Preparation of isolated rat heart models
Sixteen healthy male Sprague-Dawley (SD) rats of 2 to 3 months old, weighing 300 to 400 g at clean grade were provided by the Experimental Animal Center, Guizhou Medical University (Guiyang, Guizhou Province, China). Heparin (3%, batch number: 51606118, Jiangsu Wanbang Biochemical Medicine Co., Ltd) of 500 Iu/kg was intraperitoneally injected for anticoagulation. After administration for 10 min, 40 mg/kg of pentobarbital was intraperitoneally injected for anesthesia. The chest of the rats was opened (immediately after the anesthesia took effect) to remove the heart, which was then placed in the Krebs-Henseleit (K-H) solution at 4 °C before trimming and exposing the aorta. The aorta was fixed on Langendroff perfusion equipment (Shanghai Alcott Biotech Co., Ltd) to conduct acyclic retrograde perfusion at constant temperature (37 °C) and constant pressure (8.65 kPa) with a K-H solution saturated with 95% O2 and 5% CO2. The model for Langendroff perfusion of isolated rat hearts was regarded as having been successfully prepared if the heart rhythm (HR) was recovered within 3 min after balance perfusion and HR was greater than 180 times/min at the end of balance perfusion (Krebs-Henseleit (K-H) buffer solutions included: NaCl 118 mmol/L, MgSO4 1.2 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L, and NaHCO3 25 mmol/L, glucose 11 mmol/L, and HEPES 10 mmol/L, pH 7.35, adjusted with NaOH).
Experimental treatments
Sixteen prepared models for Langendroff perfusion of isolated rat hearts were divided randomly into two groups (n = 8 in each group): a control group (Group C) and an ischemia reperfusion (IR) group. The former was subjected to continuous perfusion for 120 min with K–H solution at 37 °C. As for the latter, after balance perfusion with K–H solution at 37 °C for 30 min, St. Thomas solution (St. Thomas’ Hospital cardioplegic solution, 20 ml/kg) at 4 °C was injected at the root of the aorta to allow 60 min of cardiac arrest. The periphery of the heart was protected with K–H solution at 4 °C. Then, St. Thomas solution (10 ml/kg) at 4 °C was perfused again after cardiac arrest had lasted for 30 min and K–H solution at 37 °C was re-perfused for 30 min after 60 min of cardiac arrest (St. Thomas’ solution included: NaCl 110 mmol/L, CaCl 1.2 mmol/L, MgCl2 16 mmol/L, KCl 16 mmol/L, and NaHCO3 10 mmol/L at pH 7.6 to 7.8. This reagent comes from the Department of Anesthesiology, Guizhou Medical University, China).
Evaluation of arrhythmia
The method used to measure the ECG was as previously described [13]. The electrode wires were connected to the signal input wires of the BL-420F biological function experiment system (Chengdu Taimeng Software Co., Ltd) respectively. The ECG was amplified and analyzed using a BL-420 biological function system. Bipolar electrograms between the atrial and ventricular electrodes were used to monitor arrhythmias [14].
The severity of arrhythmia during reperfusion that occurred in each individual heart was assessed via the Curtis and Walker scoring system as well as the Lepran scoring system [15]. A discrete and identifiable premature QRS complex was diagnosed as premature ventricular contractions (PVCs). A run of four or more PVCs was identified as VT. VF was defined as a ventricular rhythm without a recognizable QRS complex and thus an immeasurable HR.
No arrhythmia was observed in two IR rats (score 0) during reperfusion, but different types of arrhythmia can be observed in the other six rats of the IR group. One of the six rats was found with ventricular premature beats (91 PVC, score 2) and returned to normal rhythm after 1.63 min. The atrioventricular block was observed in three of the six rats and this lasted for 1.25 min (score 3), 11.7 min (score 5), and 21.83 min (score 5) respectively before returning to normal rhythm. Ventricular fibrillation accompanied by premature ventricular beats could be observed in the two remaining rats and this was maintained for 11.5 min (score 5) and 5.42 min (score 5) respectively before normal rhythm was restored. According to the two arrhythmia scoring systems, four IR rats with arrhythmia score > 3 were placed in a high risk group (IR-H), and the other IR rats with arrhythmia score ≤ 3 were placed in a low risk group (IR-L).
Experimental grouping
Rat hearts with scores higher, or not higher, than 3 were graded as having a high risk of IR (IR-H) or low risk of IR (IR-L), IR-H group+ IR-L was collectively called the ischemia-reperfusion (RA) group. Then, the hearts were divided into three groups based on the degree of arrhythmia: a Group C, Group IR-L, and Group IR-H, each containing specimens of ventricular myocardium of four rats. Myocardium of the left ventricle was removed immediately after perfusion and the hearts were frozen and transferred to a freezer to be stored at − 80 °C.
High-throughput sequencing and analysis of aberrant expression of miRNAs
The Trizol method was employed to extract the total RNA of the specimens and the RNA was treated with DNase I to eliminate DNA pollution. All these procedures followed published instructions. Using a micro-nucleic acid protein tester (Agilent Company), formaldehyde denaturing gradient gel electrophoresis (DGGE), and capillary electrophoresis, the extracted total RNA was tested to ensure that its concentration and completeness reached the requirements for sequencing. The Beijing Genomics Institute was entrusted to perform high-throughput sequencing for the aforementioned samples on the BGISEQ-500 platform. The miRNA expression of the samples was standardized as transcripts per million (TPM). For given transcripts, the gene expression was estimated by aligning the number of fragments in a gene region. The DEGseq [16,17,18] method was used to reveal changes in the expression of the gene transcripts of the three groups of samples. After obtaining the value of P, multiple hypothesis tests were conducted and the corrected P was represented by false discovery rate (FDR), satisfying FDR < 0.05. If the fold change in expression was equal to or greater than 2, the gene was deemed to have been aberrantly expressed.
Prediction of target genes of aberrantly expressed miRNAs
RNAhybrid and miRanda [17] databases were used to predict the target genes of aberrantly expressed miRNAs and those genes predicted mapped to each term in the GO database to count the number of genes that were mapped to each term [19]. Then, the P-value was corrected by using the Bonferroni method, a corrected P-value ≤ 0.05 was taken as a threshold. GO terms fulfilling this condition were defined as significantly enriched GO terms. Then, by using KEGG database (http://www.genome.jp/kegg/), target genes of aberrantly expressed miRNAs were mapped and the same calculation method was used to obtain the KEGG analysis results. Finally, the mRNA–miRNA interaction network graph was established.
Analysis of genes associated with the progression of cardiovascular diseases
All of the target mRNAs of differently regulated miRNAs were screened using RNAhybrid and miRanda. Then, the mRNA–miRNA process was analyzed to build networks of the physiological system and pathophysiology. Based on GO and KEGG cardiac function databases, the mRNAs that participated in pathophysiological process of cardiovascular diseases (CVDs), including hypertrophy, fibrosis, conduction abnormality, and arrhythmia, were selected.
Screening of the target miRNAs
Based on gene function analysis, KEGG pathway analysis, and mRNA–miRNA interaction, bioinformatics analysis was conducted on transcripts of miRNAs related to KCNJ2/GIA1 genes. The target miRNAs regulating expressions of KCNJ2 or GJA1 in the hypothermia ischemic–RA were preliminarily screened.
Reverse transcription-quantitative PCR
RT-qPCR verification was performed as described elsewhere [20]. The left ventricular myocardial tissue (apex of the left ventricle) was used to extract total RNA with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. We performed qPCR on four miRNAs with higher expression level (rno-miR-122-5p, novel_miR-17, rno-miR-429, and novel_miR-19), which were closely related to the biological processes of arrhythmias. The RNAs were reverse-transcribed to cDNA with stem-loop-like RT primers (Biofavor, Wuhan, China) that are specific for only the mature microRNA species and then quantified on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using SYBR-Green PCR Master Mix (Vazyme, Nanjing, China). The PCR reaction was carried out at 95 °C for 5 min, followed by incubation at 95 °C for 15 s, 65 °C for 15 s, and 72 °C for 32 s (repeated for 40 cycles). Each PCR was repeated at least three times. The relative expression level of each microRNA was normalized against RNU6B expression levels. The fold-change was calculated according to the 2-∆∆CT method [21].
Statistical analysis
We conducted hierarchical clustering for differentially expressed miRNAs using pheatmap, a function of R. For clustering more than two groups, we performed the intersection and union DESs between them [15]. Based on “GO Term Finder” (http://www.yeastgenome.org/goTermFinder), significantly enriched GO terms with corrected p-value ≤ 0.05 were screened. The differentially expressed transcripts were aligned to the KEGG pathway database (http://www.genome.jp/kegg/) for statistical analysis: we also removed significantly enriched KEGG pathways with corrected p-value ≤ 0.05. All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). The one-way ANOVA was used for comparisons between two groups: if there were any differences, a post hoc LSD test was conducted (wherein a value of p < 0.05 was considered to indicate a statistically significant difference).
