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>

<ol>
	<li>Dunlay, S. M., Roger, V. L., Redfield, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+heart+failure+with+preserved+ejection+fraction.">Epidemiology of heart failure with preserved ejection fraction.</a> Nature Reviews Cardiology. 14 (10), 591-602 (2017).</li><li>Redfield, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+with+preserved+ejection+fraction.">Heart failure with preserved ejection fraction.</a> New England Journal of Medicine. 375 (19), 1868-1877 (2016).</li><li>Lam, C. S. P., Voors, A. A., de Boer, R. A., Solomon, S. D., van Veldhuisen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+with+preserved+ejection+fraction:+From+mechanisms+to+therapies.">Heart failure with preserved ejection fraction: From mechanisms to therapies.</a> European Heart Journal. 39 (30), 2780-2792 (2018).</li><li>Solomon, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+receptor+neprilysin+inhibition+in+heart+failure+with+preserved+ejection+fraction:+Rationale+and+design+of+the+PARAGON-HF+trial.">Angiotensin receptor neprilysin inhibition in heart failure with preserved ejection fraction: Rationale and design of the PARAGON-HF trial.</a> JACC-Heart Failure. 5 (7), 471-482 (2017).</li><li>Cunningham, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sacubitril/valsartan+on+biomarkers+of+extracellular+matrix+regulation+in+patients+with+HFpEF.">Effect of sacubitril/valsartan on biomarkers of extracellular matrix regulation in patients with HFpEF.</a> Journal of the American College of Cardiology. 76 (5), 503-514 (2020).</li><li>Concei&#231;&#227;o, G., Heinonen, I., Louren&#231;o, A. P., Duncker, D. J., Falc&#227;o-Pires, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+heart+failure+with+preserved+ejection+fraction.">Animal models of heart failure with preserved ejection fraction.</a> Netherlands Heart Journal. 24 (4), 275-286 (2016).</li><li>Noll, N. A., Lal, H., Merryman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+heart+failure+with+preserved+or+reduced+ejection+fraction.">Mouse models of heart failure with preserved or reduced ejection fraction.</a> American Journal of Pathology. 190 (8), 1596-1608 (2020).</li><li>Schwarzl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+hypertensive+cardiomyopathy:+Implications+for+heart+failure+with+preserved+ejection+fraction.">A porcine model of hypertensive cardiomyopathy: Implications for heart failure with preserved ejection fraction.</a> American Journal of Physiology-Heart and Circulatory Physiology. 309 (9), 1407-1418 (2015).</li><li>Reiter, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-stage+heart+failure+with+preserved+ejection+fraction+in+the+pig:+A+cardiovascular+magnetic+resonance+study.">Early-stage heart failure with preserved ejection fraction in the pig: A cardiovascular magnetic resonance study.</a> Journal of Cardiovascular Magnetic Resonance. 18 (1), 63 (2016).</li><li>Silva, K. A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+small+heat+shock+protein+20+activation+is+not+associated+with+traditional+autophagy+markers+in+Ossabaw+swine+with+cardiometabolic+heart+failure.">Tissue-specific small heat shock protein 20 activation is not associated with traditional autophagy markers in Ossabaw swine with cardiometabolic heart failure.</a> American Journal of Physiology-Heart and Circulatory Physiology. 319 (5), 1036-1043 (2020).</li><li>Ponikowski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2016+ESC+Guidelines+for+the+diagnosis+and+treatment+of+acute+and+chronic+heart+failure:+The+Task+Force+for+the+diagnosis+and+treatment+of+acute+and+chronic+heart+failure+of+the+European+Society+of+Cardiology+(ESC)Developed+with+the+special+contribution+of+the+Heart+Failure+Association+(HFA)+of+the+ESC.">2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC.</a> European Heart Journal. 37 (27), 2129-2200 (2016).</li><li>Tsutsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JCS+2017/JHFS+2017+guideline+on+diagnosis+and+treatment+of+acute+and+chronic+heart+failure+-+Digest+version.">JCS 2017/JHFS 2017 guideline on diagnosis and treatment of acute and chronic heart failure - Digest version.</a> Circulation Journal. 83 (10), 2084-2184 (2019).</li><li>Yousefi, K., Dunkley, J. C., Shehadeh, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+model+for+phenogroup+3+HFpEF.">A preclinical model for phenogroup 3 HFpEF.</a> Aging (Albany NY). 11 (13), 4305-4307 (2019).</li><li>Zheng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aged+monkeys+fed+a+high-fat/high-sugar+diet+recapitulate+metabolic+disorders+and+cardiac+contractile+dysfunction.">Aged monkeys fed a high-fat/high-sugar diet recapitulate metabolic disorders and cardiac contractile dysfunction.</a> Journal of Cardiovascular Translational Research. 14 (5), 799-815 (2021).</li><li>Shirakabe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drp1-dependent+mitochondrial+autophagy+plays+a+protective+role+against+pressure+overload-induced+mitochondrial+dysfunction+and+heart+failure.">Drp1-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure.</a> Circulation. 133 (13), 1249-1263 (2016).</li><li>Zhabyeyev, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-overload-induced+heart+failure+induces+a+selective+reduction+in+glucose+oxidation+at+physiological+afterload.">Pressure-overload-induced heart failure induces a selective reduction in glucose oxidation at physiological afterload.</a> Cardiovascular Research. 97 (4), 676-685 (2013).</li><li>Tan, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+heart+failure+with+preserved+ejection+fraction+induced+by+chronic+pressure+overload+characterized+by+cardiac+fibrosis+and+remodeling.">A porcine model of heart failure with preserved ejection fraction induced by chronic pressure overload characterized by cardiac fibrosis and remodeling.</a> Frontiers in Cardiovascular Medicine. 8, 677727 (2021).</li><li>Beznak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+heart+weight+and+blood+pressure+following+aortic+constriction+in+rats.">Changes in heart weight and blood pressure following aortic constriction in rats.</a> Canadian Journal of Biochemistry and Physiology. 33 (6), 995-1002 (1955).</li><li>Bikou, O., Miyashita, S., Ishikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+model+of+increased+cardiac+afterload+induced+by+ascending+aortic+banding.">Pig model of increased cardiac afterload induced by ascending aortic banding.</a> Methods in Molecular Biology. 1816, 337-342 (2018).</li><li>Hiemstra, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+low-intensity+exercise+attenuates+cardiomyocyte+contractile+dysfunction+and+impaired+adrenergic+responsiveness+in+aortic-banded+mini-swine.">Chronic low-intensity exercise attenuates cardiomyocyte contractile dysfunction and impaired adrenergic responsiveness in aortic-banded mini-swine.</a> Journal of Applied Physiology. 124 (4), 1034-1044 (2018).</li><li>Massie, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+high-energy+phosphate+and+substrate+metabolism+in+swine+with+moderate+left+ventricular+hypertrophy.">Myocardial high-energy phosphate and substrate metabolism in swine with moderate left ventricular hypertrophy.</a> Circulation. 91 (6), 1814-1823 (1995).</li><li>Melleby, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+high+precision+aortic+constriction+that+allows+for+generation+of+specific+cardiac+phenotypes+in+mice.">A novel method for high precision aortic constriction that allows for generation of specific cardiac phenotypes in mice.</a> Cardiovascular Research. 114 (12), 1680-1690 (2018).</li><li>Charles, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+heart+failure+with+preserved+ejection+fraction:+magnetic+resonance+imaging+and+metabolic+energetics.">A porcine model of heart failure with preserved ejection fraction: magnetic resonance imaging and metabolic energetics.</a> ESC Heart Failure. 7 (1), 92-102 (2020).</li><li>Olver, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western,+diet-fed,+aortic-banded+ossabaw+swine:+A+Preclinical+model+of+cardio-metabolic+heart+failure.">Western, diet-fed, aortic-banded ossabaw swine: A Preclinical model of cardio-metabolic heart failure.</a> JACC Basic to Translational Science. 4 (3), 404-421 (2019).</li></ol>

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 inf...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;Medical&#160;Instruments (Group)&#160;Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery</td>
			<td>Shanghai&#160;Hutong Medical Instruments (Group)&#160;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&#160;Medical&#160;Instruments (Group)&#160;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&#160;Medical&#160;Instruments (Group)&#160;Co., Ltd.,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Deli&#160;Manufacturing&#160;Company, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handles</td>
			<td>Shanghai&#160;Medical&#160;Instruments (Group)&#160;Co., Ltd.,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (g)</td>
			<td>Shanghai&#160;Medical&#160;Instruments (Group)&#160;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 d...

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

Research

JoVE Journal

Medicine

1.4K 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

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 ...

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...

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 ...

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...