This study was funded by Quark Pharmaceuticals Inc where S.A. and E.F. are employees. This study was also supported by the National Research, Development and Innovation Office of Hungary (NKFIA; NVKP-16-1-2016-0017 National Heart Program), and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic Development thematic program of the Semmelweis University. GB.B. was supported by EFOP-3.6.3-VEKOP-16-2017-00009 and Gedeon Richter Plc. Scholarship. Z.G. was supported by a J&#225;nos Bolyai Research Scholarships of the Hungarian Academy of Sciences and by the &#218;NKP-19-4 New National Excellence Program of the Ministry of Human Capacities.

13 healthy and sexually mature female G&#246;ttingen minipigs (age between 12 and 14 months) and 10 healthy and sexually immature female Landrace pigs (age between 2 and 3 months) were housed in pig stalls conforming to the size recommendations of the most recent Guide for the Care and Use of Laboratory Animals DHEW and EU Guidelines 63/2010. Animals were not spayed. The temperature of the animal rooms was controlled, and animals were kept at a 12-hour light/dark cycle and vermin-free. Ad libitum feeding leads to overt weight gain in both G&#246;ttingen minipigs and Landrace pigs, therefore, pigs from both the breeds were fed with a restricted diet regimen. G&#246;ttingen minipigs were put on restricted diet as early as they arrived to the animal facility and for the whole study duration. Special Diet Services pig chow 180-220 g/meal/animal was given twice daily according to &#34;Taking good care of Ellegaard G&#246;ttingen Minipigs&#34; guideline (revision date: 13 March, 2013) in the first 2 days. Between day 3 and 12 animals were fed 50% Special Diet Services pig chow and 50% maintenance minipig diet. From day 14 until the end of the study animals were fed a maintenance minipig diet. Landrace pigs received pregnant sow chow, 1.5% of body weight given two times a day according to PIC Wean to Finish Manual 2008 and 2013. All the animals received food individually dispensed and food intake was monitored to avoid competition for chow. Animals with feeding difficulties were fed individually aided by tending personnel. All animals received tap water ad libitum. The experimental protocol of post-MI HF in G&#246;ttingen minipigs and in Landrace pigs is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61901/61901fig1v2.jpg" /> 
Figure 1. Experimental protocol for post-myocardial infarction-induced heart failure in Landrace pigs and G&#246;ttingen minipigs. CMRI - cardiac magnetic resonance imaging. <a href="https://www.jove.com/files/ftp_upload/61901/61901fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Baseline CMRI
<ol>
	<li>Withdraw food from animals at least 12 hours before the start of anesthesia but secure access to water to prevent dehydration.</li>
	<li>Anesthesia
	<ol>
		<li>Induce anesthesia of the animals with ketamine hydrochloride (12 mg/kg), xylazine (1 mg/kg), and atropine (0.04 mg/kg) as an intramuscular injection to the neck region.</li>
		<li>Measure the body weight and length of animals. The calculation of the body surface areas (BSA) formulas were described by Itok et al. for G&#246;ttingen minipigs (BSA [m2] = (7.98 &#215; BW [kg]2/3)/100)17 and by Swindle et al. for Landrace pigs (BSA [m2] = (7.34 &#215; BW [kg]0.656)/100)18.</li>
		<li>Intubate animals, maintain anesthesia with isoflurane (2% isoflurane, 2 L/min oxygen). The size of the endotracheal tube depends on the individual anatomic characteristics of each animal and ranges between 6.0- to 7.5-mm.</li>
		<li>Cannulate the ear vein with 18 G needle and start&#160;administration of 5% glucose in Ringer solution (1&#160;L/hour).</li>
	</ol>
	</li>
	<li>CMRI
	<ol>
		<li>Transfer animal to CMRI facility and administer 0.4-0.5 mg/kg atracurium besylate i.v.. Atracurium besylate is a nondepolarizing, skeletal muscle relaxant that is used to avoid respiratory artefacts during CMRI measurements. Start positive pressure ventilation (16/min frequency, 350 mL volume, 25-30&#160;mmHg positive pressure).</li>
		<li>Position the animals in the supine position. Place flexible coils on the chest and 32-channel coils are placed in CMRI bed. Perform non-contrast cardiac MRI with a 1.5T scanner, using a phased array coil and a vector electrocardiogram (ECG) system to assess the cardiac function and morphology (ejection fraction (EF), cardiac output (CO), chamber and wall dimensions). Acquire cine MRI images using a retrospectively ECG-gated, steady-state free precession cine MRI technique in short-axis and long-axis views of the heart using 1.2 ms echo time, 40 ms repetition time, 50-degree flip angle, 300&#160;mm field-of-view, 8 mm slice thickness, and at least 256x256 image matrix.</li>
		<li>Quantify left and right ventricular end-diastolic (LVEDV and RVEDV) and end-systolic volumes (LVESV and RVESV), stroke volumes (LVSV and RVSV), EF-s (LVEF and RVEF) and masses by manual planimetry of end-diastolic (LVED mass) and end-systolic (RVED mass) short-axis cine images. Quantify left atrial volume by tracings on the two- and four-chamber cine images. Correct the left atrial volumes to BSA to get left atrial volume indexed to body surface area (LAVi). Assess the presence of pulmonary oedema on the localizer images.</li>
		<li>For calculation of cardiac index (CI) use BSA and cardiac output.</li>
		<li>Terminate the anesthesia by withdrawal of isoflurane. When spontaneous breathing returns, extubate the animal, remove i.v. cannula and return it to its cage.</li>
	</ol>
	</li>
</ol>
2. Premedication, vascular access and coronary artery occlusion
<ol>
	<li>Premedication
	<ol>
		<li>One day prior to surgical procedure administer 500&#160;mg of acetyl salicylic acid and 300 mg of clopidogrel orally.</li>
		<li>Apply analgesia (meloxicam 0.4 mg/kg body weight) and antibiotic cocktail (benzylpenicillin-procain&#160;(24.8&#160;mg/mL), benzylpenicillin-benzatine (83.6 mg/mL), dihidrostreptomycine-sulfate (156.3&#160;mg/mL), 3&#160;mL/50&#160;kg&#160;body&#160;weight) by intramuscular injections on the day of coronary artery occlusion.</li>
		<li>Repeat the steps described in 1.2.1-1.2.4 sections.</li>
		<li>Use the ear vein cannula for fluid replacement and drug administration. Administer 1g magnesium sulfate throughout the procedure via ear vein every 30 min to prevent ventricular tachycardia (VT) and ventricular fibrillation (VF).</li>
	</ol>
	</li>
	<li>Vascular access
	<ol>
		<li>Place the animal on the operating table, fix the limbs, and apply wedges to immobilize the animal in the supine position.</li>
		<li>Disinfect the surgical site with povidone-iodine. The surgical site is around the skin fold between the gracilis and sartorius muscle.</li>
		<li>Remove the hair at the surgical site with a razor.</li>
		<li>Place surface ECG electrodes in Einthoven&#39;s triangle. This triangle is formed by the two anterior limbs and left hind limb and the electrodes are placed on limbs.</li>
		<li>Start positive pressure ventilation (16/min frequency, 350 mL volume, 25-30 mmHg positive pressure).</li>
		<li>Isolate the disinfected surgical area with a surgical drape.</li>
		<li>Approach the femoral region as described in detail by K. S. Ettrup et al.19. In brief, make a longitudinal incision to the skin between the gracilis and sartorius muscles. Separate the subcutaneous tissue and fascia. Isolate the femoral artery and put two surgical sutures below it to control hemorrhage.</li>
		<li>Puncture and cannulate the femoral artery with a 6F-ACT introducer using the Seldinger technique20,21.</li>
		<li>Fix the sheath to the skin.</li>
		<li>Use the artery for blood sampling for further biochemical analyses.</li>
		<li>Administer 5000 IU heparin via the femoral sheath to secure adequate anticoagulation and prevent thrombosis during the surgical intervention. Readminister 2500 IU heparin every 60 min throughout the procedure. The animals received approximately 370-440 IU/kg heparin during the whole intervention.</li>
		<li>Attach a pressure sensor to the femoral vessel to monitor the arterial blood pressure throughout the surgical intervention.</li>
		<li>For calibration of pressure place the pressure recording system on the level of the heart of each animal. After removing the air bubbles, the zero pressure calibration is performed when the three-way stopcock is opened to the direction of the free air.</li>
	</ol>
	</li>
	<li>Coronary artery occlusion, reperfusion and intracoronary drug administration
	<ol>
		<li>Note that this intervention should be performed only by trained interventional cardiologist. Through the femoral sheath, introduce and advance the guidewire to the aortic arch and introduce the 5F guiding catheter over the guidewire. First, advance the guidewire to approach aortic root atraumatically. Perform deep intubation by a thin, 5F guiding catheter to avoid significant obstruction of the blood flow.</li>
		<li>Position the fluoroscope in antero-posterior position.</li>
		<li>Ensure that there is no thrombus or air bubble within the catheter with the aspiration of at least 5 mL of blood, the volume of the catheter, with the syringe connected to the catheter.</li>
		<li>Connect the outer portion of the catheter to a syringe filled with radiocontrast agent (iobitridol 1.1 mL/50 kg body weight).</li>
		<li>Take care that the syringe is held elevated to prevent infusion of air bubbles into the coronary artery.</li>
		<li>To perform baseline angiography, intubate separately and fill with contrast agent selectively the ostia of right coronary artery and left main coronary artery. For more technical details, refer to catheterization textbooks20,21.</li>
		<li>Perform BARI (Bypass Angioplasty Revascularization Investigation Myocardial Jeopardy Index) scoring after the baseline angiography. A score to all terminal arteries (terminal portion of the left anterior descending, left circumflex, and right coronary artery, as well as the ramus, diagonals, obtuse marginals, posterior descending and posterolateral branches) is assigned based on their length and caliber according to specific criteria22,23. A value of 0 represents an almost insignificant vessel size. In contrast, a value of 3 defines a large-sized artery with a length of two thirds the distance between the base and cardiac apex. Do not take right ventricular marginals and posterior descending artery septal branches into account.
		<ol>
			<li>Calculate the final BARI score (% of the left ventricle at risk) by dividing the total value from the infarct-related artery by the total values of all arteries (Figure 2A-D) supplying the LV. Choose the occlusion site on the left anterior descendent (LAD) coronary artery to achieve approximately 25-30% myocardium at risk as assessed by BARI scoring.</li>
		</ol>
		</li>
		<li>Insert the percutaneous transluminal coronary angioplasty (PTCA) guidewire through the guiding catheter. Position it distally to the planned site of the occlusion under fluoroscopic guidance, and check angiography for potential complications (e.g., coronary dissection, perforation).</li>
		<li>Determine by visual estimation the optimal balloon size based on coronary artery diameter.</li>
		<li>Place the balloon catheter (balloon diameter 2.5 mm and balloon length 12 mm) over the PTCA guidewire and advance it to the planned position.</li>
		<li>Fill the balloon with contrast agent and check the position of the balloon catheter by angiography.</li>
		<li>Inflate the balloon below the nominal pressure (7-9&#160;atmospheres) of the balloon to develop the soft touch between the balloon side-wall and the surface of the vessel. Soft-touch is defined as interaction of balloon side-wall that is enough to occlude the vessel without causing injury to the vessel wall.</li>
		<li>Confirm the occlusion (TIMI 0) with angiography by visualizing the stop of the contrast flow. Keep in place the guidewire and the balloon and pull back the guiding catheter from the ostium of the coronary artery to avoid diffuse cardiac ischemia.</li>
		<li>Tape instruments to the surgical drape to avoid dislocation of the intracoronary balloon.</li>
		<li>Record and document the ECG sign of occlusion by ST elevation.</li>
		<li>During the whole procedure, carefully monitor the vital signs, heart rate (HR), blood pressure, core temperature using rectal probe, and pulse oximetry.</li>
		<li>Cover the animal with a heating device to maintain the core temperature.</li>
		<li>Administer 1 g of magnesium sulfate as an intravenous bolus if pulseless VT or VF occurs and start chest compressions with a frequency of 100/min immediately. Apply 300J DC shock and lidocaine 2-4 mg/kg as an intravenous bolus. Treat asystole with 1 mg of epinephrine as an intravenous bolus.</li>
		<li>Check balloon pressure every 30 min during the coronary occlusion. If there is a decrease of more than 0.5 bar in balloon pressure, set it back to initial values.</li>
		<li>Perform angiography just before the end of coronary occlusion to verify the maintained balloon placement and absence of flow distally to the occlusion site.</li>
		<li>Administer 2500 IU of heparin and 1 g of magnesium sulfate intracoronarily as a slow bolus to prevent thrombosis and arrhythmias.</li>
		<li>Initiate the reperfusion with balloon deflation after 120 min cardiac ischemia in G&#246;ttingen minipigs and after 90 min in Landrace pigs.</li>
		<li>Remove the deflated balloon.</li>
		<li>Confirm the success of reperfusion with coronary angiography to demonstrate the blood flow at the distal part of the coronary vessel (TIMI 3).</li>
	</ol>
	</li>
</ol>
3. Intracoronary drug administration
<ol>
	<li>To prevent coronary artery embolization, fill the therapeutic perfusion microcatheter with saline.</li>
	<li>Place the microcatheter over the PTCA guidewire.</li>
	<li>Advance and confirm the position of the microcatheter. The tip of the microcatheter should be placed at the level of occlusion.</li>
	<li>Remove the PTCA guidewire.</li>
	<li>Connect the microcatheter with the perfusion pump and initiate intracoronary administration 5 minutes after initiation of reperfusion.</li>
	<li>After drug administration remove the microcatheter.</li>
	<li>Make control angiography to check the TIMI 3-grade flow of the contrast and to exclude that intervention led to air emboli or coronary dissection.</li>
</ol>
4. Wound closure and post-operative care
<ol>
	<li>Remove the arterial sheath and tie down the femoral artery proximal to the puncture site. Occlusion of femoral artery following the angiographic intervention has no effect on the function of legs in pigs as assessed by daily veterinarian observations.</li>
	<li>Close the wound using continuous sutures and apply antiseptic coating.</li>
	<li>Terminate the anesthesia by withdrawal of isoflurane.</li>
	<li>Closely monitor the animals in the recovery period and inspect them every 12 hours until postoperation day 3, then every 24 hours until the end of the study. Particular attention should be given to eating and drinking behavior, lethargy, signs of infection, painful condition, weight change, mobility, and general health status. Following the procedure, the animals were transported with a van in small groups in cages to avoid unnecessary stress in early postoperative period.</li>
</ol>
5. Post-MI CMRI and its evaluation
<ol>
	<li>Anesthesia
	<ol>
		<li>Use the anesthetic protocol described in sections 1.2.1-1.2.4.</li>
	</ol>
	</li>
	<li>CMRI
	<ol>
		<li>Administer an intravenous bolus of contrast agent, 0.2 mmol/kg gadobutrol at a rate of 4 mL/sec, using a manual injector.</li>
		<li>Take delayed enhancement images using an inversion recovery-prepared, gradient-echo sequence. Obtain short-axis and long-axis images 10 to 15 minutes after the administration of contrast agent.</li>
		<li>Evaluation
		<ol>
			<li>Perform evaluation using MASS 7.6 analysis software in a blinded fashion.</li>
			<li>Assess end-diastolic segmental wall thickness on short-axis cine images.</li>
			<li>Measure scar transmurality on short-axis delayed enhancement images.</li>
			<li>Quantify myocardial necrosis with manual planimetry on the delayed contrast enhancement images by delineating the myocardium with signal intensity 5 SDs above the mean signal obtained in the remote, non-infarcted myocardium.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Statistics
<ol>
	<li>Show continuous data as mean &#177; standard error.</li>
	<li>Evaluate the difference using repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs and paired t-test in Landrace pigs. BARI scores were compared with unpaired t-test and mortality rates with the chi-square test between the two breeds.</li>
	<li>Use GraphPad Prism for data evaluation. The differences were claimed to be statistically significant if p&#60;0.05.</li>
</ol>

PF is the founder and CEO of Pharmahungary Group, a Group of R&#38;D companies

Here we described a detailed protocol highlighting the critical steps of a technique of induction of acute MI and the evaluation of post-MI HF in a closed-chest model of adult G&#246;ttingen minipigs. We also described the method of intracoronary drug administration, BARI scoring, and reported left and right ventricular cardiac morpho-functional changes in a translational post-MI HF model. 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 humans. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig to follow-up the development of 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 most of the cardiovascular drug and medical device development projects6,7,12. Indeed, pig models resemble humans in anatomy, physiology, and biochemical properties in particular in the field of MI research as they develop trans-mural infarctions due to the lack of collateral perfusion14. Therefore, pig models can serve as models for the analysis of cardioprotective therapies and their mechanisms24,25,26,27,28,29.
Here we have found that despite the equal scar sizes, mortality rates, and BARI scores in the two breeds, left ventricular dysfunction characterized by decreased LVEF was observed only in G&#246;ttingen minipigs. Here we observed a 15.4% acute mortality in G&#246;ttingen minipigs and no mortality in the follow-up period, the latter is comparable to that in clinical studies. Indeed, a patient-level meta-analysis of 10 randomized clinical trials found that the Kaplan-Meier estimated 1-year rate of all-cause mortality was as low as 2.2% following myocardial infarction30. Scar sizes reported here are comparable to those in clinical trials. In clinical trials performed by Lonborg et al and Stone et al in patients surviving ST-elevation myocardial infarction the median scar sizes, measured as % of left ventricular myocardial mass were 9.5% and 17.9% respectively30,31. Moreover, scar sizes in the present study accord with those reported in previous publications in G&#246;ttingen minipigs (12-25%)32,33,34,35,36,37 and in Landrace pigs (14-18%)38,39,40. The present finding on baseline LVEF in Landrace swine is according to data reported by others in large swine13,41,42. These values in large swine are smaller as compared to healthy human LVEF reference ranges (58-61%)43 and baseline (pre-infarction) values in G&#246;ttingen minipigs (55-73%)33,44,45. Nevertheless, it is worth noting that only the post-infarction data or delta changes of LVEF are reported in most publications46,47,48,49,50. In accordance with the present results, previous studies of either post-MI HF induced by 45 to 90 min LAD occlusion followed by reperfusion or by permanent LAD occlusion have demonstrated either no reduction or modest reduction of LVEF in Landrace or Yorkshire swine after 4-6 weeks follow up as compared to baseline (pre-infarction) LVEF51,52,53. However, Schuleri et al. compared morphofunctional parameters between G&#246;ttingen minipigs and Yorkshire swine and found that both breeds showed a decrease of LVEF 8 weeks after induction of MI by 120 to 150 min LAD occlusion-reperfusion; however, baseline LVEF values in Yorkshire swine were not reported54. In other experiments in female Dalland Landrace pigs post-MI adverse remodeling was induced by 90 min LAD occlusion, however, LVEF was not reported after 4 weeks of follow-up55. In contrast to our findings, in a study by de Jong et al., LVEF markedly decreased in Landrace pigs subjected to open chest LAD occlusion and followed by a 12-week follow-up56. This difference can be attributed to substantially longer ischemic period (150 min), which resulted in larger infarct size (23.4 &#177; 2.1% of LV). Elsewhere, 120-min closed-chest occlusion of left circumflex (LCX) coronary artery in German Landrace pigs led to a significant reduction in LVEF after eight weeks of reperfusion, suggesting that the different location of MI may also affect global left ventricular function57. Our present findings are consistent with others showing significant reduction in LVEF in post-MI HF in G&#246;ttingen minipigs after long-term follow up33,44,45.
The reduction of LVEF in G&#246;ttingen minipigs following MI is consistent with clinical data showing cardiac dysfunction as a consequence of ventricular remodeling in patients after AMI58. In conclusion, G&#246;ttingen minipigs better mimic the human conditions, since pre-infarction LVEF, scar size, post-infarction LVEF, and mortality are all comparable to these parameters found in humans.
Here we observed an 8%&#160;increase in LVED mass after six months in G&#246;ttingen minipigs and a markedly higher (97%) increase in LVED masses in Landrace pigs after two months. Similar data were reported by Schuleri et al. in Yorkshire pigs, where a 40% increase in heart weight was observed after two months. In contrast, in other experiments of closed-chest post-MI HF in G&#246;ttingen minipigs no significant changes in left ventricular masses were observed33,44. Therefore, differences between the two breeds regarding LVEF can be attributed to an intensive cardiac growth rate in Landrace pigs and thus altered cardiac remodeling.
In clinical settings, besides the LVEF, left ventricular volume provides valuable insight into long-term prognosis and mortality rate in post-MI patients59. LVESV is the primary determinant of both early and late mortality in patients after AMI60,61. Here we have shown that ventricular volume assessed by CMRI increased significantly in both breeds. Post-MI remodeling induced a more pronounced increase in LVESV than in LVEDV in G&#246;ttingen minipigs, while both LVESV and LVEDV were increased by a similar rate in Landrace pigs. Consequently, left ventricular ejection fraction (LVEF) was significantly decreased at 3 and 6 months only in G&#246;ttingen minipigs but not in Landrace pigs after 2 months. These results must be interpreted with caution in Landrace pigs, where increased LVESV, LVEDV, and LVSV (calculated as the difference between the LVESV and LVEDV) are more likely related to an intensive increase in cardiac mass. Increased LVESV and LVEDV are consistent with clinical data of patients with post-MI HF62,63,64. Moreover, adverse left ventricular remodeling was defined as an increase of 15% or more in the LVEDV in clinical studies65,66 and we found here a 28% increase after 3 months and a 42% increase after 6 months in LVEDV in G&#246;ttingen minipigs showing a clinically relevant adverse remodeling. In addition, here we have shown that LAVi increased only in G&#246;ttingen minipigs, but not in Landrace pigs. Increase of left atrial volume is an additional key structural alteration in the context of HF and is an independent predictor of death and HF hospitalization in patients surviving MI67.
Right ventricular function is rarely studied in post-MI HF models. Here we have found that right ventricular ejection fraction increased in both breeds. Although RV was practically not involved in myocardial necrosis, RVEF increased significantly in both breeds indicating RV volume overload and hence left ventricular dysfunction. Similarly, a clinical study enrolling 2008 patients with chronic systolic HF showed that 733 patients (37%) belonged to normal right ventricular function category with RVEF&#8805;40%68.
In conclusion, we have shown here that the adult G&#246;ttingen minipig model with long-term follow-up mimics functional and morphological parameters of post-MI HF comparable to humans. Our present data also show that Landrace pigs are not suitable for the evaluation of post-MI HF mainly due to consequences of the rapid increase in body and heart weight that does not allow long-term follow-up and interferes with post-MI HF pathology. Landrace pigs might be suitable to assess the consequences of acute myocardial infarction. The present comprehensive characterization of the closed-chest infarction models in Landrace and G&#246;ttingen minipigs will be useful for choosing the optimal large animal models to study post-MI HF and developing novel therapies against this pathology.
Limitations
The current experiment was performed only in female pigs, therefore, the potential effect of the different sexes on post-MI HF remains unknown in these models69. Signs of HF were assessed by CMRI, according to recommendations of a recent guideline on the relevance of rigor and reproducibility in preclinical studies on cardioprotection12. However, the use of more targeted angulation of CMRI imaging planes and more targeted sequence may result in better estimation of left atrial volumes, and pulmonary oedema. Although we have not&#160;measured biomarkers and histological signs of post-MI HF in this study, these models are suitable for analysis of any biomarkers since the availability of plasma and tissue samples. Due to the different susceptibility of the 2 breeds to ischemia/reperfusion injury, different durations of coronary occlusions were selected here that may although limit the comparison of the 2 models, however, by this approach we achieved similar infarct size. The follow-up time in the 2 breeds was different as in the Landrace pigs only 2 months follow up time can be achieved due to technical reasons, i.e. rapid increase in body weight that shows a major limitation of the Landrace model. A further limitation is the lack of different risk factors and comorbidities and thus the present large animal models do not completely mimic the clinical situation in terms of the presence of multiple risk factors including co-morbidities and their medications. However, currently, there are no established large animal models with multiple comorbidities for routine use. These large animal models cannot be powered for mortality analysis due to animal ethical reasons and the high cost of these studies.

Despite the decreasing mortality of acute myocardial infarction (MI), the incidence of post-myocardial infarction heart failure (post-MI HF) has not changed over time1. Heart failure (HF) is one of the most powerful predictors of death in MI patients2. To date, reperfusion therapy is the only available treatment option to limit myocardial infarct size and to reduce the risk of a subsequent HF3,4,5. HF and other complications may occur as a consequence of reperfusion injury; therefore, there is still an unmet need for the development of cardioprotective therapies beyond timely reperfusion6,7,8. Numerous cardioprotective therapies effective even in large animal models have been described, but only remote ischemic conditioning (RIC) seemed to improve clinical outcomes of post-MI HF in a small clinical trial9. However, this encouraging result on the efficacy of RIC was questioned in a single-blind, randomized controlled trial (CONDI-2/ERIC-PPCI) performed at 33 centers across Europe in STEMI patients, where RIC failed to improve clinical outcomes10. Potential reasons for the failed translation of the preclinical data might be the use of suboptimal post-MI HF animal models with low clinical relevance11.
Cardiovascular (patho)morphology and (patho)physiology of the pig models resemble human conditions; thus, it is widely used and accepted in translational cardiovascular research12,13,14. Pig breeds used in cardiovascular research belong to the very diverse domestic pig (Sus scrofa domestica) species that includes swine that vary in size, appearance, and genetic background15,16. Although post-MI HF has been researched in pigs extensively, no study was published with the aim of characterizing and comparing the effect of MI on the outcome of post-MI HF in Landrace pigs and G&#246;ttingen minipigs. The intensive growth rate of Landrace pigs may affect the cardiac morphofunctional outcomes; however, G&#246;ttingen minipigs with restricted growth patterns may overcome these concerns and can serve as a feasible model for long-term follow up in the assessment of post-MI HF. Moreover, a guideline on the relevance of rigor and reproducibility in preclinical studies on cardioprotection recommends the use of cardiac magnetic resonance imaging (CMRI) as a clinically relevant model for measurement of ventricular function in pigs12.
To analyze the scientific interest on post-MI HF in pigs we performed literature search on PubMed using the following search string: &#34;(pig OR swine OR porcine OR sus-scrofa OR minipig OR mini-pig OR miniature-pig OR miniature-swine) AND (infarct* OR ischem* OR ischaem* OR reperfus*) AND (heart OR cardi* OR myocard*) AND (LAD OR left-anterior* OR LCX OR left-circumflex OR RCA) AND (heart-failure OR lvef OR ejection-fraction OR infarct-size OR infarction-size)&#34; and found that pig models of cardiac ischemia/reperfusion are frequently used to study MI and post-MI HF, but only 17% (71 out of 425 articles) of studies involved minipigs and 7% (30 out of 425 articles) used G&#246;ttingen minipigs. Only about 1% (5 out of 425) of studies used G&#246;ttingen minipigs and clinically relevant protocols with long-term follow-up (1-9 months of reperfusion) and CMRI to analyze cardiac function. The small number of clinically relevant studies highlights the translational gap between basic research and clinical trials. Therefore, a comprehensive characterization of the closed-chest post-MI HF models in G&#246;ttingen minipigs and Landrace pigs with repeated assessment of left and right ventricular function and anatomy using CMRI during long-term follow up is required. Here we aimed to focus on the technical feasibility and clinical relevance of two post-MI HF models to describe standardized and reproducible experimental protocols for post-MI HF studies that may be used to assess cardioprotective drug and/or medical device therapies.
The present study is the first one in the literature to characterize a clinically relevant model of post-MI HF using adult G&#246;ttingen minipigs and to compare morphological and cardiac left and right ventricular functional parameters with that of the adolescent Landrace pigs.

<table border="0" cellpadding="0" cellspacing="0" style="width:627px;" width="628">
	<tbody>
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			<td height="42" style="height:42px;width:223px;">Special Diet Services pig chow&#160;</td>
			<td style="width:274px;">SDS, Witham, England, Hungarian distributor: Akronom Kft.</td>
			<td style="width:66px;"></td>
			<td style="width:66px;"></td>
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			<td height="22" style="height:22px;width:223px;">maintenance minipig diet&#160;</td>
			<td style="width:274px;">no. 9023, Altromin</td>
			<td></td>
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			<td height="22" style="height:22px;width:223px;">pregnant sow chow</td>
			<td style="width:274px;">Bonafarm-B&#225;bolna Takarm&#225;ny Plc</td>
			<td></td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ketamine hydrochloride</td>
			<td style="width:274px;">Richter Pharma AG</td>
			<td></td>
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		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">xylazine</td>
			<td style="width:274px;">Medicus Partner</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">atropine</td>
			<td style="width:274px;">Egis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">endotracheal tube&#160;</td>
			<td style="width:274px;">Portex</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">isoflurane</td>
			<td style="width:274px;">Abbot</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">anesthetic machine</td>
			<td style="width:274px;">Dr&#228;ger Julian</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">18 G needle</td>
			<td style="width:274px;">Anhul Kangda Medical Products Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">5% glucose in Ringer solution</td>
			<td style="width:274px;">B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">atracurium besylate</td>
			<td style="width:274px;">GSK</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">cardiac magnetic resonance machine</td>
			<td style="width:274px;">Siemens Healthineers Medical GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">acetyl salicylic acid</td>
			<td style="width:274px;">Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">clopidogrel</td>
			<td style="width:274px;">Zentiva</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">meloxicam (meloxidyl)</td>
			<td style="width:274px;">Ceva</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">antibiotic coctail (tardomyocel) comp III.</td>
			<td style="width:274px;">Norbrook</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ear vein cannula</td>
			<td style="width:274px;">B Braun Melsungen AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">magnesium sulfate</td>
			<td style="width:274px;">W&#246;rwag Pharma GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">povidone-iodine</td>
			<td style="width:274px;">Egis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ECG electrodes</td>
			<td style="width:274px;">Leonhard Lang GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">6F-ACT introducer</td>
			<td style="width:274px;">St Jude Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">heparin</td>
			<td style="width:274px;">TEVA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">arterial pressure sensor and monitoring system</td>
			<td style="width:274px;">GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:223px;">guidewire</td>
			<td style="width:274px;">&#160;PT2MS Boston Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">5F guiding catheter</td>
			<td style="width:274px;">Medtronic Launcher, 5F</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">fluoroscope, C-bow</td>
			<td style="width:274px;">Siemens Medical GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Iobitridol (Xenetix)</td>
			<td style="width:274px;">Guerbet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">balloon catheter</td>
			<td style="width:274px;">Boston Scientific, EMERGE, 2.5mm x 12mm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">heating device</td>
			<td style="width:274px;">3M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">rectal probe</td>
			<td style="width:274px;">Vatner Kft</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">pulse oxymeter</td>
			<td style="width:274px;">Comen medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">epinephrine</td>
			<td style="width:274px;">Richter Gedeon Rt.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">lidocaine</td>
			<td style="width:274px;">EGIS</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">microcatheter</td>
			<td style="width:274px;">Caravel ASAHI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">defibrillator</td>
			<td style="width:274px;">GE Marquette Responder 1100</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">perfusion pump&#160;</td>
			<td style="width:274px;">TSE system</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">antiseptic coating</td>
			<td style="width:274px;">Friedrich Huber aeronova GmbH&#38;Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">gadobutrol</td>
			<td style="width:274px;">Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MASS 7.6 analysis software</td>
			<td style="width:274px;">Medis Medical Imaging Software, Leiden</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Mortality
Out of 13 G&#246;ttingen minipigs subjected to myocardial infarction, two animals died (15.4% mortality), one during the ischemic period due to irreversible VT and one owing to asystole in reperfusion. In G&#246;ttingen minipigs, one animal was successfully resuscitated during cardiac ischemia. The mortality rate was 0% in Landrace pigs, ten out of ten animals survived, two of them required resuscitation due to VF in the ischemic period. Mortality did not differ significantly between the two breeds.
Myocardial scar sizes were comparable between the two breeds
To measure the extent of cardiac scar as a consequence of MI, CMRI was performed. Scar sizes and BARI scores were comparable between the two breeds measured at the 2nd month of follow-up in Landrace pigs, and at the 3rd and 6th month in G&#246;ttingen minipigs (Figure 2E,F). No differences were observed when scar sizes were related to the BARI scores in Landrace pigs at 2 months (0.55 &#177; 0.1) and in G&#246;ttingen minipigs at 3 months and 6 months respectively (0.75 &#177; 0.12 and 0.57 &#177; 0.08). The scars were localized in the anterior, anteroseptal, septal, anteroapical and apical segments of the heart in both breeds. The lateral wall was affected only in G&#246;ttingen minipigs. Right ventricular infarction was negligible, affected only one animal out of eleven surviving G&#246;ttingen minipigs and one out of ten Landrace pigs (2.11 &#177; 2.11 vs. 0.97 &#177; 0.97).
Increase in left ventricular mass was more pronounced in Landrace pigs during follow-up
The cardiac growth rate was measured by CMRI. LVED mass in G&#246;ttingen minipigs increased only moderately (8%) at 6 months (Figure 3A). In contrast, in Landrace pigs, LVED mass increased by almost 100% at 2 months (Figure 3B).
Left ventricular ejection fraction decreased only in G&#246;ttingen minipigs
LVEF, as the most widely used parameter of left ventricular systolic function, was measured by CMRI. MI resulted in a significant decrease in LVEF in minipigs at 3 months and 6 months (Figure 4A). In Landrace pigs, LVEF did not change after 2 months (Figure 4B).
Post-infarction LVESV and LVEDV increased significantly in both breeds (Table 1). LVESV increased by 69% and 80% in G&#246;ttingen minipigs after 3 and 6 months, respectively, and by 80% in Landrace pigs after 2 months. LVEDV showed a 28% increase after 3 months and a 42% increase after 6 months in G&#246;ttingen minipigs and an 82% increase in Landrace pigs after 2 months. LVSV of Landrace pigs increased by 85% in 2 months and LVSV of G&#246;ttingen minipigs did not increase significantly even at 6 months.
Left atrial volume indexed to body surface area increased only in G&#246;ttingen minipigs, but both the breeds developed pulmonary oedema following myocardial infarction
In order to further examine signs of HF, we performed measurement of the left atrial volume indexed to body surface area (LAVi). LAVi increased by 34% in G&#246;ttingen minipigs after 6 months (Figure 5A) and did not change significantly in Landrace pigs after 2 months (Figure 5B). Representative images show the tracing of the left atria (Figure 5C-D). Moreover, the presence or absence of pulmonary oedema was assessed by CMRI on the localizer images (Figure 5E). Pulmonary oedema was observed in both breeds as a result of cardiac decompensation. Ten out of eleven G&#246;ttingen minipigs and nine out of ten Landrace pigs showed obvious signs of pulmonary oedema.
Increase in body weight was more pronounced in Landrace pigs during follow-up
In G&#246;ttingen minipigs body weight gain was only 8% after 3 months and 30% after 6 months (Figure 6A), whereas increased heart weight was accompanied by a nearly 100% increase in body weight in Landrace pigs at 2 months (Figure 6B).
Trends in cardiac functional parameters differ between G&#246;ttingen minipigs and Landrace pigs
Coronary artery occlusion led to an almost significant decrease in mean arterial pressure (MAP) in G&#246;ttingen minipigs (57.9 &#177; 3.98 mmHg vs. 49.89 &#177; 1.24 mmHg) and decreased significantly in Landrace pigs (65.4 &#177; 5.97 mmHg vs. 45.47 &#177; 4.79* mmHg) in the early reperfusion phase as compared to the baseline (pre-infarction) values.
CI is a reliable indicator of cardiac performance, which relates left ventricular CO to BSA. In G&#246;ttingen minipigs, CI did not&#160;change at the measured time points (Figure 7A), whereas in Landrace pigs a tendency to increase was detected in cardiac index (Figure 7B).
HR of G&#246;ttingen minipigs increased significantly at 3 (20%) and 6 months (22%) after MI compared to baseline values (Table 2).
In contrast, the HR of Landrace pigs did not&#160;change significantly during the follow-up period. In G&#246;ttingen minipigs CO showed a significant 32% increase only at 6 months of follow-up, whereas CO was increased by 76% in Landrace pigs after 2 months due to a significant increase in LVSV (Table 2). BSA increased significantly in both breeds at the measured time points (Table 2). BSA increased by 4% and 19% in G&#246;ttingen minipigs after 3 and 6 months, respectively, and by 54% in Landrace pigs after 2 months.
Increase in right ventricular morphofunctional parameters were observed in both G&#246;ttingen minipigs and Landrace pigs
MI affected not only left ventricular function, but it also resulted in a significant increase of RVEF in both breeds (Figure 8) measured by CMRI, despite the negligible right ventricular scar size. RVED mass increased in Landrace pigs only (Table 3).
RVESV did not change during follow up in any of the breeds. RVEDV increased significantly by 37% only in Landrace pigs (Table 3). While RVSV in G&#246;ttingen minipigs increased significantly by 23% only after 6 months, in Landrace pigs a significant 80% increase in RVSV was observed at 2 months.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61901/61901fig2.jpg" /> 
Figure 2. Estimation of the myocardium at risk based on the BARI (Bypass Angioplasty Revascularization Investigation Myocardial Jeopardy Index) score (A-D). The total value of the infarct-related artery is divided by the sum of the 3 total values of each coronary artery, the right coronary artery (RCA), the left circumflex coronary artery (LCX), and the left anterior descending coronary artery (LAD). Left ventricular scar sizes in G&#246;ttingen minipigs and Landrace pigs measured by cardiac magnetic resonance imaging (E). Scar size is shown as a ratio of mass of infarction to the mass of left ventricle at end of diastole (LVED). BARI scores in G&#246;ttingen minipigs and Landrace pigs measured before coronary occlusion (F). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig2large.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/61901/61901fig3.jpg" /> 
Figure 3. Left ventricular end-diastolic (LVED) mass (g) of G&#246;ttingen minipigs (A) and Landrace pigs (B) measured by cardiac magnetic resonance imaging. *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig3large.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/61901/61901fig4.jpg" /> 
Figure 4. Left ventricular (LV) ejection fraction (%) of G&#246;ttingen minipigs (A) and Landrace pigs (B) measured by cardiac magnetic resonance imaging. *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Measured parameter</td>
			<td colspan="3">G&#246;ttingen minipigs</td>
			<td colspan="2">Landrace pigs</td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>3 months</td>
			<td>6 months</td>
			<td>Baseline</td>
			<td>2 months</td>
		</tr>
		<tr>
			<td>LVESV [ml]&#160;</td>
			<td>25.77 &#177; 1.73</td>
			<td>43.65 &#177; 4.53*</td>
			<td>46.28 &#177; 4.35*</td>
			<td>54.59 &#177; 2.00</td>
			<td>98.26 &#177; 8.60*</td>
		</tr>
		<tr>
			<td>LVEDV [ml]&#160;</td>
			<td>55.49 &#177; 3.14</td>
			<td>71.08 &#177; 5.25*</td>
			<td>78.81 &#177; 5.46*</td>
			<td>93.99 &#177; 3.85</td>
			<td>171.20 &#177; 11.50*</td>
		</tr>
		<tr>
			<td>LVSV [ml]&#160;</td>
			<td>29.71 &#177; 1.65</td>
			<td>27.44 &#177; 1.97</td>
			<td>32.52 &#177; 2.37</td>
			<td>39.40 &#177; 3.05</td>
			<td>72.94 &#177; 3.99*</td>
		</tr>
	</tbody>
</table>
Table 1. Left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), and left ventricular stroke volume (LVSV) at the measured time points in Landrace pigs and G&#246;ttingen minipigs. *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61901/61901fig5.jpg" /> 
Figure 5.&#160;Left atrial volume indexed to body surface area (LAVi) in mL/m2 in G&#246;ttingen minipigs (A) and Landrace pigs (B) measured by cardiac magnetic resonance imaging. Representative images of left atrial volumes, tracings were made on the two- (C) and four chamber (D) cine images. The white arrows show the presence of pulmonary oedema on the representative localizer image (E). *p&#60;0.05 vs. corresponding baseline (paired t-test in G&#246;ttingen minipigs and Landrace pigs). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig5large.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/61901/61901fig6v2.jpg" /> 
Figure 6. Body weight&#160;(kg) of G&#246;ttingen minipigs (A) and Landrace pigs (B). *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61901/61901fig7.jpg" /> 
Figure 7. Left ventricular (LV) cardiac indices (L/min/m2) of G&#246;ttingen minipigs (A) and Landrace pigs (B). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Measured parameter</td>
			<td colspan="3">G&#246;ttingen minipigs</td>
			<td colspan="2">Landrace pigs</td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>3 months</td>
			<td>6 months</td>
			<td>Baseline</td>
			<td>2 months</td>
		</tr>
		<tr>
			<td>HR [1/min]&#160;</td>
			<td>79.64 &#177; 4.03</td>
			<td>95.55 &#177; 5.34*</td>
			<td>97.00 &#177; 4.46*</td>
			<td>93.44 &#177; 2.73</td>
			<td>88.00 &#177; 2.52</td>
		</tr>
		<tr>
			<td>CO [L/min]&#160;</td>
			<td>2.37 &#177; 0.16</td>
			<td>2.58 &#177; 0.20</td>
			<td>3.12 &#177; 0.24*</td>
			<td>3.65 &#177; 0.25</td>
			<td>6.41 &#177; 0.39*</td>
		</tr>
		<tr>
			<td>BSA [m2]&#160;</td>
			<td>0.70 &#177; 0.01</td>
			<td>0.73 &#177; 0.01*</td>
			<td>0.83 &#177; 0.03*&#160;</td>
			<td>&#160;0.70 &#177; 0.01</td>
			<td>1.08 &#177; 0.03*&#160;</td>
		</tr>
	</tbody>
</table>
Table 2. Heart rate (HR), cardiac output (CO), and body surface area (BSA) of G&#246;ttingen minipigs&#160;and Landrace pigs. *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61901/61901fig8.jpg" /> 
Figure 8. Right ventricular (RV) ejection fractions (%) of G&#246;ttingen minipigs (A), and Landrace pigs (B). *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs). <a href="https://www.jove.com/files/ftp_upload/61901/61901fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Measured parameter</td>
			<td colspan="3">G&#246;ttingen minipigs</td>
			<td colspan="2">Landrace pigs</td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>3 months</td>
			<td>6 months</td>
			<td>Baseline</td>
			<td>2 months</td>
		</tr>
		<tr>
			<td>RVED mass [g]&#160;</td>
			<td>8.64 &#177; 0.68</td>
			<td>8.98 &#177; 0.76</td>
			<td>7.94 &#177; 0.77</td>
			<td>16.49 &#177; 0.90</td>
			<td>23.61 &#177; 1.40*</td>
		</tr>
		<tr>
			<td>RVESV [ml]&#160;</td>
			<td>18.27 &#177; 1.47</td>
			<td>16.91 &#177; 1.80</td>
			<td>14.57 &#177; 1.02</td>
			<td>43.59 &#177; 3.68</td>
			<td>42.65 &#177; 2.37</td>
		</tr>
		<tr>
			<td>RVEDV [ml]</td>
			<td>44.16 &#177; 2.61</td>
			<td>42.14 &#177; 2.83</td>
			<td>46.27 &#177; 3.45</td>
			<td>83.03 &#177; 3.42</td>
			<td>113.72 &#177; 5.12*</td>
		</tr>
		<tr>
			<td>RVSV [ml]</td>
			<td>25.82 &#177; 1.72</td>
			<td>25.25 &#177; 1.67</td>
			<td>31.71 &#177; 2.99*</td>
			<td>39.44 &#177; 3.52</td>
			<td>71.06 &#177; 3.38*</td>
		</tr>
	</tbody>
</table>
Table 3. Right ventricular end-diastolic (RVED) mass, right ventricular end-systolic volume (RVESV), right ventricular end-diastolic volume (RVEDV), and right ventricular stroke volume (RVSV) in G&#246;ttingen minipigs and Landrace pigs. *p&#60;0.05 vs. corresponding baseline (repeated measures one-way ANOVA followed by Fisher&#39;s LSD test in G&#246;ttingen minipigs; paired t-test in Landrace pigs).

Watch this Scientific Journal Video about Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in GÃƒÂ¶ttingen Minipigs and Landrace Pigs at JoVE.com

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

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 ...

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8.3K Views.  Semmelweis University. 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 Göttingen minipigs and the characterization of post-MI HF model in Göttingen minipigs as compared to Landrace pigs. 

Video: Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in Göttingen Minipigs and Landrace Pigs

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...

Closed Chest Biventricular Pressure-Volume Loop Recordings with Admittance Catheters in a Porcine Model

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular evaluation is often performed in preclinical research. However, the right and left ventricles exert functional interdependence due to their parallel and serial connections, encouraging simultaneous evaluation of both ventricles. Furthermore, various pharmacological interventions may affect the ventricles and their preloads and afterloads differently. 
We describe our closed chest approach to admittance-based bi-ventricular PV loop recordings in a porcine model of acute right ventricular (RV) overload. We utilize minimally invasive techniques with all vascular accesses guided by ultrasound. PV catheters are positioned, under fluoroscopic guidance, to avoid thoracotomy in animals, as the closed chest approach maintains the relevant cardiopulmonary physiology. The admittance technology provides real-time PV loop recordings without the need for post-hoc processing. Furthermore, we explain some essential troubleshooting steps during critical timepoints of the presented procedure. 
The presented protocol is a reproducible and physiologically relevant approach to obtain a bi-ventricular cardiac PV loop recording in a large animal model. This can be applied to a large variety of cardiovascular animal research.

Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction.

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...