The details of the breeding and maintenance of animals used in the present study were previously reported [17]. Most EP₄^–/– mice die postnatally as a result of patent ductus arteriosus or do not survive in the C57BL/6 background. Therefore, F2 progenies of surviving EP₄^–/– mice and their wild-type litter mates were independently maintained in a mixed genetic background of 129/Ola and C57BL/6 [18]. All experiments were performed using 7–12-week-old male mice per the guidelines of Japan’s Act on Welfare and Management of Animals and were approved by the Asahikawa Medical University Committee on Animal Research.

The study mice were anesthetized with pentobarbital (60 mg/kg body weight, intraperitoneally) and secured in a supine position with the upper and lower extremities held on a heated table under a two-lead electrocardiogram (ECG) monitoring device. Tracheal intubation with a blunt 20-gauge polyethylene tube (Terumo, Tokyo, Japan) was performed under direct visualization of the tube through the tracheal wall at the upper border of the thyroid cartilage, which was surgically exposed. The tracheal tube was connected to a mechanical ventilator (SN-480-7; Shinano, Tokyo, Japan) and the mice were ventilated using a volume-controlled ventilation mode (0.8 ml room air/breath at 110 breaths/min). After the left anterior thoracotomy, the heart was exposed and the pericardium dissected; the dissection table was tilted up and to the left to visually identify the left coronary artery. An 8–0 nylon suture was passed underneath the left anterior descending coronary artery (LAD) at a position 1 mm from the tip of the left auricle. A small length of polyethylene tube with a blunt edge (size 3; Hibiki, Tokyo, Japan) was threaded by two lines of suture and mounted vertically on the LAD, with a piece of rubber (1 g) attached to each end of the suture. The LAD was occluded by a suture supporting rubber weights and was reopened by manually releasing the weight loading. To confirm that the LAD was successfully occluded, the myocardium was checked for color change (from brick red to pale). In addition, ECG observations showed prolonged QRS duration, enlarged QRS voltage, and ST elevation after successful occlusion [19]. On Day 1, mice in the IPC group underwent the IPC treatment, consisting of four 5-min cycles of occlusion followed by 5 min of reperfusion of the LAD. Mice in the IPC sham group underwent the same surgery, apart from IPC treatment. On Day 2, 24 h after IPC treatment, the mice were subjected to 30 min of LAD occlusion followed by 2 h of reperfusion. To examine the effects of the EP₄ agonist, AE1-329 [20], I/R injury was induced by occluding the LAD for 30 min, without IPC treatment, followed by indicated times of reperfusion. AE1-329 (30 μg/kg) was injected subcutaneously 30 min before LAD occlusion.

The hearts of wild-type mice were excised 6 h after IPC or sham treatment and total RNA was prepared from ischemic and nonischemic areas using Isogen (Nippon Gene, Toyama, Japan). Total RNA (2 μg) was reverse-transcribed, as previously reported (10). The resulting cDNA was amplified by polymerase chain reaction (PCR) using primer sets corresponding to COX-2 mRNA, as follows:

sense 5′-ACACTCTATCACTGGCACCC-3′

antisense 5′-GGACGAGGTTTTTCCAC-CAG-3′

The quantity of PCR product was determined by real-time PCR analysis using Lightcycler apparatus (Idaho Technology, Idaho Falls, ID, USA) and DNA Master SYBR Green I (Roche Molecular Biochemicals, Mannheim, Germany) as previously described [20]. The values for the ischemic areas were expressed in relation to the nonischemic areas.

Hearts were excised immediately or 24 h after IPC treatment. Tissue samples were prepared from ischemic (anterior left ventricular wall) and nonischemic (posterior left ventricular wall) areas by homogenization in 0.1 M phosphate buffer containing 1 mM EDTA and 10 μM indomethacin. Prostaglandins were pre-extracted from tissue samples using silica-based octadecylsilane reverse-phase columns. The levels of PGE₂ in the samples were determined using an enzyme-linked immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA), according to the manufacturer’s instructions.

After 30 min of LAD occlusion (with or without IPC treatment) and following reperfusion of 2 h, the size of the AAR and the infarct size were determined using a double-staining technique [21]. Briefly, the right carotid artery was exposed via blunt dissection of the paratracheal muscles, and then cannulated with a catheter. AAR was determined by retrograde injection of 5.0% Evans Blue dye (300 μl) through the catheter while the LAD was occluded. By this procedure, all cardiac tissue except the AAR was stained blue. After the heart was excised and washed in ice-cold phosphate-buffered saline (PBS), it was frozen at − 80 °C for 5 min and cut into five slices. The slices were incubated with 1.0% triphenyltetrazolium chloride (TTC) at 37 °C for 5 min, followed by overnight immersion in PBS at 4 °C. Thus, the infarcted area was demarcated as a pale-to-white area, while viable tissue was stained red. AAR and infarct size were determined via planimetry using Photoshop 7.0 (Adobe, San Jose, CA, USA). The infarct size was calculated as ratio of the infarct (pale-to-white area) to AAR (all cardiac tissue except blue areas) since the perfusion territory of LAD was different in each mouse [21].

Echocardiography was performed before the I/R procedure, and after 2 h of reperfusion following 30 min of LAD occlusion, using a Vevo 660 machine (Primetech; VisualSonics, Toronto, Canada) with a 35-MHz probe. First, a B-mode image of the left ventricle (LV) was obtained in the short-axis view at the level of the papillary muscles. Then, end-diastolic and -systolic LV dimensions were measured from the M-mode tracings. LV ejection fraction (LVEF) was calculated using the equation: LVEF = (LV diastolic volume − LV systolic volume)/LV diastolic volume) × 100, where LV diastolic and systolic volumes indicate left ventricular diastolic and systolic volumes, respectively.

Hearts were excised after 15 min of reperfusion following 30 min of LAD occlusion without IPC treatment. The ischemic area was harvested, frozen in liquid nitrogen, and stored at − 80 °C until use. For detection of Ser473-phospholylated Akt (p-Akt) and Akt, the samples were homogenized in lysis buffer (20 mM Tris, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 2 mM Na₃VO₄, 2 mM NaF, 10 mM sodium pyrophosphate, protease inhibitor cocktail; pH 7.5). After centrifugation for 10 min at 12,000 rpm, protein concentrations in the supernatant were determined using the bicinchoninic acid (BCA) Protein Assay Kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA). The sample (40 μg protein) was electrophoresed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride transfer membrane (Millipore, Billerica, MA, USA) using a semidry transfer system (ATTO, Tokyo, Japan). After a blocking procedure using 5% nonfat dried milk for 1 h at room temperature, the membranes were incubated with the antibodies against p-Akt or Akt (×1000; Cell Signaling Technology) at 4 °C overnight. P-Akt and Akt were detected using horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA) and an enhanced chemiluminescence reagent. Densitometry of the bands was analyzed using Photoshop as previously described [20].

All data are expressed as mean ± standard error. The data were analyzed using the Student’s t test for unpaired samples. P values of < 0.05 were considered statistically significant.

The expression level of COX-2 mRNA in the heart of sham-operated wild-type mice was low and barely detectable. However, after IPC treatment, the expression level of COX-2 mRNA in the ischemic area increased remarkably at 6 h (Fig. 1), which indicates that the production of cardioprotective prostanoids (such as PGE₂) increases in the heart subjected to IPC treatment. Immediately after the IPC treatment, PGE₂ levels in the ischemic area were similar to those of the nonischemic control areas (Fig. 2), which indicated that there was no increase in PGE₂ production at that time point. However, the PGE₂ level in the ischemic area increased significantly at 24 h after IPC treatment, compared with that of the nonischemic control area, in both wild-type and EP₄^–/– hearts to a similar degree (Fig. 2), indicating that PGE₂ may play a role in the late phase of IPC. PGE₂ levels in the nonischemic areas at 24 h after IPC treatment did not differ significantly between wild-type and EP₄^–/– hearts (5.24 ± 0.73 pg/mg [n = 9] and 4.10 ± 0.38 pg/mg [n = 6], respectively).

To determine the role of PGE₂ via EP₄ in late-phase IPC, we performed the I/R procedure after IPC treatment in wild-type and EP₄^–/– mice (Fig. 3A). In wild-type mice, the infarct size of the heart after IPC treatment was significantly smaller than in sham-operated mice (Fig. 3B, C), indicating that the late phase of IPC works effectively in wild-type mice. In contrast, there was no significant difference in the infarct size between the IPC-treated and sham-operated groups in EP₄^–/– mice, indicating that the late phase of IPC disappeared completely in EP₄^–/– mice. In sham-operated groups, there were no significant differences in both infarct size and AAR between wild-type and EP₄^–/– mice (Fig. 3C). These results clearly show that the PGE₂/EP₄ system plays a critical role in the late phase of IPC.

To clarify whether insufficient production of PGE₂ in the heart was responsible for the lack of significant difference in infarct sizes between sham-operated wild-type and EP₄^–/– hearts, we used AE1-329 to activate EP₄ in wild-type hearts that did not receive IPC treatment. AE1-329 (30 mg/kg) was injected 30 min before the 30-min LAD occlusion and the infarct size was determined after 2 h of reperfusion. AE1-329 significantly reduced the infarct size in wild-type mice (Fig. 4), whereas no such effect was observed in EP₄^–/– mice (data not shown). In contrast to the efficient activation of EP₄ and the resultant reduction in the infarct size in the heart with the IPC treatment (Fig. 3C), this result indicates that the endogenous production of PGE₂ in hearts without IPC treatment was insufficient to activate EP₄, at least during the 2 h of the I/R procedure. AE1-329 did not significantly affect blood pressure (data not shown).

We used echocardiography to examine the effects of AE1-329 pretreatment on the function of wild-type hearts at 2 h of reperfusion following 30 min of LAD occlusion. In the hearts pretreated with AE1-329, movement of the anterior wall was well retained, compared with that of control hearts (Fig. 4C). Additionally, AE1-329 significantly prevented a reduction in the LVEF after the I/R procedure (Fig. 4D), which indicated that the activation of EP₄ protected the heart from I/R injury, both histologically and functionally.

To further investigate the cardioprotective mechanism of the PGE₂/EP₄ system, we examined the activation of Akt, a pro-survival serine/threonine kinase. We measured the levels of phosphorylated Akt (p-Akt), an activated form of Akt. Although AE1-329 alone did not alter the level of p-Akt, it significantly enhanced the I/R-induced increase in p-Akt level in wild-type hearts (Fig. 5), the enhancement of Akt activation by AE1-329 was not observed in EP₄^–/– hearts (Fig. 5). Both the molecular weight of p-Akt and Akt is 60 kD, suggesting that the upper bands in p-Akt were non-specific. The levels of total Akt were not different between wild-type and EP₄^–/– hearts, irrespective of the presence or absence of AE1-329. These results suggested that Akt signaling underlies the cardioprotective mechanism of the PGE₂/EP₄ system in I/R. Meanwhile, I/R treatment increased p-Akt/Akt ratio in EP4^−/− mice, suggesting that Akt activation might be increased via pathways other than PGE₂/EP₄ system.