Study design

The cardioprotection provided by iPost was tested in 2 different sets of experiment groups: intermediate ischemia duration (30 min) followed by reperfusion, and prolonged ischemia (40 min) fol- lowed by reperfusion. All animals underwent a multiparametric CMR scan 1 week after MI. This timing regarding the assessment of the effect of the intervention was chosen since it is the recommended one by international expert consensus.⁵ Selecting between the intermediate or prolonged ischemia time was based on our previous experiments where we saw that ischemia durations of < 30 min lead to very small IS (< 20% of the area at risk [AAR]) yielding no window for cardioprotection of established pharmacological interventions (eg, metoprolol).¹⁵ Similarly, in our experimental setting, ischemia durations > 40 min result in very large IS (> 80% of the AAR) thus reducing the window for cardioprotection.¹⁵

Following the 3Rs principle to reduce the use of animals, those on prolonged ischemia (40 min) with/without iPost (as well as the CMR data) were the ones already used in a former article with a different objective.¹⁶ In this study, the group of animals undergoing intermediate ischemia duration (30 min) with/without iPost (n = 28) were used ad hoc.

Apart from ischemia duration, the study protocol was equal for both ischemia–duration groups. For these experiments, we employed 3-month-old castrated male large white pigs weighing 30 to 40 Kg. Pigs were allocated to iPost or control before MI was induced. Myocardial AAR was assessed through multidetector computed tomography immediately after vessel occlusion following a previously published methodology.¹⁶ Seven days after the MI, all animals underwent a multiparametric CMR scan to asses IS (primary outcome measure), LVEF, and T2 and T1 relaxation times both in the AAR and in the remote area (figure 1).

Figure 1. Central illustration. Study design and flowchart. CMR, cardiac magnetic resonance; CT, computed tomography. 

Anesthesia and animal care protocol

Every test or experiment was performed under deep sedation. Sedation was induced through the intramuscular injection of ketamine (20 mg/Kg), xylazine (2 mg/Kg), and midazolam (0.5 mg/Kg); and maintained through the continuous infusion of ketamine (2 mg/Kg/h), xylazine (0.2 mg/Kg/h), and midazolam (0.2 mg/Kg/h). Buprenorphine (0.03 mg/Kg) was administered immediately before the MI experiment.

Animals were intubated and received mechanical ventilatory support with volume-control synchronized intermittent mandatory ventilation (fraction of inspired oxygen = 28%).

To avoid coronary thrombosis following balloon-induced MI induction, animals received 150 mg of clopidogrel orally on the day of the procedure and 75 mg 24 and 48 hours later. All animals were euthanized immediately after the day-7 CMR scan.

Myocardial infarction and ischemic postconditioning protocol

All animals underwent the same closed-chest ischemia-reperfusion protocol¹⁶ consisting of 30-min or 40-minute left anterior descending coronary artery occlusion with a monorail angioplasty balloon inserted percutaneously through the femoral artery. Balloon was inflated at 8 atm immediately distal to the first diagonal branch. Both the location of the balloon and the status of inflation were monitored on the angiography. A single intra-arterial bolus of 300 IU/kg of unfractionated heparin was administered right before coronary occlusion. Furthermore, to reduce the rate of fatal ventricular arrhythmias, continuous infusion of amiodarone (300 mg/h, no bolus) was initiated immediately after coronary occlusion and maintained until catheters were removed.

Pigs were allocated on a 1:1 ratio to iPost or control before MI induction. After index ischemia duration (30 min or 40 min according to the protocol), animals allocated to control underwent straight chronic reperfusion (balloon deflation) while animals allocated to iPost underwent balloon deflation but 1 min after balloon was reinflated for 1 min. iPost was induced by repeating the 1 min inflation-1 min deflation cycle 4 times. Artery patency was assessed after every inflation/deflation cycle.

Arterial enhanced multidetector computed tomography protocol and analysis

Multidetector computed tomography (MDCT) arterial phase studies were performed during ongoing ischemia on a 64-slice computed tomography scanner (Brilliance CT 64; Philips Healthcare, Cleveland, OH, United States) after the IV administration of iodinated contrast media. Since the MDCT scan was performed during ongoing ischemia (ie, while the balloon was inflated), non-enhanced regions accurately represent the ischemic region (ie, the AAR). MDCT images were analyzed using dedicated software (Extended MR Workspace 2.6; Philips Healthcare, The Netherlands) by 2 observers who remained blind to group allocation. Short axes orientation was obtained from volumetric computed tomography images through multiplanar reconstruction. AAR and remote areas were visually identified based on contrast enhancement differences, manually delineated, and expressed as a percentage of left ventricular (LV) area.¹⁵,¹⁶

Cardiac magnetic resonance protocol

CMR scans were performed 7 days after the MI on a Philips 3-Tesla Achieva Tx whole-body scanner (Philips Healthcare, The Netherlands) equipped with a 32-element phased-array cardiac coil. The imaging protocol included a standard segmented cine imaging with a steady-state free-precession sequence to provide high-quality anatomic references and assessment of the left ventricular mass, wall thickness, and LVEF, a T1-mapping sequence (modified Look-Locker inversion recovery) to assess T1 native relaxation time, a T2 mapping based on gradient-spin-echo imaging to provide precise myocardial T2 relaxation time,¹⁷ and a T1-weighted inversion relaxation turbo field echo sequence acquired 10 min to 15 min after the administration of gadolinium contrast (late gadolinium enhancement, LGE) to assess IS. CMR images were analyzed using dedicated software (MR Extended Workspace 2.6; QMassMR 7.6; Medis, The Netherlands and IntelliSpace Portal, Philips Healthcare, The Netherlands) by 2 experienced observers in CMR analysis and blinded to group allocation.

Statistical analysis

Normal distribution of data was assessed using the Shapiro-Wilk test. Quantitative variables were expressed as median (interquartile range). Categorical variables were expressed as numbers and percentage and rounded to the nearest integer. A 2-way ANOVA test was run on the overall sample with CMR performed on day 7 (29 pigs) to examine the effect of ischemia time and iPost on primary and secondary outcomes (IS, % LV mass), IS indexed to the AAR, LVEF, T2 relaxation time, and native T1 relaxation time). Regarding variables where we found a significant interaction between the duration of ischemia and iPost, we performed a post hoc analysis (Tukey's method) to confirm the differences seen. We estimated the sample size based on our previous experiments on cardioprotection with metoprolol.¹⁵ Lost animals were replaced to achieve the required sample size.

Study groups

Baseline characteristics

Both control and iPost groups were similar in body weight and baseline CMR-based characteristics except for the indexed left ventricular mass that was larger in the control group of animals with 40 min ischemia times (table 1). A non-significant trend towards larger MDCT-based AAR (% left ventricle mass) was observed in the iPost group (table 1).

Cardiac magnetic resonance results

Effect of iPost in a model of intermediate ischemia protocol

In the intermediate-duration ischemia group, iPost did not have any effects on any of the CMR-based variables (table 2; figure 2, figure 3 and figure 4). Both iPost and control animals present small IS with no differences being reported between the intervention groups (0.3% of LV mass [0.0 – 3.9] vs 0.9% [0.0 – 2.6] %LV in iPost and control, respectively, P = .378). We did not find any differences regarding the indexed IS (IS/AAR) either (table 2).

Table 2. CMR-based parameters for intermediate ischemia group 

Variable	Intermediate ischemia duration (N = 19)
	Control (N = 9)	iPost (N = 10)	P (post hoc analysis)
IS, %LV mass	0.9 (0.0-2.6)	0.3 (0.0-3.9)	.378
Indexed IS, IS/AAR (%)	3.8 (0.0-8.5)	0.9 (0.0-15.1)	.474
LVEF, %	54.0 (50.2-55.9)	52.9 (47.1-56.0)	.521
iLVEDV, mL/m2	98.7 (92.3-104.1)	107.3 (90.0-118.6)	.438
iLVESV, mL/m2	45.9 (40.4-52.4)	48.5 (41.0-55.2)	.355
T2 relaxation time AAR, ms	51.3 (49.3-54.8)	57.0 (53.2-58.5)	.583
T2 relaxation time REM, ms	45.6 (42.2-46.1)	44.7 (43.7-47.4)	.881
Native T1 relaxation time AAR, ms	1179.2 (1167.1-1266.4)	1225.9 (1170.2-1306.2)	.584
Native T1 relaxation time REM, ms	1087.8 (1075.1-1109.7)	1078.4 (1051.2-1134.8)	.925

AAR, area at risk; iLVEDV, indexed left ventricular end-diastolic volume; iLVESV, indexed left ventricular end-systolic volume; iPost, ischemic postconditioning; IS, infarct size; LV, left ventricle; LVEF, left ventricular ejection fraction; REM, remote area.

Figure 2. Differences in cardiac magnetic resonance-based infarct size (% of left ventricular mass) between control and iPost groups, in the overall population and based on ischemia duration. 

Figure 3. Differences in cardiac magnetic resonance-based LVEF (%) between control and iPost groups in the overall population and based on ischemia duration. 

Figure 4. Differences in cardiac magnetic resonance native T1 and T2 relaxation times (ms) between control and iPost groups, in the overall population and based on ischemia duration. A: native T1 relaxation time at the area at risk. B: native T1 relaxation time at remote area. C: native T2 relaxation time at the area at risk. D: native T2 relaxation time at remote area. AAR, area at risk. 

Effect of iPost on a model of prolonged ischemia protocol

The results of this experiment have already been published.¹⁶ In conclusion, iPost did not show any cardioprotective effects in terms of IS reduction (31.1% of LV mass [27.3–32.8] LV vs 27.3% [25.1– 27.5] in iPost and control respectively; P = .248). Regarding the previous group, we did not find any differences in indexed IS (IS/AAR). Differences in other CMR-based parameters were not observed except for a significantly longer AAR T1-relaxation time in the iPost group (1590.3 ms [1441.6 – 1591.4] vs 1309.7 ms [1248.1–1310.8] in iPost and control, respectively; P = .002) (table 3 and figure 2, figure 3 and figure 4).

Table 3. CMR-based parameters for the prolonged ischemia group 

Variable	Long ischemia duration (N = 10)
	Control (N = 5)	iPost (N = 5)	P (post-hoc analysis)
IS, %LV mass	27.3 (25.1-27.5)	31.1 (27.3-32.8)	.248
Indexed IS, IS/AAR (%)	98.2 (70.7-98.8)	96.2 (93.4-100)	.640
LVEF, %	37.9 (37.4-38.0)	38.2 (30.2-39.4)	.323
iLVEDV, mL/m2	150.8 (150.4-153.1)	150.9 (148.3-159.8)	.644
iLVESV, mL/m2	95.1 (94.1-96.6)	105.4 (88.8-109.8)	.234
T2 relaxation time AAR, ms	85.0 (67.0-87.0)	69.8 (55.9-88.7)	.342
T2 relaxation time REM, ms	47.0 (47.0-49.0)	47.6 (46.2-49.6)	.420
Native T1 relaxation time AAR, ms	1309.7 (1248.1-1310.8)	1590.3 (1441.6-1591.4)	.002
Native T1 relaxation time REM, ms	1209.7 (1156.8-1217.3)	1246.8 (1191.2-1260.9)	.101

AAR, area at risk; iLVEDV, indexed left ventricular end-diastolic volume; iLVESV, indexed left ventricular end-systolic volume; iPost, ischemic postconditioning; IS, infarct size; LV, left ventricle; LVEF, left ventricular ejection fraction; REM, remote area.

Study limitations

This study has some limitations that must be acknowledged. Despite being one of the most translatable, the present pig model has some differences with human IRI: tolerance to ischemia is species-dependent, and duration of ischemia in pigs is not equivalent to humans; similarly, the time-dependent progression of irreversible injury is much faster in pigs compared to humans as seen by the transmural progression of infarction between 30 min and 40 min of ischemia. Another limitation of the study is that animal allocation to iPost or control was not entirely random, but rather based on an alternative assignment; however, the person responsible for the CMR analysis validation was blind to the subjects' allocation group. In addition, as previously presented, data on the prolonged ischemia group correspond to experiments previously conducted at our center by a different operator and published elsewhere. The use of animals of different breeds, different anesthesia protocols, different material or any other environmental factors could explain, at least partially, the great differences seen on IS between the intermediate and the prolonged ischemia groups. Nevertheless, the decision to use these previously reported data was based on the principle of reducing animal use in animal research.²⁶,²⁷