Male SPF grade Sprague-Dwaly (SD) rats (weighing 250-300g) were purchased from the Department of Animal Resources of Tianjin Medical University. The experimental animals were kept under standard room conditions with a temperature of 25±1°C, the humidity of 60%, and natural light from 6 a.m. to 6 p.m. Standard rodent chow and water were given routinely. The Committee on Ethics of Biomedicine of Tianjin Third Central Hospital approved this study, which also conformed to the revised Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH) Publication Nos. 85-23 (1996).
A total of 60 rats were randomly divided into three groups, including the Sham group, the I/R group, and the I/R+Oprm1 group. The Sham group and the I/R group were administered with the intragastrical injection of saline, while the I/R+Oprm1 group was given a 30μl injection of recombinant AAV9-Oprm1 around the infarction region. The rats of the I/R group and the I/R+Oprm1 group was given the I/R surgical protocol, while the Sham group was given the same procedure except for the ligation.
2.2 Myocardial Ischemia/Reperfusion Model
Surgical protocols were conducted as previously described. After opening the chest and showing the heart via an incision in the fourth intercostal space under anesthesia, the left anterior descending coronary artery was temporarily ligated for 30 minutes with a 6–0 suture. Then the ligation was released, and the heart was checked for blood flow. Sham operation was conducted with similar without the ligature.
2.3 Infarct size assessment
As previously described, the infarct size of the heart was assessed with the TTC-staining technique and digital measurement with Image-Pro Plus software (Media Cybernetics).
As previously described, echocardiography was performed blindly four days after the surgery (VisualSonics Vevo 2100, M-mode, and 30MHz probe). The rats were under consciousness and examined at the mid-papillary level. LV dimensions in diastole and systole, especially the ejection fraction (EF) was measured with standard procedures and calculations.
2.5 Microassay analysis
We applied Mouse LncRNA Expression Array V3.0 chip (Aksomics, Shanghai, China) to examine differentially expressed lncRNA after MIRI. Total lncRNAs were extracted for chip hybridization. Fluorescence intensity values represented the lncRNA expression levels. The differential expression criteria were determined as fold change >2, P < 0.05.
2.6 Histology and TUNEL staining
Hematoxylin and eosin (HE) and TUNEL staining were conducted as previously described. Image-Pro Plus software (Media Cybernetics) was used to detect the apoptosis rates of TUNEL sections.
2.7 CSE activity and H2S concentration
As previously described, CSE activity was measured with an ELISA kit (ml037623, Shanghai Enzyme-linked Biotechnology Co., Ltd.). H2S concentration was measured with the endogenous hydrogen sulfide test kit (Nanjing Xinfan Biotechnology Co., Ltd.).
2.8 Luciferase reporter assay
As previously described, 293T cells were seeded in a 24‐well plate. After 24 hours, miR-30b-5p mimics or miR‐NC was cotransfected with Oprm1‐wt (wild type) or Oprm1‐mut (mutant) into 293T cells using Lipofectamine 3000 (Invitrogen), dual‐luciferase reporter assay system (Promega) were applied at 48 hours after transfection according to the manufacturer's instructions.
2.9 Cell culture and H/R protocol
H9c2 cardiomyocytes were purchased from Shanghai Gefen Biotechnology. The cell was cultured in DMEM supplemented with 10% FBS with standard protocols. The H/R procedure was conducted as follows: the culture plates were placed in the hypoxia chamber (1% O2, 5% CO2, and 94% N2) for 24 h, following by reoxygenation (5% CO2 and 95% air) with maintenance medium for 6 h.
2.10 Gene transfection
As previously described, lncRNA Oprm1, miR-30b-5p mimics were synthesized by Shanghai GenePharma (Shanghai, China). H9c2 cardiomyocytes were transfected with Oprm1 or miR-30b-5p mimics when cell confluence reached 70%. After transfection for 48 h, the cells were exposed to the H/R procedure.
2.11 Cell viability
The MTT assay was used to test the cardiomyocyte viability. The absorbance of the solution was determined at 570 nm at 0, 3, 6, 9, 12, 18, and 24 h after reoxygenation.
2.12 Apoptosis detection in vitro
According to the manufacturer’s instructions, flow cytometry (FACS Calibur™, BD Biosciences, CA, USA) was adopted to measure the percentage of early apoptosis of four groups after H/R procedure with the Annexin V-FITC/PI apoptosis kit (Invitrogen).
2.13 Key protein activity
According to the manufacturer’s instructions (RapidBio Lab, USA), the ROS and LDH activity was measured in the cardiac homogenates and cell supernatant. According to the manufacturer’s instructions (BioVision, USA), the Caspase-3, -8, and -9 Fluorometric Assay Kits were used to determine the enzyme’s activity of the cardiac homogenates and cell supernatant. According to the manufacturer’s instructions (Sigma-Aldrich, USA), the antioxidant enzyme of the SOD Assay kit was applied to measure activities in heart homogenates and cell supernatant.
Total RNA was extracted using TRIzol (Takara). 1000 ng total RNA was reversely transcribed into cDNA using the Prime-Script TMRT reagent kit (Takara). qRT-PCR was performed using SYBR green (Takara) and normalized to GAPDH expression. LncRNA Oprm1, miR-30b-5p, CSE were examined.
2.15 Western blotting
Western blotting was conducted as previously described. Primary antibodies were purchased from Abcam: CSE, ab96755; p-Akt 1/2/3, ab8805; Akt 1/2/3, ab179463; HIF-1α, ab51608; Bnip3 ab219609; Bcl-2, ab185002; Bax: ab-32503; Bcl-xl, ab32370; Caspase-3, ab197202; Caspase-9, ab52298; and β-actin, ab8226. Image lab software (BIO-RAD, USA) was used to quantify the grayscale value of the bands.
2.16 Statistical analysis
We used GraphPad Prism 7.0 to analyze the data. The continuous data were expressed as the mean ± standard deviation (SD). One-way ANOVA followed by Post-hoc t Turkey’s test was adopted to compare the variables of different groups. A P-value of less than 0.05 was considered as statistical significance.