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 ...

Evaluation of Exercise Tolerance of Pigs Subjected to Exercise

<ol>
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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 eva...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160; 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.

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

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

The aim of the study is to provide a reliable exercise test for pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies. Usually, cardiovascular animal studies emphasize easily quantifiable endpoints just as capillary area calculated from the histological sections. In contrast, clinical studies use more subjective endpoints such as excess tolerance.This protocol better matches the endpoints between pre-clinical and clinical studies. The previously published methods with longer running sessions aim to evaluate chronic exercise adaptations, whereas the presented method is more suitable for evaluating cardio respiratory fitness.

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

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.

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