Electroacupuncture preconditioning alleviates myocardial injury through regulating mitochondrial function.

Background Electroacupuncture is well known for its advantageous neuroanalgesic and therapeutic effects on myocardial ischemia-reperfusion injury. The purpose of the present research was to verify whether electroacupuncture can alleviate bupivacaine-induced myocardial injury. Methods Specic-pathogen-free Wistar rats were used to establish the bupivacaine-induced myocardial injury model. Western blot, PCR, transmission electron microscope and enzyme-linked immunosorbent (ELISA) methods were used to evaluate bupivacaine-induced structure injury and dysfunction of the mitochondria as well as the alleviating effects of lipid emulsion, acupoint injection, and electroacupuncture pre-treatment of the oxidase stress response. Results Bupivacaine caused structural damage, degradation, and swelling of mitochondria. Furthermore, it reduced adenosine triphosphate (ATP) synthesis and impaired energy metabolism in the mitochondria. Structural and functional impairment of the mitochondria was alleviated via lipid emulsion injection, acupoint injection, and electroacupuncture pre-treatment. Electroacupuncture pre-treatment of PC6 yielded a greater alleviating effect than others approaches. Following electroacupuncture pre-treatment of PC6 point, the number of mitochondria increased; apoptosis was reduced, enzymatic activity of cytochrome C oxidase (COX) and superoxide dismutase and expression of uncoupling protein 2, voltage-dependent anion channel 1, and Bcl 2 were upregulated and SLC25A6, MDA levels were downregulated. Additionally, our ndings indicated that electroacupuncture pre-treatment of PC6 point exerted an effect on the mitochondria via the mitochondrial-transcription-factor-A / nuclear-respiratory-factor-1 / proliferator-activated-receptor-gamma-coactivator -1 pathway. Conclusion The present study revealed that electroacupuncture pre-treatment of PC6 could effectively alleviate bupivacaine-induced myocardial mitochondrial damage, thereby providing a theoretical basis for clinical studies and applications of this treatment method.

onset of action and is relatively safe at the recommended dosage [5]. However, overdose or accidental intravasal application of bupivacaine can result in signi cant adverse effects. For every 1,000 peripheral nerve blocks, 0.04 to 1.8 cases of myocardial toxicity are likely to occur, leading to severe symptoms [6].
Bupivacaine reportedly induces muscle toxicity in skeletal muscles [5]. Bupivacaine-induced cardiotoxicity may result in cardiac arrhythmia, poor myocardial contractility and cardiac arrest due to circulatory collapse [7,8]. Sztark et al. reported that bupivacaine may directly inhibit mitochondrial respiratory chain complex I (MRCC-I), alter mitochondrial membrane structure, increase proton permeability in the mitochondrial inner membranes (MIMs), induce loss of mitochondrial calcium, and reduce mitochondrial membrane potential (MMP) [9], thereby affecting the respiratory function and energy production of the mitochondria and eventually leading to mitochondrial dysfunction.
Evidently, bupivacaine inhibits MRCC-I activity in a dose-dependent manner, accounting for alterations in mitochondrial energy uxes [10]. Bupivacaine negatively regulates fatty acid oxidation and oxidative phosphorylation and inhibits the activity of mitochondrial carnitine transferases, thereby reducing the synthesis of adenosine triphosphate (ATP) required for myocardial contractility [7]. Bupivacaine also inhibits the aerobic respiration of the mitochondria; speci cally, decoupling of oxidative phosphorylation alters MMP and inhibits mitochondrial respiration, resulting in the production of reactive oxygen species (ROS) in the mitochondria and triggering the mitochondrial pathway of apoptosis [11]. Lipid emulsion is widely used to alleviate bupivacaine-induced myocardial toxicity because this technique can enhance mitochondrial metabolism and improve myocardial contractility [12,13]. Similarly, puerarin isolated from the traditional Chinese medicine radix puerariae has demonstrated a protective effect on myocardial infarction by targeting the mitochondria [14].
Over the past three millennia, acupuncture has exhibited signi cant potential in clinical studies of various diseases in China and other Asian countries. Electroacupuncture (EA) is a combination of acupuncture and electrical stimulation [15]. Because EA allows for more precise parameter tuning, it has been widely applied in clinical studies [16]. Previous studies have shown that EA can regulate mitochondria autophagy, inhibit NLRP3 in ammasome activation, and alleviate myocardial injury [17,18]. EA can also serve as an adjuvant therapy for various types of pain with fewer side effects; glial cells may contribute to these analgesic effects [19]. Besides, EA can also affect the secretion of proin ammatory cytokines and vascular endothelial growth factor by regulating endocrine hormones [20].
Acupuncture-assisted anesthesia, such as ST36 stimulation is commonly used in clinical studies to reduce the required dose of bupivacaine and thus avoid its the side effects [21]. Similarly, acupuncture of ST40 can exert a protective effect on heart structure and function by regulating the metabolism of lipid emulsions [22]. The Neiguan point (Pericardium-6, PC6) is located on the medial surface of the forelimb between the tibia and the ulna. Researchers have veri ed that PC6 plays a vital role in cardiovascular diseases when stimulated. Reportedly, the protective effects of PC6 EA are predominantly achieved via the regulation of various signals of mitochondrial energy metabolism [23]. In present study, we explored the effects of PC6 stimulation on structure and function of mitochondria.

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 speci c-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 sacri ced 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 [24]. 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 modi cations [25]. 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 puri ed mitochondria with an appropriate proportion and the results were analyzed using a uorescence 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 [7]. Fluo-3 AM was added to resuspended mitochondria and incubated for 1h. Then, the Fluo-3 AM was removed. Fluorescence intensity was analyzed by ow 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.

ATP content
Myocardial ATP levels were measured using a commercial assay kit. Brie y, 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.
qPCR 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.

Western blot
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 uoride 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 xed 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 [26]. Five microscopic elds 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 occulent densities in their mitochondrial matrices; and Grade IV (score 4), mitochondria lacking matrix with fragmented cristae and disrupted outer membranes.

Statistical analysis
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 signi cant results.

Protective effect of different treatments on bupivacaine-induced myocardial injury
Previous studies have demonstrated that EA pre-treatment exerts an alleviating effect on bupivacaineinduced toxicity [8]. As shown in Fig. 1A, signi cant differences in the lethal dose of bupivacaine were observed between different groups, among which the EA pre-treatment of PC6 corresponded to the highest lethal dose of bupivacaine. The lethal dose of bupivacaine in EA-PC6 was higher than EA-ST36 and puerarin injection at PC6. This nding suggested that EA-PC6 had the greatest alleviating effect against myocardial toxicity. Intravenous injection with lipid emulsions yielded a relatively poor alleviating effect but still outperformed the bupivacaine group. The results showed that bupivacaine signi cantly prolonged the duration of the QRS complex. The duration of QRS complex was 26ms, 32ms, 260ms in control, arrhythmia, and lethal groups respectively. The difference between the control and arrhythmia groups was signi cant (P<0.05). The difference between the QRS complex durations of the lethal and control groups was extremely signi cant (p<0.01; Fig. 1B).
The effect of EA pre-treatment on mitochondrial structure Structural and functional damage to the mitochondria is the main mechanism underlying myocardial injury. The control group rats in the present study exhibited densely-packed myocardial bers with clearly visible sarcomeres and abundant mitochondria. Their mitochondrial structure was intact with a typical oblong shape and continuous cristae, as well as intact membranes and granules (Fig 2A). The rats in the bupivacaine group displayed mitochondrial degradation with swollen, fragmented, and even missing cristae. The lipid emulsion group, the puerarin group, and the EA pre-treatment group exhibited increasingly alleviated mitochondrial injuries. The Flameng scores showed that bupivacaine was associated with the least mitochondrial damage in the EA group rats. In addition, there were signi cant differences in the Flameng scores between different groups (P<0.01). And the therapy effect of EA-PC6 was signi cantly preceded EA-ST36. In further research, we mainly focused on electroacupuncture pretreatment of PC6 point because of its best protection effect on myocardial mitochondria. Mitochondrial abundance also varied between the different treatment groups (Fig. 2B). All the treatment groups showed lower mitochondrial abundance than the control group, and the bupivacaine group exhibited the lowest mitochondrial abundance, amounting to half of that of the control group. The lipid emulsion group, the puerarin group, and EA-PC6 group exhibited gradually increasing mitochondrial abundances, and the ATP synthesis abilities were enhanced along with these changes in mitochondrial abundance, suggesting that their mitochondrial function was gradually recovered (Fig 2C).

Functional changes in mitochondria
As shown in Fig. 3A, bupivacaine upregulated monoamine oxidases (MAO) activity, downregulated COX activity, and reduced MMP. This nding indicated that bupivacaine induces mitochondrial autophagy and dysfunction (Fig. 3A, 3B). Therapy involving lipid emulsion, puerarin and EA-PC6 pre-treatment promoted the expression of Bcl2, UCP2, and VDAC1 and decreased the expression of ANT1 and SLC25A6. After these treatments, mitochondrial dysfunction was relieved and apoptosis was reduced (Fig. 3C).
Bupivacaine induced the oxidative stress response Dysfunction of the mitochondria is closely related to ROS levels. The present results indicated that ROS production was induced by bupivacaine. All three methods of treatment increased SOD activity and decreased MDA content, playing a key role in mitochondrial protection (Fig. 4).

Effects of bupivacaine on myocardial mitochondrial biogenesis
Mitochondrial calcium uptake signi cantly improved depleted cells and regulated cytosolic calcium signal [27,28]. Our results indicated that increased Ca 2+ levels of mitochondrial signi cantly upregulate the expression of PGC-1, NRF-1, and mtTFA, is one of the signals mediating mitochondrial biogenesis.
The concentrations of Ca 2+ were lowest in bupivacaine group. However, the lipid emulsion, puerarin, and EA-PC6 pre-treatment groups exhibited signi cantly higher expression of PGC-1, mtTFA, and NRF than the bupivacaine-induced group (Fig. 5).

Discussion
Numerous studies have con rmed that acupuncture can prevent myocardial injury. Patients who have undergone cardiac surgery have shown signi cantly improved heart rates, blood pressure, and rates of recovery after receiving EA stimulation [29]. Recently, researchers found that EA pre-treatment improved the survival rate of rats with myocardial ischemia-reperfusion injury with reduced apoptosis and expressions of Cyt c and cleaved caspase 3 [30,31]. EA regulated the activation of NLRP3, polarization of macrophages, declined arrhythmia scores, improving cardiac function [18,31]. EA played a role of antiarrhythmia through reducing ventricular tachycardia and ventricular, and related to concentration of calcium in the cytoplasm [32,33]. EA pre-treatment inhibits apoptosis via the mitochondria-dependent pathway [34]. In addition, EA can effectively promote angiogenesis and protect myocardial tissue from damage [35]. Previous researches have shown that the effect of electroacupuncture treatment on different acupoints was signi cantly different. The analgesic effect of acupuncture PC6 was better than ST36 [36,37]. Acupuncture point of CV12 was better than PC6 and ST36 in the anorexia treatment [38]. And there was no difference in recovery of gastrointestinal function between acupuncture ST36 and the combination of acupuncture ST36+ST37+PC6 [39]. In present research, the results showed EA pretreatment of PC6 to prevent bupivacaine-induced myocardial injury was the best treatment due to its highest lethal dose of bupivacaine and the superior protection effect on myocardial mitochondria.
The mitochondria, which are comprise a central source of metabolism and energy production, play an important role in cellular energy metabolism. Mitochondrial injury may lead to cardiotoxicity [40]. The uncoupling of mitochondrial oxidative phosphorylation and/or inhibition of the electron transport chain leads to metabolic dysregulation in mitochondria. The cumulative release of ROS, decline in ATP synthesis, and leakage of mitochondrial Ca 2+ may result in an in ammatory respond, mitochondrial injury, which may aggravate apoptosis and lead to the death of cells surrounding the injured areas. Mitochondria-targeting treatment, considered to be an appealing strategy for the control and management of mitochondrial injury [41], has become the focus of many important research studies [42]. Previously-published data have suggested that the injection of isolated viable respiration-competent myocardial mitochondria into ischemic regions prior to reperfusion may reverse post-ischemic functional deterioration and apoptosis, thereby limiting the infarct area [43].
The present ndings indicated that Bcl-2 expression increased and ROS production was signi cantly reduced following EA pre-treatment of PC6. Similarly, calcium ions concentration of mitochondria was downregulated by bupivacaine. This is consistent with previous research [7]. We proposed that the leakage of mitochondrial calcium ions aggravated the overloaded of cytoplasmic calcium ions in arrhythmias. While, EA promoted uptake of mitochondrial calcium ions and reduced calcium overload in the cytoplasm. Moreover the increased concentration of mitochondrial calcium promoted mitochondria biogenesis, achieving the therapeutic effect.

Conclusions
EA pre-treatment of PC6 alleviated the mitochondrial damage caused by myocardial toxicity, enhanced the MMP, and altered concentration of Ca 2+ . All of these effects may contribute to the prevention of bupivacaine-induced cardiac arrhythmia and the improvement of myocardial metabolism.

Declarations Acknowledgement
We sincerely thank Regional Science Fund Project (81760892) for its support.

Consent for publication
Not applicable.

Competing interest
The authors have no potential con icts of interest to disclose.  Table   Table 1