The primary materials used in the present study were as follows: fetal bovine serum (FBS), bupivacaine, lipid emulsions, puerarin injections, pentobarbital sodium, the RNeasy Mini Kit (QIAGEN), the QuantiNova SYBR Green PCR Kit (QIAGEN), the QuantiNova Reverse Transcription Kit (QIAGEN), Anti-UCP2 (GeneTex), Anti-NRF1 (GeneTex), Anti-SLC25A6 (GeneTex), Anti-mtTFA (GeneTex), Anti-VDAC1, Anti-PGC-1 (Abcam), Anti-Bcl-2 (Abcam) and and ATP assay kit (Abcam, ab83355). JC-1 kit (Beyotime, C2006), Fluo-3 AM (Abcam), DCFH-DA (Solarbio), Rat COX ELISA kit (CUSABIO), MDA ELISA Kit (Elabscience).
General animal and animal group treatment conditions
Animal experimentation was carried out in the present study in accordance with the requirements of Committee of the Animal Protection and Utilization Institute. This study was approved by animal experiment ethics of Gansu University of Traditional Chinese Medicine (No. 2018-018) and complied with the Declaration of Helsinki. Male specific-pathogen-free Wistar rats (8-week-old, weighing 300±10 g) were provided by Shanghai Experimental Animal Co., Ltd. (Shanghai, China). Each of these animals was anaesthetized and placed in the supine position on a surgical platform, and three needle electrodes were placed under the skin to produce a 12-lead electrocardiogram (ECG) and monitor other basic conditions, such as blood pressure and heart rate.
The experimental rats were divided into six groups as follows: control group (n=8) rats intravenously infused with 3 ml of 0.9% physiological saline solution (kg/min) and sacrificed after 30 min; bupivacaine group (n=16) rats intravenously infused with physiological saline solution for 30 min and then infused with 0.5% bupivacaine to induce cardiac arrhythmia or death (standard criteria for cardiac arrhythmia); lipid emulsion group (n=16) rats subject to continuous intravenous infusion with 3 ml of 20% lipid emulsion for 30 min, followed by infusion with bupivacaine to induce cardiac arrhythmia or death; puerarin group (n=16) rats infused with bupivacaine after being injected with 0.1 ml of puerarin at PC6 on both forelimbs; EA-PC6 group and EA-ST36 group (n=16) rats infused with bupivacaine after 30 min of EA stimulation (longitudinal wave: 2/10 Hz; current intensity: 2 mA; pulse width: 0.2 ms) at PC6 and ST36, respectively.
The rate and duration of infusion and lipid emulsion administration were determined according to a study reported by Weinberg . Cardiac arrhythmia was assessed by an electrocardiographer on the basis of premature ventricular contractions (PVC) or ventricular tachycardia (VT) with the duration of the QRS complex prolonged. At the conclusion of the experiment, the rats were euthanized and subjected to retrograde perfusion followed by immediate resection of their hearts for subsequent analyses.
Isolation of mitochondria
Myocardial mitochondria were isolated using a previously published method with some modifications . The rats were euthanized with pentobarbital sodium, and their hearts were harvested, excised into small pieces, and homogenized. This process was followed by the removal of cell debris. Then, the supernatant was centrifuged at 13,000 g for 10 min to isolate the mitochondria.
Mitochondrial membrane potentials (MMP) assay
MMP was measured with a JC-1 kit (Beyotime, C2006) according to the manufacturers’ instructions. The 1x JC-1 working solution was added to the purified mitochondria with an appropriate proportion and the results were analyzed using a fluorescence spectrophotometer. The excitation wavelength was 458nm, and the emission wavelength was 590nm.
Determination of calcium ion levels in mitochondria
Mitochondrial calcium ion concentrations were measured using Fluo-3 AM in accordance with previous study. Fluo-3 AM was added to resuspended mitochondria and incubated for 1h. Then, the Fluo-3 AM was removed. Fluorescence intensity was analyzed by flow cytometry after the mitochondria were subjected to Triton X100, calcium chloride and EDTA, respectively.
Reactive oxygen species measurement
Cardiomyocytes were isolated from the SD rats, and their ROS levels were evaluated by incubating the cells in DCFH-DA. After 30min incubation, the cells were subjected to washed, and resuspended. Subsequently, the results were analyzed using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Myocardial ATP levels were measured using a commercial assay kit. Briefly, tissues were lysed and centrifuged at 12,000 g and 4°C for 5 min. The resulting supernatant was harvested for subsequent assays. Mitochondrial ATP content was analyzed by colorimetry using phosphomolybdic acid.
Enzyme linked immunosorbent assay (ELISA)
After being homogenized in phosphate-buffered saline, the tissue samples were centrifuged to obtain the supernatant for subsequent measurements. All experimental procedures were carried out in accordance with the manufacturers’ instructions provided with the commercial kits. Standard samples and test samples were placed in wells intended for blank, standard, and test samples, respectively. After being incubated at 37°C for 30 min, each well was washed and incubated with the enzyme-labelling reagent. Then, the wells were washed again and subjected to color development. After the reaction was terminated, the absorbance value of each well was measured to estimate the sample concentration. This experiment was repeated three times.
Cardiac muscle tissues were rapidly harvested and immersed in liquid nitrogen to extract RNA for subsequent analyses. Experimental procedures were carried out in accordance with the instructions provided with the commercial kits. The purity and quality of the resulting RNA samples were determined alongside the removal of DNA, followed by a reverse transcription PCR (RT-PCR) assay. Primer sequences for the target genes are listed in Table 1.
After the tissue samples were homogenized, the supernatant was obtained for the purpose of isolating total proteins using a protein extraction kit. After the protein concentration was measured, the total proteins were loaded in equal amounts and separated via sodium dodecylsulphate polyacrylamide gel electrophoresis. Subsequently, the proteins were transferred onto a polyvinylidene fluoride membrane, which was then incubated with special primary antibodies followed by secondary antibodies for 1 h at 37℃. Western blot images were obtained via the enhanced chemiluminescence method. Grayscale analysis was performed on target protein bands using Image J software, and the results were analyzed statistically.
Transmission electron microscopy
Myocardial tissues were sequentially fixed with 2.5% glutaraldehyde and 1% citric acid then dehydrated in an acetone gradient and embedded in resin. Then the embedded tissues were dried rapidly prior to sectioning. Next, tissue sections were stained and imaged under an electron microscope. Mitochondrial injuries were assessed using the Flameng score . Five microscopic fields were randomly selected to obtain the mean Flameng score for each group and the mitochondria were graded according to the following criteria: Grade 0 (score 0), mitochondria with normal ultramicrostructure and intact granules; Grade I (score 1), mitochondria with basically normal ultramicrostructure and partial loss of granules; Grade II (score 2), swollen mitochondria with transparent matrices; Grade III (score 3), mitochondria with transparent matrices and fragmented cristae or formation of flocculent densities in their mitochondrial matrices; and Grade IV (score 4), mitochondria lacking matrix with fragmented cristae and disrupted outer membranes.
The statistical analyses of the present study were performed using SPSS 20.0 software, and the data were expressed as means of the scores yielded by triplicate experiments. Multiple comparisons between groups were carried out using one-way analysis of variance (ANOVA). P-values <0.05 were considered to indicate statistically significant results.