The animal experimental protocol in this study was approved by the Animal Care Committee of Air Force Medical University. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Research from US National Institutes of Health (National Institutes of Health Publication No. 8523, revised 1996). Male C57BL/6 mice aged 10~12 weeks and weighing 22~26 g were obtained from the Laboratory Animal Center of Air Force Medical University. All mice were housed at 20–25°C under a 12-h light/dark cycle and received a standard diet and water ad libitum.

MI/R injury mouse model was established according to the previous study (20). Briefly, mice were anesthetized with 1–2% isoflurane via an isoflurane vaporizer (Matrx, Orchard Park, NY, USA). A skin scar was cut in the left chest, and a tiny hole was made at the fourth intercostal space. Afterwards, the heart was smoothly squeezed out of the thoracic cavity. The left anterior descending (LAD) coronary artery was ligated by a 6-0 silk suture for 30 min, and then the slipknot was released. The reperfusion phase lasted for 2–4 h, and the heart samples were collected for protein expressions analysis. The cardiac function, myocardial infarct size, cell apoptosis, and lactate dehydrogenase (LDH) as well as creatine kinase-MB (CK-MB) were assessed following a 24-h reperfusion. The same procedure except ligation of LAD was performed in the mice of sham group.

EA pretreatment was conducted by using the Hwato Electronic Acupuncture Treatment Instrument (Suzhou Medical Appliances, Suzhou, China). Briefly, mice were anesthetized and maintained by inhalation of 1–2% isoflurane via an isoflurane vaporizer. The needles connected to the electrodes were inserted into 2- to 3-mm depth of muscle layers at the Neiguan acupoint (PC6) of both forelimbs, which are located between the palmar tendon and flexor carpi ulnaris (21). Mice were stimulated at the density of 1 mA with a frequency of 2/15 Hz for 30 min once a day for 3 days. The MI/R surgery or sham surgery was performed within the 30 min after the last EA treatment. Mice from Sham and Sham+EA groups were anesthetized for 30 min to avoid the effects of isoflurane between different groups.

The mice were divided into the following four groups with 12–15 mice for each: sham group (Sham), sham group with EA pretreatment (Sham+EA), MI/R injury group (MI/R), and MI/R group with EA pretreatment (MI/R+EA). Mice in the Sham+EA or MI/R+EA groups received EA preconditioning for 3 consecutive days followed by sham or MI/R surgery, while mice in the Sham or MI/R group underwent the sham or MI/R surgery, respectively.

Mice were anesthetized and maintained by inhalation of 1–2% isoflurane after 24-h reperfusion. Cardiac function was evaluated by Doppler echocardiography with a 15-MHz linear transducer (Visual Sonic Vevo 2100, Toronto, ON, Canada). Mice were placed on a heating pad to maintain the body temperature during the whole procedure. M-mode echocardiography was recorded and used to assess cardiac function. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were obtained by using Vevo LAB 3.1.0 software.

The serum was obtained by centrifugation of mouse blood at 3,000 rpm for 10 min after 24-h reperfusion and used for LDH and CK-MB determination. The assay was conducted according to the manufacturer's instruction (Jiancheng Bioengineering, Nanjing, China). The activities of LDH and CK-MB were calculated based on the methods described in the manufacturer's instruction.

The mice were anesthetized with 1–2% isoflurane, and the LAD was occluded at the same position following 24-h reperfusion. The 3% Evans blue solution was injected into the hearts via the aorta to stain the non-ischemic area of the heart. The whole hearts were then collected and frozen on dry ice for 10 min and cut into four slices transversally from the bottom of the hearts. The slices were stained in 1.5% triphenyltetrazolium chloride (TTC) in phosphate solution (pH 7.4) and incubated at 37°C for 20 min. Then the slices were fixed in 4% paraformaldehyde for 12 h, and the images were obtained by using a digital camera. The infarct size was calculated by the ratio of white area to white and red areas. The size was determined by using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).

Myocardial apoptosis and cell apoptosis were determined by an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously described (22). In brief, at the end of the experiment, the myocardial tissues and H9c2 cells were fixed in 4% paraformaldehyde for at least 24 h. After the paraffin-imbedded sections were prepared, the manufacturer's instruction for TUNEL staining was followed. The apoptotic myocardial cells and H9c2 cells were stained with TUNEL staining solution, and nuclei were visualized by DAPI staining. Then the images were obtained with an Olympus FV10i microscope (Olympus, Tokyo, Japan); and an apoptotic rate was presented as the count of TUNEL-positive cardiomyocytes to the total number of cells.

H9c2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; HyClone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO₂ air incubator. The experiments included four groups: (1) the cells in the CON group were cultured in the serum-free DMEM. (2) H9c2 cells in stimulated I/R (SI/R) group were cultured in serum-free DMEM for 12 h and then subjected to the ischemic buffer (10 mM of deoxyglucose, 137 mM of NaCl, 12 mM of KCl, 0.49 mM of MgCl₂, 0.9 mM of CaCl₂·2H₂O, 0.75 mM of sodium dithionate, 20 mM of lactate, and 4 mM of Hepes, pH 6.5) for 2 h. The cells were then cultured in normal DMEM at 37°C in an incubator (5% CO₂, 95% air) for 4 h to establish in vitro SI/R model. (3) The cells in XI group (XBP1 inhibitor, 4μ8C) were cultured in the serum-free DMEM and then incubated with 5 μM of 4μ8C for 2 h. (4) The cells in SI/R+XI group were incubated 5 μM of 4μ8C for 2 h and then subjected to ischemic medium for 2 h. Then the cells were cultured in normal DMEM at 37°C in an incubator (5% CO₂, 95% air) for 4 h. The supernatant of cell culture was collected for LDH and CK-MB activities measurements following the protocol described above.

The proteins were isolated from the heart left ventricles including the infarct zone and broader zone and H9c2 cells for western blotting detection. The proteins were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred on to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA); subsequently, the membrane is incubated with 5% non-fat milk in TBST. Western blotting was then performed with antibodies against XBP1 (Cat. 83418, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), GRP78 (Cat. 3183, 1:1,000; Cell Signaling Technology), p-Akt (Cat. 4060, 1:1,000; Cell Signaling Technology), Akt (Cat. 4691, 1:1,000; Cell Signaling Technology), Bcl-2 (Cat. ab196495, 1:1,000; Abcam, Cambridge, UK), Bax (Cat. 2772, 1:1,000; Cell Signaling Technology), cleaved Caspase 3 (Cat. 9664, 1:1,000; Cell Signaling Technology), and GAPDH (Cat. AT0002, CMC TAG, 1:5,000). After that, the proteins were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized by a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Then the relative quantification of proteins was presented as the ratio of target proteins to GAPDH.

All data were presented as mean ± SD. Data were analyzed with the GraphPad Prism Software version 7.0 (GraphPad Software, San Diego, CA, USA). Normality analysis of data was performed by the Shapiro–Wilk test. Statistical significance (P < 0.05) was estimated by one-way ANOVA followed by Bonferroni correction for post-hoc t-test.

To evaluate the effects of EA on cardiac function following MI/R injury and EA treatment, echocardiography was performed. M-mode images were obtained to measure LVEF and LVFS so as to evaluate cardiac contractile function. As shown in Figure 1, MI/R significantly reduced LVEF and LVFS compared with sham group, while EA pretreatment for 3 consecutive days greatly increased LVEF and LVFS compared with MI/R group. These data demonstrated that MI/R resulted in compromised cardiac function, while EA pretreatment improved cardiac function, which was impaired by MI/R injury. However, cardiac function in EA+sham group was not significantly altered compared with sham group, implying no obvious effects on cardiac function by EA pretreatment in sham-operated mice. These results data together revealed that EA pretreatment elevated MI/R-induced reduction of LVEF and LVFS, thus protecting hearts from MI/R injury.

To further investigate whether EA pretreatment could affect MI/R-induced myocardial infarct size and myocardial cell death, TTC/Evans blue double staining was used to assess infarct size; and LDH and CK-MB activities were determined to evaluate myocardial cell death. Our results showed that MI/R led to a significant increase of myocardial infarct size compared with sham group, while EA pretreatment dramatically reduced myocardial infarct size compared with MI/R group as shown in Figure 2. The above result clearly showed that EA pretreatment significantly reduced MI/R-induced myocardial infarct size. Furthermore, the activities of LDH and CK-MB in the serum were increased in response to MI/R injury compared with sham group, while EA pretreatment decreased the activities of LDH and CK-MB in EA+MI/R group compared with MI/R group as shown in Figure 2. These data revealed that myocardial cell death caused by MI/R injury was inhibited following EA pretreatment. Taken together, these results indicated that EA pretreatment decreased myocardial infarct size and LDH and CK-MB release to protect hearts from MI/R injury.

To elucidate the role of cell apoptosis in EA protection against MI/R injury, cell apoptosis among different groups was determined. As shown in Figure 3, MI/R significantly enhanced myocardial apoptosis compared with sham group. Moreover, EA pretreatment decreased MI/R-induced cell apoptosis by 27.6% compared with MI/R group, implying the protective role of EA on reducing cell apoptosis during MI/R injury.

To further investigate the potential molecular mechanisms underlying EA protection against MI/R injury, the expressions of XBP1/GRP78/Akt signaling pathway and apoptotic proteins were assessed. As shown in Figure 4, our data demonstrated that MI/R injury resulted in increase of XBP1, GRP78, and phosphorylated Akt (p-Akt) expressions, while the pro-apoptotic proteins including Bax and cleaved Caspase 3 were upregulated compared with sham group. The results showed that the expressions of XBP1, GRP78, and Akt were further increased, and the expressions of Bax and cleaved Caspase 3 were decreased following EA pretreatment. These results indicated that the molecular mechanism of EA protection against MI/R injury was at least partly via activation of XBP1/GRP78/Akt signaling pathway, therefore ultimately reducing cell apoptosis.

To further investigate the role of XBP1-mediated signaling pathway in SI/R-induced cell injury, the inhibitor 4μ8C was used in in vitro study. The results in Figure 5 revealed that XBP1 inhibitor 4μ8C significantly exacerbated SI/R-induced H9c2 cell injury manifested by the increase of LDH and CK-MB activities and the elevation of cell apoptosis compared with the SI/R group. However, 4μ8C alone did not significantly affect the activities of LDH and CK-MB or cell apoptosis in normal conditions. These results showed that XBP1 inhibition could deteriorate SI/R-induced cell injury via increase of cell death.

To demonstrate the effects of XBP1 inhibition on GRP78/Akt signaling pathway, we further explored the effects of 4μ8C on the XBP1/GRP78/Akt pathway. The western blotting in Figure 6 demonstrated that the XBP1 inhibitor 4μ8C downregulated XBP1, GRP78, Akt, and Bcl-2 expressions and elevated the expressions of Bax and cleaved Caspase 3 compared with MI/R group. Our in vitro results favored the notion that the inhibition of XBP1 could worsen SI/R-induced cell injury via regulation of GRP78/Akt pathway.