The author would like to thank Minna T&#246;rr&#246;nen, Riikka Ven&#228;l&#228;inen, Heikki Karhunen, and Inkeri Niemi from the National Laboratory Animal Center for their assistance in animal work. This study is supported by Finnish Academy, ERC, and CardioReGenix EU Horizon grant.

All experiments are approved by the Animal Experiment Board of the University of Eastern Finland. This protocol describes a clinically relevant treadmill exercise test for pigs to evaluate the safety and efficacy of novel therapies for heart diseases. Female domestic pigs weighing 25-80 kg were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Setting up the running track
<ol>
	<li>Set up the running track so that animals can only move one way. Use gates and hatches to prevent the animals from moving back. The floor plan of the running track is shown in Figure 1, and an example of a running track is in Figure 2.</li>
	<li>Ensure the treadmill (see Table of Materials) has enough space to allow incline changes.</li>
	<li>Ensure the treadmill has an adjustable width to prevent the animal from turning during the run.</li>
	<li>Use transparent plastic to make the front wall of the treadmill. This prevents the animal from running away from the treadmill, but still allows the animal to see through the wall. 
	NOTE: It is essential that the animals can see through the front wall, as our experience suggests that pigs are more motivated to run if they see their fellow pigs on the other side of the wall.</li>
	<li>Place an ECG monitor and defibrillator (see Table of Materials) next to the treadmill. 
	NOTE: Fatal arrhythmias may occur during the stress test, especially if the pig has myocardial ischemia14,15,16.</li>
	<li>Ensure the running track includes a water point where the animals can drink and cool off after the run.</li>
</ol>
2. Acclimation period of the pigs before the test
<ol>
	<li>House the animals for 2 weeks before starting the experiments.</li>
	<li>During the 1st week of acclimation, ensure the animals get accustomed to their handlers and new housing environment, excluding the running track.</li>
	<li>During the 2nd week of the acclimation period, ensure the animals get accustomed to the running track.</li>
	<li>Start accustoming so that the animals get familiar with the running track. First, keep all the gates open, so the animals can walk freely on the track and explore the environment.</li>
	<li>When the animals are more familiar with the track, turn on the treadmill and let the animal run for short periods at a time, such as 7 min. The length of the running times must be extended daily. 
	&#8203;NOTE: Remember to reward the animals during the acclimation period. For example, the pigs were rewarded with unsalted popcorn in the present study.</li>
</ol>
3. The exercise test
NOTE: Pigs should be fasted at least 2 h before the exercise test or given only a tiny portion of food before the run.
<ol>
	<li>Turn on the treadmill and set the incline to 5%-10%.</li>
	<li>As soon as the animal is on the treadmill, start the treadmill with a starting speed of 2 km/h.</li>
	<li>Increase the speed by 0.5 km/h every 60 s until 5 km/h is reached. The total running time is 15 min.</li>
	<li>In case the animal cannot run the entire time at the maximum speed, perform the steps below.
	<ol>
		<li>If the pig is not running as fast as a selected speed, gently push it from the back, as this may give the animal the feeling that it needs to run faster without slowing down.</li>
		<li>Try gently pushing the animal a maximum of three times; after that, slow down the speed by 0.5 km/h at a time until the pig can handle the speed. Do not slow down to below 2 km/h.</li>
		<li>If the animal refuses to run even at a slow speed, turn off the treadmill and stop the test.</li>
	</ol>
	</li>
</ol>
4. ECG monitoring during the exercise test
<ol>
	<li>Place ECG electrodes (see Table of Materials) in anatomical locations which have minimal movement during the run, such as scapulas or the chest. 
	NOTE: Use ECG electrodes designed for exercise tests to achieve better adhesion to the skin. Remember to shave hair from the area where the ECG electrodes will be placed.</li>
	<li>Record the heart rate changes during the run. 
	&#8203;NOTE: Our experience suggests that ST segment analyses are often complicated due to movement and other artifacts. Rhythm monitoring can also be done using an implantable loop recorder or pacemaker.</li>
</ol>
5. Data collection
<ol>
	<li>Record the run distance, total time, and speed every time the speed is changed. 
	NOTE: Modern treadmills may collect a lot of other data, so it is essential to familiarize yourself with the treadmill manual to utilize the equipment&#39;s full potential.</li>
	<li>Note possible changes in animal behavior, such as limping. 
	NOTE: If needed, contact a veterinarian and make sure that the animal receives necessary analgesia. Remove the animal from future exercises until it fully recovers.</li>
</ol>
6. Post-procedural care
<ol>
	<li>Ensure the animal has access to the water point.</li>
	<li>Reward the animal, for example, with treats or toys.</li>
	<li>Monitor the animal for 30 min after the run for possible adverse effects.</li>
</ol>

Evaluation of Exercise Tolerance of Pigs Subjected to Exercise

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+anatomic+and+physiologic+overview+of+the+porcine+heart.">A comparative anatomic and physiologic overview of the porcine heart.</a> Journal of the American Association for Laboratory Animal Science. 53 (5), 432-438 (2014).</li><li>Korpela, 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=AAV2-VEGF-B+gene+therapy+failed+to+induce+angiogenesis+in+ischemic+porcine+myocardium+due+to+inflammatory+responses.">AAV2-VEGF-B gene therapy failed to induce angiogenesis in ischemic porcine myocardium due to inflammatory responses.</a> Gene Therapy. 29 (10-11), 643-652 (2022).</li><li>Swindle, 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=.">.</a> Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques. 2nd edition. , (2007).</li><li>Poole, D. C., 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=Guidelines+for+animal+exercise+and+training+protocols+for+cardiovascular+studies.">Guidelines for animal exercise and training protocols for cardiovascular studies.</a> American Journal of Physiology. Heart and Circulatory Physiology. 318 (5), H1100-H1138 (2020).</li></ol>

The authors declare no conflicts of interest.

This large animal exercise test mimics the test used in clinics, reducing the gap in endpoints between the preclinical studies and clinical trials. It can be applied to evaluate the efficacy of new treatments for severe cardiovascular diseases, such as arteriosclerosis obliterans, heart failure, and ischemic heart diseases. The time points applied in this protocol may vary depending on the tested treatment. This protocol has been standardized based on a long experience of working with large animals and can be used to evaluate the safety and efficacy of cardiovascular gene therapy and other novel therapeutic approaches.
The pig heart and cardiovascular system are similar to human physiology, anatomy, and function. Therefore, pigs have often been used to model cardiovascular disease mechanisms and therapeutic procedures17. The follow-up time in our pig studies has been up to 12 months18; however, handling the animals becomes increasingly challenging as they grow during the long follow-up periods.
This method consists of various critical steps, which are essential for the success of the test and impossible to correct afterward. First, pigs have individual differences in their running motivation. Getting animals motivated to run and maintaining sufficient motivation throughout the test is essential. This assures that all time points are comparable. Maintaining pigs&#39; running motivation requires specific knowledge of their individual behavioral characteristics. Animals must be acclimated to the treadmill and testing environment before the exercise test. Pigs are taught to go on the treadmill, and their successful performances are rewarded. Another way to increase their running motivation is to keep other test animals in the field of vision of the runner.
Avoiding typical anatomical flaws, such as different leg problems, is important. The most common anatomical flaws are hoof diseases, congenital limb deformities, and problems caused by accidents, such as lacerations, scapular injuries, fractures, and wounds. These are mostly due to habitats, accidents, hereditary factors, and aberrations in feeding19. A weakness in the legs leads to uncoordinated walking, making attending exercise tests impossible. Also, if leg weakness appears during the study, the animal must be excluded from the test. Leg problems can be avoided by choosing pigs with intact leg structures. During the research, leg injuries can be prevented by having good conditions in the piggery. Hard, corrosive surfaces must be avoided, and general hygiene must be maintained. Pigs must be fed moderately so that they do not gain weight too rapidly, since this strains their legs. In addition, pigs must be placed in their pens carefully to avoid accidents, and they should have enough stimuli, such as toys, so that their chewing is not directed at other pigs.
During the exercise test, the ECG was recorded with a 3-lead ECG or implantable loop recorder. It is not as accurate as a 12-lead ECG, but can still assess multiple variables, such as arrhythmias and heart rate. Several types of errors and disturbances can falsify ECGs. For example, incorrectly connected electrodes, poor contact between the skin and electrodes, and skeletal muscle contractions can cause errors. The electrodes must remain firmly in place throughout the test. This is challenging as the skin heats up and sweats during running. Contact between the skin and electrodes can be improved by shaving hair, disinfecting, and removing dead skin cells. Also, the movement of muscles causes artifacts that affect ECGs13. This can challenge the interpretation of ST segments. In addition, ECG leads can interfere with running. However, these issues can be reduced by tapping the ECG leads firmly on the skin. ECGs can also be registered with an implantable loop recorder or pacemaker. Using an implantable loop recorder solves many problems that 3-lead ECG usage has. However, installing the implantable loop recorder is an invasive operation with risks, such as infections.
Researchers must observe animal behavior throughout the test to ensure the procedure&#39;s overall safety. For example, exhaustion, severe fatigue, nausea, loss of consciousness, severe dyspnea, or cyanotic skin are reasons for terminating the exercise test. Also, researchers must observe changes in the ECG, such as arrhythmias. However, with well-trained personnel and enough experience working with large animals, the current exercise protocol can be routinely utilized in preclinical studies to produce clinically relevant data that should make the clinical transition of new therapeutic approaches more successful regarding clinical benefits to the patients.
Poole et al.20 have published guidelines for animal exercise and training protocols for cardiovascular studies. In these protocols, pigs exercise on a treadmill for approximately 30 min after warming up. During this 30 min, the target heart rate zone for the test animals is 65%-75% of the maximum heart rate. The heart rate is modified either by changing the speed or the incline of the treadmill. The protocol of Poole et al. and the 15 min exercise test presented in this manuscript have multiple similarities, such as the acclimation period, treadmill requirements, the weight of selected test animals, and positive reinforcement by rewarding the animal after the exercise. In both protocols, the test animals can outgrow the treadmill&#39;s capacity, limiting the follow-up time.
The main difference between the protocol described by Poole et al. and the exercise test presented in this manuscript is the purpose of the testing. The protocol described by Poole et al. is designed to elicit classical training adaptations noted in humans. Therefore, it focuses on moderate-intensity exercise, whereas the 15 min exercise test method aims to make a near-maximal effort to better evaluate cardiorespiratory fitness. This is achieved when the subjective level of exertion is approximately 90% of the maximum heart rate13. A 15 min exercise test mimics the test used in clinics by gradually increasing the level of exertion until it is near maximal. Due to the difference in the aims of the protocols, the exercise frequency of the test animals differs. Poole et al. describe that pigs can run up to four times a week to achieve better cardiovascular adaptations caused by exercising. The 15 min exercise test evaluates the functional efficacy of gene therapy and other novel therapies, which is why the required frequency is significantly lower and depends on the treatment&#39;s requirements. An example of these requirements has been described in Figure 4.

Chronic cardiovascular diseases are significant causes of mortality and morbidity worldwide1,2. Although current treatments are effective for the majority of patients, many still cannot benefit from the current therapies due to, for example, diffuse chronic disease or comorbidities. In addition, in some patients, cardiac symptoms are not relieved by the available treatments, and their cardiovascular disease progresses despite optimal medical therapy3. Thus, there is a clear need to develop novel treatment options for severe cardiovascular diseases.
During the past several years, new molecular pathways and ways to manipulate these targets have been discovered, making gene therapy, cell therapy, and other novel therapies a realistic option for treating severe cardiovascular diseases4. However, after promising preclinical results, many cardiovascular applications have failed to fulfill expectations in clinical trials. In spite of the poor efficacy in clinical trials, several trials have established good safety profiles of novel therapies5,6,7,8,9. Thus, bringing new cardiovascular therapies to patients will require improved approaches and better preclinical models, study settings, and endpoints in preclinical studies that can predict clinical efficacy.
In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of capillary vessels calculated from histological sections or parameters from left ventricle imaging at rest and under pharmacological stress. In clinical trials, many endpoints have been more subjective, such as exercise tolerance or symptom relief4. Thus, it is likely that the endpoints in preclinical studies and clinical trials measure different aspects of the applied therapy. For example, an increase in the quantity of blood vessels does not always correlate with better perfusion, cardiac function, or exercise tolerance. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches10.&#160;Still, the main goal is always to alleviate symptoms and to improve the patient&#39;s prognosis and quality of life. To achieve this, endpoint measurements must be better matched between preclinical and clinical studies4.
Cardiorespiratory fitness reflects the ability of the circulatory and respiratory systems to provide oxygen during sustained physical activity, and thus it quantifies the functional capacity of an individual. Functional capacity is a key prognostic marker as it is a strong independent predictor for the risk of cardiovascular and all-cause mortality11. Improvements in cardiorespiratory fitness are associated with a reduced risk of mortality12. Exercise tests are suitable for evaluating aerobic performance and treatment responses in cardiovascular diseases. Depending on the availability, tests are performed on a bicycle ergometer or a treadmill. A gradual increase in the workload per minute is usually used, and abrupt increases are avoided; this leads to a linear physiological response. The most important variables in the exercise tests include the total exercise time, metabolic equivalents (METs) achieved, heart rate, and changes on an electrocardiogram (ECG) line between the QRS complex (Q, R, and S waves) and T-wave (ST segment). Clinical stress tests have low costs and are easily accessible13. For these reasons, stress tests, such as the 6 min walking test, have been widely used in clinics and should also be used in the preclinical evaluation of new therapies.
To our knowledge, there are no well-described large animal models for evaluating the functional efficacy of gene therapy or other novel therapies. Therefore, the clinically relevant exercise test provides an excellent perspective for evaluating the efficiency of these new therapies in the preclinical setting.

<table><tbody><tr><td>Defibrillator</td><td>Zoll M series</td><td>TO9K116790</td><td>All portable defribrillators will work</td></tr><tr><td>Defibrillator pads</td><td>Philips</td><td>M3713A</td><td>All pads work, as long as the pads are compatible with the defibrillator</td></tr><tr><td>ECG electrodes</td><td>Several providers</td><td></td><td>Prefer ECG electrodes designed for exercise tests</td></tr><tr><td>Loop recorder</td><td>Abbott Oy</td><td>DM3500</td><td>Optional for rhythm monitoring</td></tr><tr><td>Patient monitor</td><td>Schiller Argus LCM Plus</td><td>7,80,05,935</td><td>All portable ecg monitors will work</td></tr><tr><td>Pigs</td><td>Emolandia Oy</td><td></td><td></td></tr><tr><td>Treadmill</td><td>NordicTrack</td><td></td><td>All treadmills with adjustable incline and speed are suitable for the exercise test.&amp;nbsp; The treadmill should be as long and wide as possible.</td></tr><tr><td>Ultrasound system</td><td>Philips EPIQ 7 ultrasound</td><td></td><td></td></tr><tr><td>Various building materials</td><td>Several providers</td><td></td><td>For building fences, ramps and gates according to the Figure 1 and Figure 2</td></tr><tr><td>Various treats for the animals</td><td></td><td></td><td></td></tr></tbody></table>

exercise test for evaluation of the functional efficacy of the pig cardiovascular system

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based therapeutic angiogenesis is a promising approach for treating patients with significant symptoms, despite optimal pharmacological therapy and invasive procedures. However, many promising cardiovascular gene therapy techniques have failed to accomplish expectations in clinical trials. One explanation is a mismatch between preclinical and clinical endpoints used to measure efficacy. In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of the capillary vessels calculated from histological sections. Apart from mortality and morbidity, endpoints in clinical trials are subjective, such as exercise tolerance and quality of life. However, the preclinical and clinical endpoints likely measure different aspects of the applied therapy. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches. In clinics, the main goal is always to alleviate patients' symptoms and improve their prognosis and quality of life. To achieve better predictive data from preclinical studies, endpoint measurements must be better matched to those in clinical studies. Here, we introduce a protocol for a clinically relevant treadmill exercise test in pigs. This study aims to: (1) provide a reliable exercise test in pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies, and (2) better match the endpoints between preclinical and clinical studies.

One must have experience working with large animals to succeed with this protocol. Researchers need to be able to evaluate whether an animal stops running due to fatigue or lack of motivation. Recording the speed and distance may help to evaluate this, as usually, animals lacking motivation stop running totally, whereas fatigued animals keep running after slowing down the speed (Figure 3). If necessary, the protocol can be repeated the next day if the results seem unreliable.
A representative timeline for adeno-associated virus (AAV)-treated animals is shown in Figure 4. The timeline may vary depending on the study setting, especially regarding the sacrification timepoint. Note the acclimation period when planning the experiments.
The results can be compared to other organ structure and function measurements, such as cardiac echo, to see how exercise tolerance relates to these other measurements. For example, the change in running distance correlates with the change in ejection fraction. With a low ejection fraction, an animal cannot run at full speed throughout the exercise test (Figure 5). Analyzed variables may differ depending on the study settings. This protocol enables the comparison of total running distance, speed variation, METs, heart rate variation, and arrhythmias.
The ECG is recorded during the exercise test (Figure 6). ST segment analysis is difficult due to artifacts. Changes in heart rate intervals can be measured from the ECG throughout the exercise test.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65233/65233fig01.jpg" /> 
Figure 1: Floor plan of the running track. The place for animals not running is marked with (A). One animal at a time is guided to the treadmill [zone (C)] through a corridor (B). The gate between zones (A) and (B) is closed to ensure that only one animal at a time goes to the running track and the other animals stay in zone (A). It is essential that other animals stay in zone (A), as the animal on the treadmill can see the other animals in zone (A), which motivates them to run. The gate between the treadmill and zone (B) is closed to ensure that the animal cannot back off from the treadmill. The treadmill is operated from zone (D), and the animal is returned to zone (A) through zone (D) after the run. In the present case, a water point where animals can drink and cool off after the run is installed in zone (A). Symbols: black arrows indicate the direction of rotation, and quarter circles indicate gates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig01large.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/65233/65233fig02.jpg" /> 
Figure 2: Representative images of the running track. (A) The importance of only one available route for the animals. (B) The treadmill, which should have an adjustable width to prevent animals from turning during the run. (C) An enclosed space meant for another animal in addition to the running animal. Animals are more motivated to run when they see a member of their species. (D) An example of a water point for the animals where the animals can cool off and drink after the stress test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig02large.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/65233/65233fig03.jpg" /> 
Figure 3: Running distance and speed. (A) Representative data from the total running distances of four healthy pigs. The mean total running distance for the test animals was 970 m, and the standard deviation of the total distances was 80 m. (B) Data on speed variation between pigs. The speed is slowed down by 0.5 km/h intervals until the pig can handle the speed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig03large.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/65233/65233fig04.jpg" /> 
Figure 4: Representative timeline. Note that time points may vary between the studies. However, it is notable that animals should arrive at the laboratory animal center 3 weeks before starting the experiment due to the acclimation period. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig04large.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/65233/65233fig05.jpg" /> 
Figure 5: Correlation of the running distance with the change in ejection fraction. Correlation of the change in running distance with the change in ejection fraction. The ejection fraction at rest was measured by the Biplane Simpon method. The change in ejection fraction from the baseline correlates with the change in the running distance of pigs with pacemaker-induced heart failure, with r = 0.2831, p = 0.0284, and R2 = 0.0801. Despite the low r and R2, the change in left ventricular ejection fraction percentage (LVEF%) tends to affect running distances. It is important to note that several factors influence the measured variables, affecting the results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig05large.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/65233/65233fig06.jpg" /> 
Figure 6: Representative ECG tapes of a healthy pig. The top ECG panel shows an ECG 3 min after the beginning of the exercise test. The bottom ECG strip shows an ECG after running for 10 min. The ECG can be used to evaluate the differences in the heart rate of the test animals. The heart rate of the top ECG panel is 176 beats per minute, and on the bottom ECG strip is 250 beats per minute. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65233/65233fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System at JoVE.com

Author Spotlight: Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System  

The present protocol describes a large animal exercise test model to assess the functional capacity of the cardiovascular system for evaluating the efficiency of new therapies in the preclinical setting. It is comparable to a clinical exercise test.

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based ...

exercise-test-for-evaluation-functional-efficacy-pig-cardiovascular

Nanyang Technological University

Research

JoVE Journal

Genetics

1.3K Views.  University of Eastern Finland. Het doel van de studie is om een betrouwbare inspanningstest voor varkens te bieden die kan worden gebruikt om de veiligheid en functionele werkzaamheid van gentherapie en andere nieuwe therapieën te evalueren. Gewoonlijk benadrukken cardiovasculaire dierstudies gemakkelijk kwantificeerbare eindpunten, net als capillair gebied berekend op basis van de histologische secties. Klinische studies gebruiken daarentegen meer subjectieve eindpunten zoals overmatige tolerantie.Dit protocol kom...

Video: Auteur in de schijnwerpers: inspanningstest voor evaluatie van de functionele werkzaamheid van het cardiovasculaire systeem van varkens  

Myocardial Infarction and Functional Outcome Assessment in Pigs

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

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

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

Medicine

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

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 Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

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 ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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 ...

Measuring Cardiac Output in a Swine Model

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume and cardiac output are essential in this type of research. Contrast ventriculography, thermodilution, and pressure-volume loop (PV-loop) catheters can be used to accurately obtain cardiac performance data depending on which resources and expertise are available. For this study,five Yorkshire swine were anesthetized and intubated. Central venous and arterial access was obtained to place the necessary measurement instruments.A temperature probe was placed in the aortic root. A cold saline bolus was delivered to the right atrium and temperature deflection curve was recorded. Integration of the area under the curve allowed for the calculation of the current cardiac output.A pigtail catheter was percutaneously placed in the left ventricle and 30 mL of iodinated contrast was power injected over 2 seconds. Digital subtraction angiography images were uploaded to volumetric analysis software to calculate the stroke volume and cardiac output. A pressure volume-loop catheter was placed into the left ventricle (LV) and provided continuous pressure and volume data of the LV, which allowed the calculation of both stroke volume and cardiac output.All three methods demonstrated good correlation with each other. The PV-loop catheter and thermodilution exhibited the best correlation with a 3% error and a Pearson coefficient of 0.99, with 95% CI=0.97 to 1.1, (p=0.002). The PV-loop catheter against ventriculography also showed good correlation with a 6% error and a Pearson coefficient of 0.95, 95% CI=0.96 to 1.1 (p=0.01). Finally, thermodilution against ventriculography had a 2% error with r=0.95, 95% CI=0.93 to 1.11, (p=0.01). In conclusion, we state that the PV-loop catheter, contrast ventriculography, and thermodilution each offer certain advantages depending on the researcher's requirements. Each method is reliable and accurate for measuring various cardiac parameters in swine such as the stroke volume and cardiac output.

Thermodilution, pressure-volume loop catheters, and contrast ventriculography are reliable and accurate methods for determining cardiac physiology such as stroke volume and cardiac output in a laboratory setting in swine.

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume ...

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 ...

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

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