into 5 &#181;m slices using a microtome for histological examination.</li>
	</ol>
	</li>
	<li>Cell survival
	<ol>
		<li>Detect the engraftment of the transplanted cells by immunohistochemical staining with anti-human nuclear antigen (HNA) according to the protocol provided by the manufacturer.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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...

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 MI4,5. 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.

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

establishing a swine model of post-myocardial infarction heart failure for stem cell treatment

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.

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 &#62; 40% of baseline). As a result, 16 animals completed the whole study protocol.
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Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

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.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

establishing-swine-model-post-myocardial-infarction-heart-failure-for

Research

JoVE Journal

Medicine

6.8K Views.  University of Hong Kong. Dit varkens hartfalen model, veroorzaakt door linker circumflex slagader blokkade en snelle pacing, kan worden gebruikt om de effecten van directe intramyocardial stamcel injectie op hartregeneratie te beoordelen. Ons varkenskolonie hartfalen model is vrij stabiel. De nieuwe infarct grootte van dit model is ongeveer 16% van de linker ventrikel en linker ventrikel injectie infarct verminderd met ten minste 40%ten opzichte van baseline.Met het experimentele varken vast in de supine posit...

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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Bioengineering

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

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

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