Correspondence: Chunyan Jian (E-mail: jianchunyan666@163.com)

Remifentanil protects against myocardial ischemia/reperfusion injury via miR-205-mediated regulation of PINK1

Lu Cheng1, Yifan Wu2, Jiayu Tang3, Chao Zhang1, Huan Cheng4, Qi Jiang1 and Chunyan Jian2

1Department of Cardiovascular Internal Medicine, The Affiliated Cardiovascular Hospital of Qindao University,

Qingdao City, Shandong Province, 266071, China2Department of Cardiovascular Internal Medicine, Central People’s Hospital of Zhanjiang,

Zhanjiang City, Guangdong Province, 524045, China3Department of Medical Laboratory, Central People’s Hospital of Zhanjiang,

Zhanjiang City, Guangdong Province, 524045, China4Department of Uitrasound, The Affiliated Hospital of Qindao University,

Qingdao City, Shandong Province, 266000, China

(Received December 17, 2020; Accepted February 22, 2021)

ABSTRACT — Myocardial ischemia/reperfusion (I/R) injury could lead to severe cardiovascular ischemic disease, including myocardial infarction and contractile dysfunction. Remifentanil demonstrated protective effect on myocardial I/R injury. The underlying pathophysiological mechanism was then inves-tigated in this study. In the current study, primary cardiomyocytes were isolated from rats, and then pre-conditioned with remifentanil. Rats, tail vein injected with miR-205 antagomir, were subjected to infusion of remifentanil, and then subjected to regional ischemia followed by reperfusion. The results demonstrat-ed that cell viability of hypoxia/reoxygenation-induced cardiomyocytes was increased post remifenta-nil, while the apoptosis was decreased accompanied with reduced cleaved caspase-3 expression. Hypox-ia/reoxygenation treatment increased miR-205 and decreased PINK1 (PTEN induced putative kinase 1) expression. However, preconditioning with remifentanil reduced miR-205 and enhanced PINK1. Moreo-ver, over-expression of miR-205 decreased PINK1 expression and counteracted the effects of remifenta-nil-induced increase of cell viability and decrease of cell apoptosis in hypoxia/reoxygenation-induced car-diomyocytes. Injection with miR-205 antagomir improved remifentanil-induced decrease of infarct size and LDH (lactic acid dehydrogenase) activity in rat model with I/R injury. In conclusion, miR-205 might participate in the protective effect of remifentanil in rat myocardial I/R injury via regulation of PINK1, providing a potential target for amelioration of cardiovascular ischemic disease. Key words: miR-205, PINK1, Remifentanil, I/R injury

INTRODUCTION

Myocardial ischemia, with the blockage of blood and oxygen supply to heart, could lead to serious complica-tions, such as myocardial infarction (Toldo et al., 2018). Congestive heart failure and malignant arrhythmia caused by myocardial infarction could result in cardiovascular diseases, that have a high incidence and mortality glob-ally (Jiao et al., 2017). Reperfusion of occluded coro-nary artery is regarded as the “gold standard” for the treatment of myocardial ischemia (E et al., 2019). How-

ever, the adverse effect of reperfusion could cause myo-cardial ischemia/reperfusion (I/R) injury, such as systol-ic function decline (Dorweiler et al., 2007). Therefore, to improve myocardial ischemia, it is imperative to identify strategies to ameliorate myocardial I/R injury during rep-erfusion.

Remifentanil is a newly synthesized piperidine deriva-tive and widely used as component of anesthesia, that has been shown to protect the uterus (Atalay et al., 2015) or ovary (Atalay et al., 2016) against ischemia-reperfusion injury. Pretreatment with remifentanil could confer car-

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dioprotection through opioid receptor (Yu et al., 2007). During coronary artery bypass surgery, anesthesia regi-men with remifentanil reduced biomarkers of myocardial damage, including creatine kinase and cardiac troponin I (Wong et al., 2010). A study has shown that intravenous infusion of remifentanil could effectively repress hypox-ia-reoxygenation-induced injury in cardiomyocytes and reduce the myocardial infarction area in rats in a dose-dependent pattern (Dou et al., 2016). Moreover, remifen-tanil was reported to attenuate oxidative injury in cardi-omyocytes (Lei et al., 2018) and activate anti-apoptotic pathways to sustain survival of myocardium in ischemia-reperfused rats (Kim et al., 2009). However, the under-lying pathophysiological mechanism of remifentanil-mediated protective effect against myocardial I/R injury remains unclear.

Previous studies have demonstrated that the cardiopro-tective effect of diazoxide (Zhang et al., 2020a) or dex-medetomidine (Zhang et al., 2020b) during myocardi-al I/R injury or hypoxia-reoxygenation injury relied on regulation of miR-10a or lncRNA H19/miR-29b-3p axis, respectively. Administration of the anesthetic, propofol, during reperfusion alleviated myocardial I/R injury with reduced cardiomyocyte apoptosis and infarct size through up-regulation of miR-451 (Li et al., 2019). MiR-205 was up-regulated in the plasma and heart of mice post imatin-ib mesylate and doxorubicin treatment (Hanousková et al., 2019), and down-regulation of miR-205 could reduce myocardial apoptosis during chronic heart failure (Xuan et al., 2017). Therefore, this study hypothesized that remifentanil might prevent myocardial I/R injury through regulation of miR-205.

The alteration of miR-205 level was evaluated in hypoxia-reoxygenation-induced cardiomyocyte and rat model with myocardial I/R injury post remifentanil. The effects of miR-205 on myocardial apoptosis and infarc-tion size induced by hypoxia-reoxygenation and I/R were also determined in this study.

MATERIALS AND METHODS

AnimalsNeonatal rats (1 to 2 days) and Sprague-Dawley rats

(6-8 weeks old, 300-350 g weight) were purchased from Experimental Animal Centre of Soochow University (Suzhou, China). All animal experiments were approved by the Ethics Committee of The Affiliated Cardiovascular Hospital of Qindao University for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.

Cell isolation and cultureNeonatal rats were anesthetized with 40 mg/kg pento-

barbital, and the hearts were harvested and then cut into 2 mm3 pieces. The pieces were digested with 0.1% col-lagenase type II (Sigma Aldrich, St. Louis, MO, USA), and the supernatants of the digestion were collected by centrifugation at 1500 x g for 10 min. The supernatants were cultured in DMEM-F12 medium containing 10% fetal bovine serum (Beyotime Institute of Biotechnology, Haimen, China). Cardiomyocytes were separated from the fibroblasts via differential wall adhesion method, and cultured in DMEM-F12 medium containing 10% fetal bovine serum and 0.1 mM 5-bromodeoxyuridine in a 37°C incubator.

Hypoxia-reoxygenation injury modelThe isolated primary cardiomyocytes were plat-

ed and pretreated with remifentanil (1, 5 or 10 μM) for 10 min. Thirty minutes later, cells were incubated with 4 mM Na2S2O4 for 1 hr. After removing Na2S2O4, cells were then cultured in the normal DMEM-F12 medium for another 12 hr to generate hypoxia-reoxygenation inju-ry model. For over-expression or knockdown of miR-205, the isolated primary cardiomyocytes were transfect-ed with 50 nM miR-205 mimic or inhibitor (GenePharm, Shanghai, China) via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Two days later, cells were conduct-ed with or without hypoxia-reoxygenation injury model.

Cell viability and apoptosis assaysMTT was used to detect cell viability. Primary car-

diomyocytes with indicated treatment and transfec-tion were seeded in plates, and incubated with 20 µL MTT reagent (Sigma Aldrich) for 4 hr. The medi-um was removed and cells were incubated with dime-thyl sulfoxide, and the absorbance at 490 nm was measured by microplate spectrophotometer (Ther-mo Scientific, Waltham, MA, USA). TUNEL was used to detect cell apoptosis. Primary cardiomyocytes with indicated treatment and transfection were fixed and then stained with TUNEL and DAPI (Roche, Man-nheim, Germany). A microscope (Zeiss Axio Observer Z1, Zeiss, Tokyo, Japan) with software Zen 2011 was used to capture images and detect TUNEL-positive cells.

Luciferase reporter assaySequences of wild type or mutant PINK1 3’ UTR

(165 bp) were cloned into pMIR-GLO™ Luciferase vec-tor (Promega, Madison, WI, USA) and named as PINK1-WT and PINK1-MUT. HEK293T was co-transfected with miR-205 mimic or miR-NC (negative control), miR-205

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inhibitor of NC inh and PINK1-WT or PINK1-MUT. For-ty-eight hours later, cells were conducted with a Dual-Luciferase Reporter Assay system (Promega).

Myocardial I/R injury modelSixty Sprague–Dawley rats were randomly separated

into six groups: sham, acute myocardial infarction (AMI), AMI with remifentanil preconditioning (RPC), AMI + RPC with NC antagomir, AMI + RPC with miR-205 antagomir and AMI with miR-205 antagomir. For AMI models, rats were anesthetized with 80 mg/kg pento-barbital. A 16-gauge cannula, connected to a ventilator (Harvard Rodent Ventilator Model 683, Holliston, MA, USA) was intubated into the trachea of rats. A 24-gauge catheter, connecting to pressure transducer, was cannu-lated into right carotid artery. Drug administration was performed through the 24-gauge catheter installed in the left jugular vein. The chest was opened and the heart was exposed. The first branch of the anterior descending cor-onary artery was placed with a 6-0 prolene loop, and a snare for reversible occlusion was formed via thread through the ends of the suture. For AMI with RPC group, rats were administered three times with 20 μg/kg per min-ute remifentanil for five minutes. For antagomir groups, rats were tail vein injected with 80 mg/kg miR-205 antag-omir or NC antagomir (GenePharm) for 3 days. Rats were then conducted with the AMI model and remifent-anil administration. For AMI models, hearts of rats were subjected to regional ischemia for 30 min, followed by reperfusion for 2 hr. Rats in the sham group were con-ducted with the surgery without the regional ischemia and reperfusion.

Measurement of infarct sizeThe hearts were excised and then transferred to Lan-

gendorff apparatus after the reperfusion. The hearts were perfused with normal saline, and then injected with 0.25% Evan blue dye to stain the perfused region in hearts. Hearts were sliced into 2-mm slices and stained with 2% triphenyl-tetrazolium chloride (Sigma Aldrich) for 20 min. After immersion in 10% formalin, the infarct size in hearts was determined by computerized plan-imetry technique (SigmaScan 4.0, Systat Software, Richmond, CA, USA).

Measurement of lactate dehydrogenaseAt the end of reperfusion, the blood sample was col-

lected from the carotid artery of rats. Samples were cen-trifuged at 5000 x g for 10 min to isolate serum. Activity of lactate dehydrogenase in the serum was then meas-ured LDH assay kit (Nanjing Jiancheng Bioengineering

Institute, Nanjing, China).

qRT-PCRTotal RNAs, extracted from primary cardiomyo-

cytes or heart tissues were conducted with complemen-tary DNA synthesis. FastStart Essential DNA Green Master kit (Roche Life Sciences, Indianapolis, IN, USA) was used for qRT-PCR analysis of miR-205 and U6 with primers: miR-205 (forward; 5’-CTTGTC-CTTCATTCCACCGGA-3’ and reverse: 5’-TGCCGC-CTGAACTTCACTCC-3’) and U6 (forward: 5’-CTG-GTTAGTACTTGGACGGGAGAC-3’ and reverse: 5’-GTGCAGGGTCCGAGGT-3’).

Western blot analysisLysates of primary cardiomyocytes or heart tissues

were separated by electrophoresis and transferred to pol-yvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). After blocking in 5% skim milk, membranes were incubated with primary antibod-ies against Pink1 (1:2000; Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3 (1:2500; Cell Signaling Technology) and β-actin (1:3000; Cell Signaling Technology). The membranes were incubated with horseradish peroxidase-linked secondary antibody (1:5000; Cell Signaling Technology). ChemiDoc MP sys-tem (Bio-Rad, Hercules, CA, USA) was used to detect signals, and Image J software was used for densitomet-ric analysis.

Statistical analysisData were presented as mean ± standard deviation, and

conducted with SPSS 11.5 statistical software. Statistical analysis was performed via Student’s t-test or one-way analysis of variance. The p < 0.05 was considered signif-icant.

RESULTS

Preconditioning with remifentanil suppressed hypoxia/reoxygenation-induced cell apoptosis of cardiomyocytes

To establish in vitro cell model of myocardial I/R inju-ry, primary cardiomyocytes were isolated from rats and then conducted with hypoxia/reoxygenation treatment. Hypoxia/reoxygenation treatment decreased cell viabili-ty (Fig. 1A) and promoted cell apoptosis (Fig. 1B) of car-diomyocytes. However, cardiomyocytes preconditioned with different concentrations of remifentanil (1, 5 or 10 μM) attenuated hypoxia/reoxygenation-induced decrease of cell viability (Fig. 1A) and increase of cell

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apoptosis ( Fig. 1B ), suggesting that preconditioning with remifentanil suppressed hypoxia/reoxygenation-induced cell apoptosis of cardiomyocytes. Hypoxia/reoxygena-tion-induced enhance of cleaved caspase-3 was down-regulated by remifentanil preconditioning ( Fig. 1C ), con-fi rming the suppressive eff ect of remifentanil on hypoxia/reoxygenation-induced cell apoptosis of cardiomyocytes.

Preconditioning with remifentanil reduced hypoxia/reoxygenation-induced increase of miR-205 in cardiomyocytes

Transfection efficiency of miR-205 mimic or inhibi-tor into cardiomyocytes is shown in Fig. 2A . To unrav-el involvement of miR-205 in myocardial I/R injury, expression level of miR-205 in hypoxia/reoxygenation-induced cardiomyocytes was determined by qRT-PCR analysis. The results showed that miR-205 was up-regu-lated in hypoxia/reoxygenation-induced cardiomyocytes ( Fig. 2B ). Moreover, preconditioning with remifentanil reduced hypoxia/reoxygenation-induced increase of miR-205 in cardiomyocytes ( Fig. 2B ), showing that miR-205 might participate in myocardial I/R injury.

MiR-205 was involved in protective effect of remifentanil preconditioning against hypoxia/reoxygenation-induced injury

To investigate the role of miR-205 in myocardial I/R injury, cardiomyocytes were transfected with miR-205 mimic or inhibitor, and then conducted with remifenta-nil preconditioning and hypoxia/reoxygenation treatment. Result showed that transfection with miR-205 mim-ic or inhibitor did not aff ect cell viability of cardiomyo-cytes without hypoxia/reoxygenation treatment ( Fig. 3A ). However, knockdown of miR-205 increased cell viability of hypoxia/reoxygenation-induced cardiomyocytes pre-conditioned with remifentanil ( Fig. 3B ), but repressed the cell apoptosis ( Fig. 3C ) and decreased protein expression of cleaved caspase-3 ( Fig. 3D ). Moreover, over-expres-sion of miR-205 demonstrated reversed effects on cell viability ( Fig. 3B ), apoptosis ( Fig. 3C ) and cleaved cas-pase-3 expression ( Fig. 3D ) in hypoxia/reoxygenation-induced cardiomyocytes preconditioned with remifent-anil, indicating that remifentanil preconditioning might reduce miR-205 to protect against hypoxia/reoxygena-tion-induced injury in cardiomyocytes.

Fig. 1 . Preconditioning with remifentanil suppressed hypoxia/reoxygenation-induced cell apoptosis of cardiomyocytes. (A) Remifentanil preconditioning increased cell viability of hypoxia/reoxygenation-induced cardiomyocytes. N = 3. (B) Remifentanil preconditioning decreased cell apoptosis of hypoxia/reoxygenation-induced cardiomyocytes. N = 3. (C) Remifentanil preconditioning decreased protein expression of cleaved caspase-3 in hypoxia/reoxygenation-induced cardio-myocytes. N = 3. * p < 0.05; ** p < 0.01. H/R: hypoxia/reoxygenation, RPC-1, -5, -10: 1,5, 10 μM remifentanil.

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Fig. 2 . Preconditioning with remifentanil reduced hypoxia/reoxygenation-induced increase of miR-205 in cardiomyocytes. (A) Transfection effi ciency of miR-205 mimic or inhibitor into cardiomyocytes was identifi ed by qRT-PCR. (B) Remifentanil preconditioning decreased expression of miR-205 in hypoxia/reoxygenation-induced cardiomyocytes. N = 3. * p < 0.05; ** p < 0.01. H/R: hypoxia/reoxygenation, RPC-1, -5, -10: 1,5, 10 μM remifentanil.

Fig. 3 . MiR-205 was involved in protective eff ect of remifentanil preconditioning against hypoxia/reoxygenation-induced injury. (A) Transfection with miR-205 mimic or inhibitor did not aff ect cell viability of cardiomyocytes without hypoxia/reoxygen-ation treatment. N = 3. (B) Knockdown of miR-205 increased cell viability of hypoxia/reoxygenation-induced cardiomyo-cytes preconditioned with remifentanil, and over-expression of miR-205 decreased the cell viability. N = 3. (C) Knockdown of miR-205 repressed the cell apoptosis of hypoxia/reoxygenation-induced cardiomyocytes preconditioned with remifen-tanil, and over-expression of miR-205 promoted the cell apoptosis. N = 3. (D) Knockdown of miR-205 decreased protein expression of cleaved caspase-3 in hypoxia/reoxygenation-induced cardiomyocytes preconditioned with remifentanil, and over-expression of miR-205 increased the expression. N = 3. * p < 0.05; ** p < 0.01. H/R: hypoxia/reoxygenation.

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miR-205/PINK1 axis in remifentanil-mediated I/R injury

MiR-205 suppressed PINK1 expression Data from Targetscan ( http://www.targetscan.org/

vert_72/ ) showed that miR-205 could bind to 3’UTR of PINK1 ( Fig. 4A ). Luciferase activity assay revealed that over-expression of miR-205 decreased luciferase activi-ty of Pink1-WT ( Fig. 4B ), while knockdown of miR-205 increased the activity ( Fig. 4B ). However, mutation at the binding site between miR-205 and PINK1 abolished the

eff ect of miR-205 on luciferase activity of Pink1-MUT ( Fig. 4B ), suggesting that miR-205 bind to PINK1. Trans-fection with miR-205 mimic decreased protein expres-sion of PINK1 in cardiomyocytes ( Fig. 4C ), while PINK1 was increased in cardiomyocytes transfected with miR-205 inhibitor ( Fig. 4C ). Protein expression of PINK1 was reduced in hypoxia/reoxygenation-induced cardiomyo-cytes ( Fig. 4D ), while preconditioning with remifentanil

Fig. 4 . MiR-205 suppressed PINK1 expression. (A) Potential binding site between miR-205 and 3’UTR of PINK1. N = 3. (B) Over-expression of miR-205 decreased luciferase activity of Pink1-WT, knockdown of miR-205 increased the activity. N = 3. (C) Transfection with miR-205 mimic decreased protein expression of PINK1 in cardiomyocytes, and PINK1 was increased in cardiomyocytes transfected with miR-205 inhibitor. N = 3. (D) Remifentanil preconditioning increased protein expression of PINK1 in hypoxia/reoxygenation-induced cardiomyocytes. N = 3. ** p < 0.01. H/R: hypoxia/reoxygenation, RPC-1, -5, -10: 1,5, 10 μM remifentanl.

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increased PINK1 ( Fig. 4D ), demonstrating that remifent-anil preconditioning might regulate miR-205/PINK1 axis to protect against hypoxia/reoxygenation-induced injury in cardiomyocytes.

Knockdown of miR-205 enhanced protective eff ect of remifentanil preconditioning against myocardial I/R injury

To establish in vivo model of myocardial I/R injury, rats were conducted with regional ischemia and reper-fusion treatment. Rats with myocardial I/R injury mod-el (AMI) showed higher expression of miR-205 com-pared with the sham group ( Fig. 5A ), while tail vein injection with miR-205 antagomir decreased miR-205 expression in AMI rats ( Fig. 5A ). However, preadmin-istration of remifentanil decreased miR-205 expression ( Fig. 5A ), and injection with miR-205 antagomir aggra-vated remifentanil-induced decrease of miR-205 expres-

sion ( Fig. 5A ). Lower expression of PINK1 and high-er expression of cleaved caspase were detected in AMI rats compared to the sham group ( Fig. 5B ), while injec-tion with miR-205 antagomir increased PINK1 and decreased cleaved caspase-3 expression in AMI rats ( Fig. 5B ). Moreover, preadministration of remifenta-nil increased PINK1 and decreased cleaved caspase-3 expression ( Fig. 5B ), and injection with miR-205 antag-omir aggravated remifentanil-induced increase of PINK1 and decrease of cleaved caspase-3 ( Fig. 5B ). Regional ischemia and reperfusion treatment induced myocardial I/R injury in rats with increased infarct size ( Fig. 5C ) and LDH activity ( Fig. 5D ). However, injection with miR-205 antagomir ameliorated the injury through decrease of infarct size ( Fig. 5C ) and LDH activity ( Fig. 5D ). More-over, preadministration of remifentanil decreased infarct size ( Fig. 5C ) and LDH activity ( Fig. 5D ), and injection with miR-205 antagomir aggravated remifentanil-induced

Fig. 5 . Knockdown of miR-205 enhanced protective eff ect of remifentanil preconditioning against myocardial I/R injury. (A) Ex-pression of miR-205 was increased in rats with myocardial I/R injury model (AMI), tail vein injection with miR-205 or preadministration of remifentanil antagomir decreased miR-205 expression, and injection with miR-205 antagomir aggra-vated remifentanil-induced decrease of miR-205 expression. N = 10. (B) Expression of PINK1 was decreased and cleaved caspase-3 was increased in rats with myocardial I/R injury model (AMI), tail vein injection with miR-205 or preadministra-tion of remifentanil antagomir increased PINK1 expression and decreased cleaved caspase-3, and injection with miR-205 antagomir aggravated remifentanil-induced increase of PINK1 and decrease of cleaved caspase-3. N = 10. (C) Infarct size was increased in rats with myocardial I/R injury model (AMI), tail vein injection with miR-205 or preadministration of remifentanil antagomir decreased the infarct size, and injection with miR-205 antagomir aggravated remifentanil-induced decrease of infarct size. N = 10. (D) LDH activity was increased in rats with myocardial I/R injury model (AMI), tail vein injection with miR-205 or preadministration of remifentanil antagomir decreased the LDH activity, and injection with miR-205 antagomir aggravated remifentanil-induced decrease of LDH activity. N = 10. ** p < 0.01.

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decrease of infarct size (Fig. 5C) and LDH activity (Fig. 5D). These results revealed that knockdown of miR-205 enhanced the protective effect of remifentanil precon-ditioning against myocardial I/R injury.

DISCUSSION

Anesthesia, such as with sevoflurane and propofol, has been implicated in cardioprotective effect against myo-cardial I/R injury (Lotz et al., 2020). Sevoflurane anesthe-sia could repress miR-135b-5p to reduce cardiomyocyte necrosis and apoptosis during myocardial I/R injury (Xie et al., 2017). Since remifentanil was widely used as anes-thesia, and demonstrated protective effect against myo-cardial I/R injury (Zhang et al., 2004). MiRNAs involved in remifentanil-mediated myocardial I/R injury were then evaluated in this study to unravel the underlying mecha-nism.

Hypoxia/reoxygenation could induce cell damage and inflammation in cardiomyocytes, and represent an in vit-ro cell model of myocardial I/R injury (Gao et al., 2017). This study firstly isolated primary cardiomyocytes from neonatal rats, and then treated the cells with hypox-ia/reoxygenation. Cell viability of cardiomyocytes was decreased while apoptosis was increased post hypoxia/reoxygenation treatment, confirming the cytotoxic effect. However, in line with previous study that precondition-ing with remifentanil could attenuate hypoxia/reoxygena-tion-induced injury in cardiomyocytes (Dou et al., 2016), this study also revealed that remifentanil preconditioning increased cell viability of hypoxia/reoxygenation-induced cardiomyocytes and decreased the apoptosis via reduce of cleaved caspase-3.

Data from qRT-PCR analysis demonstrated higher expression of miR-205 in hypoxia/reoxygenation-induced cardiomyocytes and hearts post regional ischemia and reperfusion treatment, suggesting potential relation between miR-205 and myocardial I/R injury. A previ-ous study has shown that miR-205 was decreased in plas-ma and heart samples of imatinib mesylatemice or dox-orubicin-induced cardiotoxicity in mice (Hanousková et al., 2019), and the decreased miR-205 protects cardio-myocytes via repression of myocardial apoptosis (Xuan et al., 2017). Here, knockdown of miR-205 increased cell viability of hypoxia/reoxygenation-induced cardiomyo-cytes preconditioned with remifentanil, while repressed the cell apoptosis and decreased protein expression of cleaved caspase-3. Moreover, injection with miR-205 antagomir aggravated remifentanil-induced decrease of infarct size and LDH activity in rat model with myocar-dial I/R injury, indicating that remifentanil precondition-

ing might decrease miR-205 to protect against myocardial I/R injury. Moreover, remifentanil could reduce inflam-mation and oxidative stress to attenuate myocardial inju-ry (Chen et al., 2020), inflammatory response and oxi-dative stress contribute to myocardial I/R injury (Liu et al., 2020). Since miR-205 was involved in high glucose-induced inflammation and oxidative stress in mesangial cells (Chen et al., 2019), remifentanil might also decrease miR-205 to regulate inflammation and oxidative stress in cardiomyocytes and demonstrate protective effect against myocardial I/R injury.

PINK1 was reported to regulate mitochondrial dys-function and oxidative stress, and participate in cardiac dysfunction and heart failure (Billia et al., 2011). PINK1 was reduced in hypoxia/reoxygenation-induced H9c2 cells, and forced PINK1 expression could alleviate myo-cardial I/R injury through regulation of mitochondrial dysfunction (Li et al., 2017). Our results also demonstrat-ed that remifentanil preconditioning enhanced PINK1 via reduce of miR-205 to repress hypoxia/reoxygenation-in-duced cell damage and myocardial I/R injury. The direct binding ability between miR-205 and PINK1 was validat-ed by luciferase activity assay. PINK1 was validated as target gene of miR-421-mediated mitochondrial fragmen-tation during myocardial infarction (Wang et al., 2015), as well as target gene of miR-27a/b (MiR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1)). In addition, circular RNA ACR could suppress autophagy through up-regulation of PINK1 to attenuate myocar-dial I/R injury (Zhou et al., 2019). Whether miR-205/PINK1 axis could regulate mitochondrial dysfunction and autophagy to participate in remifentanil-mediated myo-cardial I/R injury should be further investigated. Adminis-tration with PINK1 interference should also be performed in rat model with myocardial I/R injury post remifen-tanil to strength functional role of miR-205/PINK1 axis in remifentanil-mediated myocardial I/R injury.

In summary, this study provided evidence that remifen-tanil preconditioning could reduce miR-205 to enhance PINK1, repress hypoxia-reoxygenation-induced cell inju-ry in cardiomyocytes and myocardial I/R injury in rats. Although the downstream pathway remains elusive, results in this study shed new light on the development of potential target for ischemic cardiac injury.

Conflict of interest---- The authors declare that there is no conflict of interest.

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