The experimental protocol which delivered the results described in this article has been approved by the regional Ethics Committee for Laboratory Animal Experiments at the Medical University of Vienna and the Austrian Federal Ministry of Education, Science and Research (BMWFW-66.009/0023-WF/V/3b/2016). All experiments conform with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

NOTE: 10−12-week-old male Sprague Dawley rats of 250−300 g body weight (BW) are used. As the following procedures and treatments are performed in a sterile environment of an operating room (OR), wear scrubs, gloves, facemasks and hoods when handling animals. Before entering the OR, ensure that hands are washed and disinfected. If the intention is to operate on several animals in a surgical session, either wash and disinfect, or autoclave the instruments in between operations. These hygienic guidelines are valid for all procedures presented in the protocol section.

1. Preoperative preparation and anaesthesia

1. Initiate preoperative anaesthesia by injecting a mixture of xylazine (4 mg/kg BW) and ketamine (100 mg/kg BW) intraperitoneally.
2. Intubate the rats with a 14 G tube and volume-controlled ventilation with a mixture of O₂, air and isoflurane (1−2.5%) at 75−85 strokes/min, 100 mL/stroke/BW (Figure 1A). If necessary, for a better view while intubating: apply Xylocain via a cotton-wool tip on the lower pharynx to achieve local relaxation.
3. Place the rats on a heated operating table in a supine position and fix the forelimbs with tape (Figure 1B).
4. Measure rectal temperature with a probe.
   NOTE: It should be maintained between 37.5−38.5 °C.
5. Shave the thorax and clean the operating area with antiseptic povidone iodine solution. Apply eye ointment to the rat to prevent drying of the eyes.
6. Administer intraoperative analgesia by injecting piritramide (0.1 mL/kg BW) intraperitoneally. 
7. Place ECG probes subcutaneously in the extremities of the animal.
8. Check tail and toe reflexes prior to initiating the surgical procedure.

2. Surgical procedure―induction of myocardial ischemia

1. Perform skin incision using a scalpel. Ensure to start 2 mm parasternal on the left thorax at the level of the 3rd intercostal space and continue to the anterior axillary line at the level of the 5th intercostal space (Figure 1C).
2. Replace the superficial muscles gently to make the ribs visible (Figure 1D).
3. In the case of minor bleeding, use a cauter to obliterate or to disconnect the surrounding tissue.
4. Perform the thoracotomy at the level of the 4th intercostal space and insert a retractor to gain visibility of the heart and the lung (Figure 1E). Carefully open the pleura to avoid bleeding.
5. Temporarily occlude the LAD using a tourniquet to induce ischemia/reperfusion (MIR) over a defined time; or permanently (MI) occlude it by making 6−7 knots using a 6-0 suture to close the ligation (Figure 1F,G).
   NOTE: The right spot for occlusion of the LAD is located about 2−3 mm beneath the left auricle on the ventral/left lateral margin of the heart. Successful occlusion is associated with ECG changes (ST-segment elevation) and macroscopic changes in the LV as paling.
6. In the case of the ischemia/reperfusion model, reopen the LAD by removal of the tourniquet after 30 min of occlusion.
7. Close the thorax with three single button sutures using a 4-0 single monofilament suture (Figure 1H). Prior to tightening the last suture, remove any residual air from the thorax with a 10 mL syringe to prevent a pneumothorax (Figure 1I).
8. Reposition the muscles and turn off the volatile anaesthesia.
9. Suture the skin with a continuous suture using a 4-0 suture (Figure 1J).
10. Administer an antiseptic spray to protect against infections and biting of the suture by rats.

3. Postoperative treatment and exclusion criteria

1. Keep the rats on the heating table until they wake up. Extubate the rats as soon as they commence breathing spontaneously.
2. Put the extubated rats in a cage under a heating lamp to prevent them from cooling.
3. Return rats to the animal houses under standardized conditions when they commence behaving normally again.
4. Add 2 ampules of piritramide and 30 mL of 5% glucose to 250 mL of water for post-operative analgesia for three days.
5. Check the fitness and behavior of rats with the checklist and exclusion criteria (Table 1). Observe the animals twice a day for the following week, then twice a week.
   NOTE: In accordance with international standards, present any suffering animals, or animals that gain up to 6 points in the evaluation with the checklist, to veterinarians to make therapy-related decisions. Any animals that gain 7 or more points must be immediately sacrificed with an overdose of ketamine and xylazine.

Table 1: Checklist and exclusion criteria. This table contains the examinations that must be observed and the corresponding score. Accordingly, the post-operative treatment of the animal must be adapted, or a veterinarian must be consulted.

4. Echocardiography measurements

NOTE: Echocardiography is usually performed twice, prior to the induction of MI and before the organs are harvested.

1. Inject rats with a mixture of xylazine (4 mg/kg BW) and ketamine (100 mg/kg BW) intraperitoneally.
2. Place the rats in a supine position on a heating tray. Apply echo gel to the chest, which helps the ultrasound waves travel better and reduces signal interferences.
3. Obtain parasternal short axis views of the LV cavity at the level of the papillary muscle.
4. Perform M-mode echocardiography in order to measure left ventricular ejection fraction and morphology.

5. Organ harvesting (without working heart)

1. Administer xylazine (4 mg/kg BW) and ketamine (100 mg/kg BW) intraperitoneally prior to organ harvesting. Ensure that the reflexes are negative.
   NOTE: No intubation is required as the procedure does not last longer than 1 min.
2. Use a scalpel to make a skin incision under the xiphoid and extend it parallelly to the ribs on both sides using scissors.
3. Cut the ribs in the frontal axillary line and grab the xiphoid to lift the chest up (Figure 2A).
4. Remove anatomical or fibrotic tissue adhesions by carefully rupturing the tissue with two pairs of forceps.
5. Take blood samples (for blood gas evaluation or molecular analyses) from the vena cava inferior with a 5 mL syringe.
6. Perform the excision of the whole heart at the inlet and outlet level (Figure 2B). If necessary, proceed with working heart evaluation as described in section 6.
7. Harvest organs, shock frost them in liquid nitrogen and store in -80 °C for further molecular analyses, or in formaldehyde for histological purposes.

6. Ex vivo hemodynamic measurements via a working heart system

NOTE: The general setup and the components of the apparatus has been previously described¹¹. The following protocol describes the handling of the animal’s heart and the necessary steps to evaluate LV function.

1. Anesthetize rats as described in step 5.1 and inject 200 IU of heparin intravenously (femoral vein).
2. Open the thorax via an incision beneath the costal arch with a scalpel and extend it to both anterior axillary lines with scissors and elevate the sternum.
3. Cut the great vessels near their outlet or inlet to the heart to excise it (Figure 2B).
4. Immerse the heart in ice-cold Krebs-Henseleit buffer and mount it on the erythrocyte-perfused isolated heart system via cannulating the aorta (Figure 3A).
5. Start with the LD mode with a constant afterload of 60 mmHg (stabilization period).
6. After 15 min of LD mode, switch to the WH mode. Therefore, cannulate the left atrium via a pulmonary vein (Figure 3B). Then, change the flow direction in the system by opening the clip that occludes the atrial cannula. This results in a perfusion of the left atrium and a physiological blood flow in the left heart¹¹.
7. Record hemodynamic measurements for 20 min in the WH mode.
8. Collect blood drops of the coronaries with a 2 mL syringe to measure coronary flow (CF, mL/min) every 5 min.
   NOTE: CF is measured as the difference between left atrial flow (LAF) and aortic flow (AF).
9. Perform continuous measurements of LAF (equivalent to cardiac output) and AF with a flow probe.
   NOTE: The probe is inserted via the WH apparatus into the LV. All data are continuously registered.
10. If the ongoing protocol requests, insert a high-fidelity catheter retrogradely via the aortic valve into the LV and measure the left ventricle systolic pressure (LVSP).
11. To assess the pressure–volume work performed per minute, calculate stroke volume as cardiac output divided by heart rate.
12. Calculate external heart work (EHW) according to the following formula: CO x LVSP (g x m/min) normalized to heart weight.