Name: a surgical model of heart failure with preserved ejection fraction in tibetan minipigs
Uploaded: 2022-02-18T14:30:00.000+00:00
Duration: 7 min 9 s
Description: Watch this Scientific Journal Video about A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs at JoVE.com

This work was supported by the Guangdong Science and Technology Program (2008A08003, 2016A020216019, 2019A030317014), the Guangzhou Science and Technology Program (201804010206), the National Natural Science Foundation of China (31672376, 81941002), and the Guangdong Provincial Key Laboratory of Laboratory Animals (2017B030314171).

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
<ol>
	<li>Acclimate the animals to the facility for 14 days before surgery.</li>
	<li>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 &#60;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).</li>
	<li>Fast the animals for more than 12 h before anesthesia by not feeding on the day of the surgery.</li>
	<li>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).</li>
</ol>
2. Sedation, tracheal intubation, and vein cannulation
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Perform the echocardiography (step 5) and collect 2 mL of blood samples.</li>
	<li>Intubate the minipigs with an endotracheal tube connected to a veterinary anesthesia ventilator station (Figure 3A) (see Table of Materials).</li>
	<li>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.</li>
	<li>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).</li>
	<li>Connect the animal to a veterinary monitor.</li>
</ol>
3. Surgical procedure
<ol>
	<li>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).</li>
	<li>Place sterile drapes over the surgical area.</li>
	<li>Administer propofol (5 mg/kg) (see Table of Materials) by intravenous injection to maintain general anesthesia.</li>
	<li>Mark the incision (~15 cm long) along the 4th intercostal space prior to skin incision with electrocautery.</li>
	<li>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.</li>
	<li>Use a rib retractor to spread the ribs (Figure 3D).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use a drainage chest tube to evacuate the air and excess fluids in the chest cavity.</li>
	<li>Close the chest wall in layers, reapproximate the ribs&#160;and divided muscles with absorbable sutures.</li>
	<li>Check for any bleeding and ensure good hemostasis.</li>
	<li>Apply a bottle of benzylpenicillin (800,000 units) (see Table of Materials) to the operation area post-surgery.</li>
	<li>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.</li>
	<li>Return the animal to its housing room and leave it to wake up automatically.</li>
</ol>
4. Post-surgery care
<ol>
	<li>Apply benzylpenicillin daily for 1 week (20,000 U/kg).</li>
	<li>Apply 1 mg/kg of flunixin meglumine (see Table of Materials) daily for 1 week. 
	NOTE:&#160;Opioid and NSAID analgesics should be administered intra- and post-operatively.</li>
</ol>
5. Transthoracic echocardiography
<ol>
	<li>Sedate the animal with 1 mg/kg of zoletil.</li>
	<li>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.</li>
	<li>Shave the left chest of the animal.</li>
	<li>Place fingers on the left-center of the chest to feel the apical pulse. Apply the ultrasonic gel to the surrounding area.</li>
	<li>Place the ultrasound system&#39;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.</li>
	<li>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.</li>
	<li>Turn the transducer head 90&#176; 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.</li>
	<li>Use the workstation provided by the manufacturer of the ultrasound system to assess the cardiac structure and function.</li>
</ol>

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The authors declare that they have no competing interests.

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 inflammation were observed in DAC hearts. 
The combination of open chest operation and aortic constriction has been used to develop heart failure models in large and small animals. For example, rodent aortic constriction-induced hypertension was reported as early as the 1950s18. Constriction of the ascending aorta in pigs induced mild left ventricular hypertrophy in 2-4 weeks old pigs. Regarding the operation site for locating the ascending aorta, a few studies selected the third intercostal space19,20, while another study selected the fourth intercostal space for the lateral thoracotomy21. It was found that constriction at the descending aorta was practical in adult Tibetan minipigs. The descending aortic segment was located right under the fourth intercostal space and surrounded by little connective tissue. 
The degree of constriction can be crucial for inducing key features of HFpEF. Melleby et al. reported that a smaller ring size accelerated hypertrophy, while larger ring sizes led to preserved EF for 8-20 weeks in mice with ascending aortic constriction22. Massie et al. set a pressure gradient of 20 mmHg for open-chest surgery in pigs to induce ventricular hypertrophy21. Charles et al. adopted progressive cuff inflation to generate HFpEF in female Yorkshire-Landrace pigs23. In the current study, a 20% increase in pressure at the descending aorta for 12 weeks led to HFpEF. Researchers have also combined aortic constriction techniques with deoxycorticosterone acetate or Western diet to induce HFpEF in female Ossabaw swine10,24. The constriction degrees are typically estimated by the pressure measured using a micro manometer catheter or echocardiography. A tool had been modified to measure the aortic pressure. A catheter with disposable blood pressure transducers connected to a patient monitor was used to record the pressure at the descending aorta.
Our previous study presented typical parasternal long-axis images of the HFpEF hearts in minipigs17; here, representative parasternal short-axis images are added. Consistent with the earlier results, the minipig DAC model displayed two distinct stages of cardiac remodeling, concentric hypertrophy, and dilation, during the 12-week observation period. These phenotypes are consistent with the clinical symptoms of HFpEF. New histological findings in the HFpEF model are also revealed in this work. Cardiomyocyte hypertrophy in the atria, ventricular septum, and ventricles are found. In addition, severe inflammatory cell infiltration in the left ventricle, right atrium, and aortic wall are obtained. This complements the previous findings, which demonstrated upregulation of interleukins -6 and -1&#946;, NF&#954;B, and cytokine production in the DAC myocardium17. The muscle layer disappeared in the mitral valve of the HFpEF pig, suggesting that abnormalities in the mitral valve contributed to cardiac dysfunction.
Establishing an aseptic surgical procedure is critical for obtaining successful and stable pig models. The aortic constriction surgery in pigs requires more operators than that in rodents. It usually requires an experienced surgical team of two surgeons, one anesthesiologist, two operating room nurses. These roles can be taken by veterinarians, human surgeons, and/or well-trained technicians. Compared with a rodent surgery that takes about 30 min to complete an aortic constriction procedure, it may take more than 3 h to complete a similar procedure in pigs. In practice, insufficient facilities and skilled personnel for large animal surgery limit the application of pig surgical models.

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 HFpEF4,5. 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 factors6,7. For example, deoxycorticosterone acetate alone or combined with a high fat/sugar diet induces HFpEF in pigs8,9. 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 EF15,16. 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.

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

a surgical model of heart failure with preserved ejection fraction in tibetan minipigs

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 &gt;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.

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 during the observation period, suggesting that hypertrophic remodeling was present in the left ventricle of the DAC minipigs (Figure 4B,C). The LVID at end-diastole decreased in weeks 4 and 6 and then gradually increased after week 8, suggesting that the ventricles underwent concentric hypertrophy before dilation (Figure 4D). The LVEF of the model hearts was maintained at &#62;50% during the 12-week period (Figure 4E).
Morphology and heart failure marker 
After week 12, the hearts were harvested as previously described17. Compared with those of the sham hearts, enlargement of the DAC hearts was observed (Figure 5A). The serum concentration of cardiac troponin I (cTnI) was determined using an enzyme-linked immunosorbent assay kit at weeks 0, 4, 8, and 12 following the manufacturer&#39;s instructions (see Table of Materials). The optical density was measured at 450 nm using a microplate reader. The heart failure marker cTnI was significantly higher at weeks 4, 8, and 12 in the DAC group than in the sham group at the corresponding time points (Figure 5B).
Histological examination 
Tissues from the free walls of the left and right ventricles, ventricular septum, left and right atrium, mitral valve, and aorta were collected and fixed with 4% paraformaldehyde. The tissues were embedded, sliced into sections, and stained with hematoxylin and eosin (H &#38; E) solution following the previous report17. Hypertrophic cardiomyocytes, fibrosis, inflammatory cells, pyknotic nuclei, and other structures were identified with a light microscope. Cardiomyocytes in the atria, ventricular septum, and ventricles displayed hypertrophy with pyknosis (Figure 6A). Muscular layers were reduced in the mitral valve (Figure 6B), and vascular endothelial hyperplasia was observed in the aorta (Figure 6C). Moreover, DAC induced extensive fibrosis in the myocardium of the minipigs (Figure 7A), accompanied by infiltration of inflammatory cells in the left ventricles, right atrium, and aortic walls (Figure 7B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63526/63526fig1v2.jpg" /> 
Figure 1: Experimental design. The experimental plan was made collaboratively by the principal investigator, surgeons, laboratory technicians, and animal care staff. The minipigs underwent health examinations, including biochemical tests and echocardiography. Following the surgery, anti-inflammatory and analgesic procedures were performed. Echocardiography, histological examination, and biomarker test evaluated heart failure phenotypes. The number of animals, n = 3 each, was for the sham and DAC groups. <a href="https://www.jove.com/files/ftp_upload/63526/63526fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63526/63526fig2v2.jpg" /> 
Figure 2. Surgical devices. The necessary devices (A) for the DAC surgery included aspirator (a), surgical table (b), Veterinary monitor (c), LED surgical lights (d), and aesthesia ventilator station (e). A veterinary ultrasound system was used to evaluate the structure and function of the animal hearts before and after surgery (B). The surgical tools included a laryngoscope (C) and various forceps, scalpel handles, and scissors (D). <a href="https://www.jove.com/files/ftp_upload/63526/63526fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63526/63526fig3v2.jpg" /> 
Figure 3: Surgical procedure. After sedation, the animal was intubated with an endotracheal tube (A), and the intravenous cannulation was established through an ear vein (B). The surgical site was at the left chest of the animal (C). After exposing the descending aorta (D,E), the constriction site (SB) and invasive sites for pressure monitoring (SA, SC) were determined (F,G), and the aortic pressure was measured using a patient monitor (H). A cartoon displays the overview of the constriction strategy (I). <a href="https://www.jove.com/files/ftp_upload/63526/63526fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63526/63526fig4v2.jpg" /> 
Figure 4: Transthoracic echocardiography evaluation. The representative B-mode and M-mode images of the pressure overload hearts from week 0 to week 12 are displayed in (A).&#160;The M mode images recorded for 4 s are shown. The pink scale bar indicates the record length of 1 s.The ventricular septum thickness (VST) at end-diastole increased in the DAC hearts (B). In contrast, the posterior wall thickness (PWT) at end-diastole gradually increased and decreased during the observation period (C). The left ventricular internal dimension (LVID) at end-diastole decreased in week 4 and week 6 and then gradually increased after week 8 (D). The LVEF of the model hearts was maintained at &#62;50% during the 12-week period (E). The number of animals, n = 3 each, was for the sham and DAC groups. Unpaired t-tests were used to determine the differences between the groups. *P &#60; 0.05 vs. the sham group. <a href="https://www.jove.com/files/ftp_upload/63526/63526fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63526/63526fig5v2.jpg" /> 
Figure 5: Heart morphology and serum cTnI. The size of the heart appeared to increase (A). The heart failure marker cTnI was significantly higher at weeks 4, 8, and 12 in the DAC group than in the sham group at the corresponding time points (B). The number of animals, n = 3 each, was for the sham and DAC groups. Unpaired t-tests were used to determine the differences between the groups. *P &#60; 0.05 vs. the sham group. <a href="https://www.jove.com/files/ftp_upload/63526/63526fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63526/63526fig6v2.jpg" /> 
Figure 6: Histology of myocardium, mitral valve, and aortic wall. H &#38; E staining was used to examine the cardiac tissue at the end of the experiment. Cardiomyocytes in atria, ventricular septum, and ventricles displayed hypertrophy (arrows in green; A), accompanied by pyrosis (arrows in yellow; A). Muscular layers were reduced in the mitral valve (arrows in blue; B). Vascular endothelial hyperplasia was observed in the aorta (area within the blue lines; C). Red asterisks: examined tissues; L. ventricle, left ventricle; R. ventricle, right ventricle; L. atrium, left atrium; R. atrium, right atrium. <a href="https://www.jove.com/files/ftp_upload/63526/63526fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63526/63526fig7v2.jpg" /> 
Figure 7: Fibrosis and inflammation in the DAC hearts. Histological examination showed extensive myocardial fibrosis in DAC minipigs. A fibrotic area in the left ventricle was displayed (asterisks and arrows in yellow; A). Infiltration of inflammatory cells was observed in the left ventricles, right atrium, and aortic walls (asterisks in green; B). Red asterisks: examined tissues; arrows in blue, eosinophils; L. ventricle, left ventricle; R. atrium, right atrium. <a href="https://www.jove.com/files/ftp_upload/63526/63526fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs at JoVE.com

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

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.

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

a-surgical-model-heart-failure-with-preserved-ejection-fraction

Nanyang Technological University

Research

JoVE Journal

Medicine

1.5K Views.  Guangdong Laboratory Animals Monitoring Institute. 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.

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

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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 ...

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.

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 ...

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 ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

Sterile Pericarditis in Aachener Minipigs As a Model for Atrial Myopathy and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only target the electrical abnormalities and not the underlying atrial myopathy. For the development of novel therapies, a reproducible large animal model of atrial myopathy is necessary. This paper presents a model of sterile pericarditis-induced atrial myopathy in Aachener minipigs. Sterile pericarditis was induced by spraying sterile talcum and leaving a layer of sterile gauze over the atrial epicardial surface. This led to inflammation and fibrosis, two crucial components of the pathophysiology of atrial myopathy, making the atria susceptible to the induction of AF. Two pacemaker electrodes were positioned epicardially on each atrium and connected to two pacemakers from different manufacturers. This strategy allowed for repeated non-invasive atrial programmed stimulation to determine the inducibility of AF at specified time points after surgery. Different protocols to test AF inducibility were used. The advantages of this model are its clinical relevance, with AF inducibility and the rapid induction of inflammation and fibrosis-both present in atrial myopathy-and its reproducibility. The model will be useful in the development of novel therapies targeting atrial myopathy and AF.

We describe a sterile pericarditis model in minipigs to study atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only ...

Echocardiography Recording in Awake Miniature Pigs

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and monitoring. The miniature pig, or minipig, is increasingly being used as a model of cardiac disease in medical research. Pigs are notoriously difficult to restrain and handle safely, and, therefore, research echocardiography in this species is almost always performed under anesthesia or heavy sedation. Anesthetics and sedatives universally affect cardiovascular function and may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow. Therefore, sedated or anesthetized echocardiography may not accurately depict the progression of cardiac disease in large animal models, thereby limiting the translational value of these important studies. This paper describes a novel device that allows for standing awake echocardiography in minipigs. In addition, training techniques used to teach pigs to tolerate this painless and non-invasive procedure without the need for hemodynamic-altering anesthetics are described. Standing awake echocardiography represents a safe and feasible way to perform the most common cardiac monitoring test in minipigs for cardiovascular research.

A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images.

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 &#181;m and severe: 250 &#181;m). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...