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

The overall goal of the current study is to present the techniques of induction of myocardial infarction and post-myocardial infarction induced heart failure in closed-chest adult Gottingen minipigs. We also aim to characterize the post-myocardial infarction induced heart failure model in minipigs and Landrace pigs. First to assess myocardial function and morphology at the beginning of the study, baseline cardiac magnetic resonance imaging is performed one day prior to induction of myocardial infarction.

Then myocardial infarction is induced by intraluminal occlusion of the left anterior descending coronary artery. After balloon deflation, reperfusion of the myocardium is induced, and intercoronary drug therapy, in this case, thecal, is administered by using microcatheter. As a consequence of myocardial infarction, the pigs will develop heart failure.

Chronic morpho-functional consequences of acute myocardial infarction are evaluated by cardiac magnetic resonance imaging at three and six months in Gottingen minipigs and at two months in Landrace pigs. The Landrace pigs are terminated after two months and Gottingen minipigs after eight months of reperfusion. The clinical outcome of post-myocardial infarction induced heart failure could be further improved by the development of new cardioprotective therapies.

Such cardioprotective therapies effective in preclinical animal models failed in clinical translation so far for several reasons. A former Hungary group, together with academic and other partners, provides wide range of innovative preclinical research and development services focusing on, but not limited to, novel drugs and medical devices for cardiovascular diseases, for example, myocardial infarction and heart failure. Furthermore, we are developing several cardioprotective technologies in-house.

So the failure of clinical development of cardio-protective therapies can be attributed at least in part due to the low translational value of myocardial infarction models in small animals. Large animal models with good translational value are essential for the successful development of cardio-protective therapies. In contrast to clinical trials of cardio-protective therapies in rodents and Landrace pigs, the most value as large animal model, the cardiac functional outcome can be assessed up to only one to two months in these animals.

Use of other minipigs enables us to implement longer, fuller periods up to several months, that better reflect the clinical situation. After anesthesia and body weight measurement, cardiac magnetic resonance imaging is performed for evaluation of baseline cardiac morphology, such as chamber and wall dimensions and cardiac function, such as ejection fraction and cardiac output. Induce anesthesia of the animals with ketamine hydrochloride xylazine as an intramuscular injection to the neck region.

Use the E-vein cannula for food replacement and drug administration to prevent or treat ventricular arrhythmias. When the surgery room is prepared, place the animal on the operating table. Fix the limbs and apply wedges to immobilize the animal in supine position.

Disinfect the surgical site with povidone iodine. The surgical site in this case is around the skinfold between the gracilis and sartorius muscle. Remove the hair at the surgical site with a razor.

Place surface ECG electrodes in Einthoven's triangle. Set the anesthesia machine and start positive-pressure ventilation. Isolate the disinfected surgical area with a surgical drape.

Approach the femoral region and make a longitudinal incision to the skin between the gracilis and sartorius muscles. Dissect the subcutaneous tissue and fascia. Isolate the femoral artery.

Put two surgical sutures below the femoral artery to control the hemorrhage. Puncture the femoral artery. Cannulate the femoral artery using Seldinger technique.

Fix the sheath to the skin. Use the artery for blood sampling for further biochemical analysis. Administer 5, 000 IU heparin via the femoral sheath to secure adequate anticoagulation and prevent thrombosis during the surgical intervention.

Attach pressure sensor to the femoral vessel to monitor the arterial blood pressure throughout the surgical intervention. 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 open to direction of the free air.

After reparation of the animal, myocardial infarction is induced by intraluminal occlusion of the left anterior descending coronary artery Through the femoral sheath, introduce and advance the guide wire to the aortic arch and introduce the guiding catheter over the guide wire. After positioning the fluoroscope in anteroposterior position, ensure that there is no thrombus or air bubble within the catheter, with aspiration of at least five milliliter of blood, the volume of the catheter, the syringe connected to the catheter. Connect the other portion of the catheter to syringe filled with radiocontrast agent.

Take care that the syringe is held elevated to prevent infusion of air bubbles into coronary artery. Perform baseline angiography by selective filling of the right and the left coronary arteries with contrast agent. Perform BARI scoring after the baseline angiography, which finally permits estimation of the percentage of myocardial muscle at risk.

Insert the percutaneous transluminal coronary angioplasty guide wire through the guiding catheter. Position it distally to the planned site of the occlusion. Check the position of the PTCA guide wire by angiography.

Determine by visual estimation the optimal balloon size based on coronary artery diameter. Place the balloon catheter over the PTCA guide wire and advance it to the planned position. Check the position of the balloon catheter by angiography.

Inflate the balloon and confirm the total occlusion by visualizing the stop of the contrast flow. Tape instruments to the surgical drape to avoid dislocation of the intracoronary balloon. Record and document the ECG sign of occlusion by ST elevation.

Cover the animal with a heating device to maintain the core temperature. In case of ventricular arrhythmias, follow the protocol as described in the text. Check balloon pressure every 30 minutes during the two hours of coronary occlusion.

If there is a decrease of more than 0.5 bar in balloon pressure, set it back to initial values. Perform control angiography just prior to the end of coronary occlusion to verify the maintained balloon placement and absence of flow distally to occlusion site. Administer 2, 500 IU heparin and one gram magnesium sulfate intracoronarily as a slow bolus to prevent thrombosis and arrhythmias.

Initiate the reperfusion with balloon deflation. Remove the deflated balloon. Confirm the success of complete reperfusion with coronary angiography to demonstrate the blood flow at the distal part of the coronary vessel.

Prepare for intracoronary drug administration after initiation of myocardial reperfusion. To prevent coronary artery embolization, fill the therapeutic perfusion microcatheter with saline. Place the microcatheter over the PTCA guide wire.

Advance and confirm the position of the microcatheter. The tip of the microcatheter should be placed at the level of occlusion. Remove the PTCA guide wire.

Connect the microcatheter with the perfusion pump. Initiate intercoronary administration. After drug administration, remove the microcatheter and perform control angiography to exclude that intervention led to air emboli or coronary dissection.

Remove the arterial sheath and tie down the femoral artery proximal to the puncture site. Close the wound using continuous sutures. Apply antiseptic coating.

Terminate the anesthesia via withdrawal of isoflurane. Chronic morpho-functional consequences of acute myocardial infarction are evaluated by cardiac MRI at three and six months in Gottingen minipigs and two months in Landrace pigs. Mortality rate did not differ significantly between the two breeds.

Cardiac scar sizes and BARI scores were comparable between the two breeds. Left ventricular end diastolic masses at baseline and during follow-up period differ between the two breeds. LVED mass in Gottingen minipigs increased only moderately at six months.

In contrast, in Landrace pigs, LVED mass increased by almost 100%at two months. Myocardial infarction resulted in significant decrease of ejection fraction in minipigs at three months and six months. However, in Landrace pigs, ejection fraction did not change after two months.

Differences between the two breeds regarding ejection fraction can be attributed to an intensive cardiac growth rate in Landrace pigs, and thus altered cardiac remodeling. Increased heart weight was accompanied by nearly 100%increase in body weight in Landrace pigs at two months. Whereas in Gottingen minipigs, body weight gain was only 8%after three months and 30%after six months.

In order to further examine signs of heart failure, we performed measurements of the left atrial volume indexed to body surface area. LAVi increased by 34%in Gottingen minipigs after six months but did not change significantly in Landrace pigs after two months. The presence or absence of pulmonary edema was assessed by cardiac MRI on the localizer images.

Pulmonary edema was observed in both breeds as a result of cardiac decompensation. In Gottingen minipigs, cardiac index didn't show significant changes at the measured time points. Whereas in Landrace pigs, cardiac index increased almost significantly.

Myocardial infarction effected not only left ventricular function, but it also resulted in a significant increase of right ventricular ejection fraction in both strains. We have shown that the ideal Gottingen minipig model mimics functional and morphological post-myocardial infarction heart failure parameters comparable to humans. This is a feasible, producible, and translational model to assess efficacy of treatment of both acute and chronic myocardial infarction and of their consequences.

We also showed method for local drug delivery to the coronaries using microcatheters. The comprehensive characterization of the closed-chest infarction models in Landrace and minipigs will be useful for choosing the most suitable large animal models to develop novel therapies for post-infarction heart failure.