Twenty-four intact mature female Pannon minipig (Source of supply: Farm registered in the Hungarian state Farm Animal Identification System (ENAR)) from authority-monitored breeders were included in the study. The mean body weights were 60 kg at the mean age of 9 months at order. The animals were free from Aujeszky disease, PRRS, Leptospirosis, and Brucellosis, approved by the EU and Hungarian authority monitoring system. The animals were placed in groups of 6–7 animals. By keeping them in a traditional pen, we provided the possibility of expressing natural behaviors, preventing social stress caused by isolation. Abundant space and environmental enrichment were also provided to allow for more physical activity in the daily activity pattern of these animals, compared to those kept in individual cages. Throughout the experiment, the animals were fed a special diet containing 88.2% dry matter, 14.01% crude protein, 2.57% crude fat, 10.13% crude fiber, and 11.35 MJ/kg digestible energy (“Agroszász” Experimental Pig Food; Szászvár, Hungary). The minipigs were fed an amount equal to 1.5% of their body weight twice per day, with drinking water available ad libitum.

Animals were observed and checked for signs of illness, distress, or deviation from normal (activity, food intake (qualitative), etc.). No abnormalities and/or unexpected observations were reported in individual animal records. The animals were observed individually before and after the procedure (on day 0). Daily farm-side observations were made to check the animals, with a focus on behavioral changes or illness. The animals were placed in groups of 6–7 animals. During the 30-day follow-up period, the animals were under regular veterinary control, and no interventions were needed. The animals were transported to the Institute in small groups 1 day before the study started. In the research area, they were randomly placed in individual cages and signed with a simply growing number sequence. The animals were kept individually until the third day of the experiment, to perform the individual medications and treatments. The isolation time was 4 days. Blinding was not considered during the study. The animals underwent the same procedure without treatment, and the experimental unit was the single animal. At the endpoint of the experiment on day 30, the animals were gently anesthetized by inhalation of a 5% volume isoflurane and were euthanized by administration of 30 mg/kg of body weight potassium chloride into the jugular vein and then dissected.

Cardiac MRI examinations were performed 24–48 h before the induction of the myocardial infarction. The animals were taken to the institute 1 day before the MRI, which was performed after 12 h of fasting. Before the MRI scan, the minipigs were sedated with a combination of 12 mg/kg ketamine hydrochloride (Narkamon 100 mg/mL inj., Bioveta; Ivanovice na Hané, Czechia), 1 mg/kg xylazine (CP-Xylazin2% inj., CP-Pharma GmbH; Burgdorf, Germany), and 0.04 mg/kg atropine sulfate (Atropine sulfuricum-EGIS 1 mg/mL inj., Egis; Budapest, Hungary) administered intramuscularly, followed by the placement of an endotracheal tube (Chanelmed Ltd.; diameter: 5.0–6.0). Imaging was performed under inhalation anesthesia with 1.5–2.5 m/m% isoflurane (Isoflutek 1,000 mg/g, Karizoo; Barcelona, Spain) and 2 L/min oxygen with a MAGNETOM Vision Plus 1.5 T MRI scanner (Siemens AG; Erlangen, Germany) with ECG control. To improve image quality, cine MRI images in short-axis and long-axis views of the heart were acquired following the intravenous administration of 1.5 mg/kg atracurium (Tracrium 10 mg/mL inj., GlaxoSmithKline; Brentford, United Kingdom) and mechanical ventilation (10 mL/kg 15/min). The animals were kept in single cages until the control MRI scans on day 3. After the MRI scan on day 3, the animals were transported to their usual keeping place and kept in groups. The control MRI scans were performed on day 30, and the animals were transported back to the Institute 1 day before the procedure. Preparation and method of examination were the same for the MRI imaging performed on days 3 and 30. At these check points, gadobutrol (Gadovist 1 mmoL/mL inj., Bayer; Leverkusen, Germany) was administered intravenously at a dose of 0.16 mmol/kg for the examination of the myocardium, the determination of left ventricular function, and the measurement of perfusion and viability.

The day before AMI induction, the animals were treated with 600 mg clopidogrel and 300 mg aspirin per os in the drinking water, followed by a daily dose of 100 mg acetylsalicylic acid (Aspirin protect 100 mg tablets, Bayer; Leverkusen, Germany) and 75 mg clopidogrel (Trombex 75 mg tablets, GlaxoSmithKline; Brentford, United Kingdom) from day 1, administered per os. Induction of AMI was performed on day 0 under general anesthesia as described for the acquisition of MRI images. Surgical access was provided to the femoral artery and the jugular vein, and a sheath (St. Jude Medical introducer set) was inserted (6–7 F diameter, according to the size). After blood was drawn, 5,000 IU heparin was administered via the sheath following blood sampling. AMI induction was performed under invasive hemodynamic monitoring and by placing a balloon catheter (2.5–2.75 mm diameter, 8–15 mm length) in the left anterior descending coronary artery (LAD) after the origin of the second diagonal branch and inflating the balloon with 5–6 atm for 90 min, followed by reperfusion. A Siemens Cios Alpha C-arm, a Siemens Axiom Sensis hemodynamic unit, and a Comen C-50 V patient monitor were used during the angiographic procedure. The contrast material used was Xenetix 350 mg I/ml (iobitridol); Guerbet, and contrast volumes range from 50 to 100 mL/pig (245–370 mg/kg with a short injection duration).

Reperfusion and ventricular function were confirmed by angiography, and a second blood sample was taken from the sheath. The sheath was then removed, the femoral artery was ligated, and the wound was closed. The surgical site was treated with an aluminum-containing polymer spray and a broad-spectrum antibiotic (procain-benzylpenicillin and benzathine-benzylpenicillin 5-5-mg/kg and dihydrostreptomycin 10 mg/kg) (Shotapen inj., Virbac; Carros, France), and a long-acting, non-steroidal anti-inflammatory and pain killer drug (0.4 mg/kg meloxicam) (Meloxidyl inj., Ceva; Marseille, France) was administered. The animals were allowed to recover under continuous monitoring and were extubated when the swallowing reflex returned. After extubation, the animals were placed in individual cages in a climate-controlled (23°C) surgical preparation room until full recovery.

Blood samples were obtained from the animals from the jugular vein at different stages of the myocardial infarction (0: before infarction-induction; 1: post-reperfusion; 2: 72 h; 3: day 30). The experimental design is shown in Figure 1. Under anesthesia, 20 mL of blood was drawn aseptically from the sheath in the jugular vein and equally distributed into blood collection tubes, one of them containing ethylenediaminetetraacetic acid (EDTA) and the other without any additives (serum tube). The baseline samples were collected immediately after the introducer implantation, ca. 15–30 min before the AMI induction. The follow-up samples were taken after the MR imaging procedure.

The control MRI was followed by surgical preparation as described for the AMI induction procedure, and 20 mL of blood samples were obtained again from the animals through a sheath. Blood samples were centrifuged for 10 min at 4,000 rpm, and the plasma or serum was transferred to storage tubes and stored at −20°C in quality-controlled refrigerators. The blood samples were tested at the veterinary laboratory Praxis Lab Ltd., and the following biochemical parameters were determined: AST, ALT, LDH, CK, AST/ALT ratio, and high-sensitivity troponin I. Indicators of sample quality (hemolysis, icterus, and lipemia) were evaluated. Measurement of AST, ALT, LDH, and CK was carried out on a Beckman Coulter AU480 autoanalyzer using dedicated reagent kits (Beckman Coulter, Brae, CA, United States). Troponin I was determined using a high-sensitive human electro-chemiluminescent immunoassay (Acces hsTnI; B52699) on a Beckman Coulter Access 2 analyzer. The used materials and reagents were validated for swine species.

Basic hematological and biochemical data were collected prior to the study, analyzing blood samples collected from 35 healthy pigs of the herd. The results were identical to the meat-type Landrace pig’s laboratory normal values.

The animals were included in the study with a successful LAD occlusion-reperfusion process and evaluable MR images at all three time points. Animals were excluded when were not suitable for the procedure or died before or during the catheterization.

To observe differences between the time points, linear mixed effect models were used. The individual ID of the animals was used as a random factor, and no other factors were used for adjusting. For comparison, marginal means were estimated using the restricted maximum likelihood (REML) fitting method. For p-value calculation, Satterthwaite’s method was applied. Due to the non-normal nature of the examined blood parameters, the data were logarithmized to perform the calculations. When the results are reported, the data are transformed back and reported as mean (M) and standard deviation (SD). Bonferroni–Holm post-hoc test was used to compare different time points to control the family-wise error rate (FWER). A result was considered significant if p < 0.05. All calculations were made using R statistical software¹ (14) and packages lme4 (15) and lmerTest (16).

The mean AST levels were within the reference range (10–45 U/L) at baseline (M = 16.89 U/L, SD = 15.61). The post-reperfusion (M = 27.38 U/L, SD = 19.56) and 72 h (M = 37.4 U/L, SD = 71.87) blood results did not differ statistically (p = 0.532, p = 0.884, respectively). The day 30 values were lower than the baseline (M = 8 U/L, SD = 7.66), and the difference was statistically significant (p = 0.011) (Figure 2).

ALT levels were within the physiological reference range values (5–40 U/L) in the baseline samples taken before AMI induction (M = 18.6 U/L SD = 9.54) and in the samples taken immediately following reperfusion (M = 19.3 U/L, SD = 10.99). The mean value increased not significantly with great individual variation (M = 36.6 U/L, SD = 54.52, p = 0.873) for the samples obtained at 72 h. The mean of the results obtained from blood samples collected on day 30 was statistically lower (p = 0.001) than the baseline values (M = 11.7 U/L, SD = 9.08) (Figure 3). In the AST/ALT ratio was a statistically significant (p = 0.005) difference between the baseline and day 30 values (Figure 4).

LDH mean serum levels were markedly increased in the 72-h samples (M = 2,334 U/L, SD = 2,970) (p < 0.001) compared to baseline (M = 601 U/L, SD = 208) and post-reperfusion (M = 768 U/L, SD = 264) and were above the reference range values (50–985 U/L) for porcine. LDH values dropped to a level significantly below the baseline values on day 30 (M = 420 U/L, SD = 210) (p = 0.013) (Figure 5).

The CK levels were above the reference range values already at the first blood sampling time point (M = 1,606 U/L, SD = 832). An increasing trend was observed for the post-reperfusion (M = 2,400 U/L, SD = 844, p = 0.389) and the 72-h (M = 2,349 U/L, SD = 2,300, p = 0.021) samples. The mean of the day 30 values (M = 374 U/L, SD = 201) was lower (p < 0.001) than the baseline values. The CK reference range for swine is 20–200 U/L (Figure 6).

The baseline troponin I mean value was 12.1 pg./mL. Troponin I mean values were significantly increased immediately following AMI (M = 237.2 pg./mL, SD = 354.7, p < 0.001) and increased manifold by the 72 h (M = 2,933 pg./mL, SD = 4,573, p < 0.001). By day 30, the troponin levels dropped to a normal level, to almost zero (M = 19.8 pg./mL, SD = 70.4, p = 0.426), and did not differ from the results of the baseline level (Figure 7). The detailed statistical analysis is demonstrated in the Supplementary material.