Remifentanil preconditioning alleviates myocardial ischemia/reperfusion injury in rats via activating Jagged-1/Notch signaling pathway

: Ischemic heart diseases have emerged as great threats to human health. Nowadays, restoration of cardiac blood flow supply is widely regarded as a feasible treatment choice for ischemic heart diseases; however, this intervention would contradictorily elicit reperfusion injury. Recently, myocardial ischemia/reperfusion injury (MI/RI) has aroused widespread public concerns. Remifentanil, an ultra-short acting opioid analgesic, is frequently used for surgical anesthesia. Previous studies have demonstrated the cardioprotective effects of remifentanil preconditioning in clinical practice and in vitro experimental models; however, its exact mechanisms remain largely unclear. This study aimed to further evaluate the protective effects of remifentanil preconditioning against MI/RI and elucidate the potential molecular mechanisms. Rat models of MI/RI were successfully established via ligation of left anterior descending coronary artery for 30 minutes and restoration of blood flow for 2 hours. Herein, animal experiments displayed that remifentanil preconditioning could alleviate myocardial damage in rat models of MI/RI. Consistently, cell model experiments implied that remifentanil preconditioning attenuated hypoxia/reoxygenation exposure-induced injury in rat cardiomyocytes. Moreover, our findings verified the involvement of Notch signaling pathway in the protective effects of remifentanil preconditioning. In addition, mechanistic studies revealed that remifentanil preconditioning could up-regulate Jagged-1 expression and that Jagged-1 mediated the cardioprotective effects of remifentanil preconditioning through activating Notch signaling pathway. Taken together, our data indicate that remifentanil preconditioning ameliorates myocardial damage in rat MI/RI models via Jagged-1-mediated Notch signaling pathway activation. Thus, this study may offer some novel clues for understanding the cardioprotective mechanisms of remifentanil preconditioning against MI/RI.

Abstract: Ischemic heart diseases have emerged as great threats to human health.
Nowadays, restoration of cardiac blood flow supply is widely regarded as a feasible treatment choice for ischemic heart diseases; however, this intervention would contradictorily elicit reperfusion injury. Recently, myocardial ischemia/reperfusion injury (MI/RI) has aroused widespread public concerns. Remifentanil, an ultra-short acting opioid analgesic, is frequently used for surgical anesthesia. Previous studies have demonstrated the cardioprotective effects of remifentanil preconditioning in clinical practice and in vitro experimental models; however, its exact mechanisms remain largely unclear. This study aimed to further evaluate the protective effects of remifentanil preconditioning against MI/RI and elucidate the potential molecular mechanisms. Rat models of MI/RI were successfully established via ligation of left anterior descending coronary artery for 30 minutes and restoration of blood flow for 2 hours. Herein, animal experiments displayed that remifentanil preconditioning could alleviate myocardial damage in rat models of MI/RI. Consistently, cell model experiments implied that remifentanil preconditioning attenuated hypoxia/reoxygenation exposure-induced injury in rat cardiomyocytes. Moreover, our findings verified the involvement of Notch signaling pathway in the protective effects of remifentanil preconditioning. In addition, mechanistic studies revealed that remifentanil preconditioning could up-regulate Jagged-1 expression and that Jagged-1 mediated the cardioprotective effects of remifentanil preconditioning through activating Notch signaling pathway. Taken together, our data indicate that remifentanil preconditioning ameliorates myocardial damage in rat MI/RI models via Jagged-1-mediated Notch signaling pathway activation. Thus, this study may offer some novel clues for understanding the cardioprotective mechanisms of remifentanil

Introduction
In recent decades, ischemic heart diseases have emerged as great threats to human health all over the world [1][2][3]. It is reported that a vast number of patients are suffering from ischemic heart diseases, which brings huge pressures on living quality [4,5]. Furthermore, it is well known that myocardial ischemia is closely associated with morbidity and mortality of patients with coronary artery disease and acute myocardial infarction [6,7]. Nowadays, immediate restoration of cardiac blood flow supply is widely considered as a feasible clinical practice to ameliorate ischemic heart diseases; however, this intervention usually brings some unwanted side effects and would paradoxically elicit reperfusion injury [8][9][10].
It is widely acknowledged that myocardial ischemia/reperfusion injury (MI/RI) may facilitate the deterioration of cardiac functions via suppressing ventricular contraction as well as triggering arrhythmia, stroke and heart failure [11][12][13]. Hence, there is an urgent demand to develop effective pharmacological agents and unveil the potential mechanisms underlying the cardioprotective effects. Furthermore, mounting evidence has manifested that ischemic preconditioning is very beneficial to mitigating MI/RI in mice and rats, which could restrain infarction size, inhibit oxidative stress, enhance cardiomyocyte viability, and avoid inducing lethal arrhythmia [14][15][16][17]. It is well documented that remifentanil, a fentanyl derivative, could be rapidly metabolized and degraded by nonspecific esterases in the blood and tissues, making it attractive for intraoperative titration and fast recovery [18,19]. Nowadays, remifentanil is frequently used as an ultra-short acting opioid analgesic in surgical anesthesia due to its unique pharmacokinetic characteristics [20,21].
Notably, previous researches have demonstrated that remifentanil preconditioning exhibits a powerful cardioprotective capability in clinical practice and in vitro experimental models [22][23][24][25][26]. Nonetheless, the exact mechanisms underlying the cardioprotective effects are still not fully understood.
The current study aimed to further evaluate the cardioprotective effects of remifentanil preconditioning and illuminate the potential molecular mechanisms involved. Herein, we investigated the impacts of remifentanil preconditioning in both experimental rat models and hypoxia/reoxygenation-exposed cardiomyocytes, and assessed the possible involvement of Notch signaling pathway in the cardioprotective effects. Collectively, our findings indicate that remifentanil preconditioning attenuates MI/RI in rats via Jagged-1-mediated activation of Notch signaling pathway.
Hence, the present study may offer some novel clues for understanding the mechanisms underlying the cardioprotective effects of remifentanil preconditioning.

Animals
Male Sprague-Dawley rats ( fixed on an operation table after being anesthetized via an intraperitoneal injection of 2% pentobarbital sodium. Absence of corneal reflex was considered as an indicator of successful anesthesia. The hearts were exposed and a suture was passed around the left anterior descending (LAD) coronary artery. The LAD coronary was occluded for 30 min through tightening the snare, followed by 2 hours' blood flow restoration. As for the sham model, a suture was placed around the LAD coronary without ligation.

Adeno-associated virus vector (AAV) construction
Recombinant adeno-associated virus vectors carrying Jagged-1 (AAV-Jagged-1) were constructed by Shanghai GeneChem Co. Ltd (Shanghai, China). Besides, adeno-associated virus vectors carrying green fluorescent protein (AAV-Con) served as the negative control. treatment. As for the sham group and I/R group, rats were given normal saline. In the second animal experiment, rats were randomly assigned into 4 experimental groups (n=4): (i) sham group; (ii) I/R group; (iii) I/R+AAV-Con; (iv) I/R+AAV-Jagged-1. For I/R+AAV-Con group and I/R+AAV-Jagged-1 group, rats were administrated 100 μl of AAV-Con and AAV-Jagged-1 (5 × 10 9 PFU/mL) via tail intravenous injection in 72 hours before I/R treatment, respectively. At the end of animal experiments, rats were intraperitoneally injected with 2% pentobarbital sodium for euthanasia, then blood and heart samples were collected for subsequent analysis.

Measurement of myocardial infarction size via TTC staining
Myocardial infarction size was evaluated via 2,3,5-triphenyltetrazolium chloride

Western blotting analysis
Total proteins were extracted in a RIPA buffer containing proteinase inhibitor

In vitro hypoxia/reoxygenation (H/R) experiments
In order to induce I/R injury, H9c2 cells were incubated in hypoxic medium and exposed to hypoxic conditions (95% N2 + 5% CO2) provided by a hypoxia chamber   System (BD Biosciences).

Statistical analysis
Experimental data were expressed as the mean ± standard deviation. Statistical analysis was conducted via SPSS 22.0 software (SPSS Inc., Chicago, IL, USA).
Differences among three or more groups were evaluated using one-way analysis of variance (ANOVA) and Duncan's post-hoc test. Differences were considered to be statistically significant when P-value < 0.05.

Remifentanil preconditioning alleviates myocardial damage and activates Notch signaling pathway in rat models of MI/RI
Herein, MI/RI models were successfully established via ligation of left anterior descending coronary for 30 minutes and subsequent restoration of blood flow for two hours (Figure 1A and 1B). Firstly, we investigated the effects of remifentanil preconditioning on MI/RI in rat models. As shown in Figure 1A, higher LDH activity was observed in the coronary effluent of rats from the I/R group compared with the sham group; remifentanil preconditioning significantly reversed the increase in LDH activity triggered by ischemia/reperfusion (I/R) treatment. Furthermore, TTC staining demonstrated that remifentanil preconditioning remarkably mitigated the myocardial infarction of I/R-treated rats (Figure 1B), suggesting the protective effects of remifentanil preconditioning against I/R-induced myocardial damage. Furthermore, the levels of HIF-1 in the cardiac tissues were determined before and after remifentanil preconditioning. Notably, remifentanil preconditioning triggered a significant increase in HIF-1 level ( Figure 1C). Subsequently, the involvement of Notch signaling pathway in the alleviating effects of remifentanil preconditioning was evaluated via Western blotting analysis. As evident from Figure 1D, remifentanil preconditioning led to a dramatic increase in the expression levels of NICD and Hes1 proteins, indicating the activation of Notch signaling pathway. Taken together, the above findings imply that remifentanil preconditioning ameliorates myocardial damage and activates Notch signaling pathway in rat MI/RI models.

Remifentanil preconditioning promotes Jagged-1 expression in damaged rat myocardial tissues and cardiomyocytes
In order to further evaluate the possible involvement of Notch signaling pathway in the protective effects of remifentanil preconditioning, the expression patterns of its two crucial ligands, namely Jagged-1 and Jagged-2, were determined using qPCR analysis and Western blotting. As presented in  Figure 2C). To sum up, these results suggest that remifentanil preconditioning facilitates Jagged-1 expression in damaged rat myocardial tissues and cardiomyocytes.

Remifentanil preconditioning ameliorates H/R-induced injury in rat cardiomyocytes
Herein, we explored whether remifentanil preconditioning could exert protective effects against H/R-induced injury in an in-vitro cell model. As determined by CCK8 assays, remifentanil preconditioning dramatically ameliorated the decrease in H9c2 cell viability induced by H/R treatment ( Figure 3A). As shown in Figure 3B,

remifentanil preconditioning triggered a notable decrease in ROS accumulation in
H9c2 cardiomyocytes in comparison with the H/R group. Furthermore, a much lower proportion of apoptotic H9c2 cells was observed in the RPC+H/R group compared with that in the H/R group ( Figure 3C). Western blotting analysis revealed that a lower cleaved caspase-3 expression level and a higher Bcl2 expression level were found in the RPC+H/R group in comparison with the H/R group (Figure 3D), which was consistent with the results about flow cytometry ( Figure 3B). In addition, the effects of remifentanil preconditioning on Jagged-1/Notch signaling pathway in H/R-challenged H9c2 cardiomyocytes were also evaluated. As illustrated in Figure 3D, remifentanil preconditioning markedly elevated the protein expression levels of Jagged-1, NICD and Hes1 in H/R-treated cardiomyocytes, indicating the activation of Jagged-1/Notch signaling pathway. Collectively, the above findings demonstrated that remifentanil preconditioning could relieve H/R-induced damage in rat cardiomyocytes. On the basis of the findings mentioned above, it was speculated that Jagged-1 may serve as a crucial player in mediating the cardioprotective effects of remifentanil preconditioning. In order to elucidate the potential molecular mechanisms underlying the protective effects of remifentanil preconditioning, we then evaluated the impact of Jagged-1 over-expression on H/R-challenged H9c2 cardiomyocytes. As presented in Figure 4A and 4C, Jagged-1 over-expression significantly enhanced the viability of H/R-treated H9c2 cardiomyocytes and suppressed their apoptosis.

Jagged-1 over-expression attenuates the damage of H/R-challenged rat cardiomyocytes
Western blotting analysis showed that Jagged-1 over-expression notably repressed cleaved caspase-3 protein expression and facilitated Bcl2 protein expression ( Figure   4D). Furthermore, it was found that Jagged-1 over-expression dramatically reduced the accumulation of ROS in H9c2 cardiomyocytes ( Figure 4B). Besides, Western blotting analysis displayed that Jagged-1 over-expression promoted the activation of its downstream Notch signaling pathway ( Figure 4D). To summarize, these results imply that Jagged-1 over-expression attenuates the damage of H/R-challenged rat cardiomyocytes, suggesting that Jagged-1 over-expression exhibits similar cardioprotective effects with remifentanil preconditioning.

Inhibition of Notch signaling pathway destroys the protective effects of remifentanil preconditioning against H/R-induced cardiomyocyte injury
With the purpose of further clarifying the possible mechanisms underlying the cardioprotective effects of remifentanil preconditioning, the influence of specific inhibition of Notch signaling pathway on H/R-exposed H9c2 cardiomyocytes was assessed. As evident from CCK8 assays, specific inhibition of Notch signaling pathway significantly reversed the promoting effects of remifentanil preconditioning on H/R-exposed H9c2 cell viability ( Figure 5A). As presented in Figure 5B and 5C,

Jagged-1 over-expression exerts protective effects against myocardial injury in rat models
In rat MI/RI models, we explored the impact of Jagged-1 over-expression on damage. As displayed in Figure 6A, Jagged-1 over-expression dramatically attenuated the increase in LDH activity of the coronary effluent from I/R-challenged rats. Besides, TTC staining showed that Jagged-1 over-expression significantly alleviated the myocardial infarction of I/R-treated rats ( Figure 6B). Additionally, Western blotting analysis demonstrated that Jagged-1 over-expression markedly elevated the expression levels of NICD and Hes1 proteins in the myocardial tissues of I/R-challenged rats, suggesting the activation of Notch signaling pathway ( Figure 6C).
Taken together, these results indicate that Jagged-1 over-expression exerts protective effects against myocardial damage in rat models, which is in consistency with the findings in vitro. Furthermore, the key findings that remifentanil preconditioning attenuates MI/RI in rats via activating Jagged-1/Notch axis have also been summarized in the proposed schematic presentation (Figure 7).

Discussion
In recent decades, ischemic heart diseases have already become increasingly prevalent worldwide and have imposes heavy burdens on public health [27,28]. It is widely acknowledged that ischemic heart diseases remain one of the leading causes of cardiovascular disorder-related deaths all around the world. Up to now, restoration of cardiac blood flow supply is still a routine treatment approach for myocardial ischemia; nevertheless, this reperfusion therapy strategy usually results in some severe adverse effects, such as stroke, ventricular contraction dysfunction, and myocardial infarction [29,30]. In addition, accumulating studies have demonstrated that ischemic preconditioning could effectively ameliorate MI/RI in experimental rat models. Zhang et al. found that dexmedetomidine preconditioning attenuated MI/RI in rats via inhibiting the HMGB1/TLR4/MyD88/NF-κB pathway [14].
Liang et al. revealed that coenzyme Q10 preconditioning alleviated MI/RI and improved cardiac function in rats through regulation of oxidative stress and myocardial apoptosis [15]. Therefore, it is of vital necessity to develop effective ischemic preconditioning measures for cardioprotection and illustrate the relevant action mechanisms.
It is worth noting that remifentanil preconditioning has been demonstrated to display potent cardioprotective activity and improve heart function in both clinical application and experimental studies [22,23,26]. Nevertheless, the exact molecular mechanisms for the cardioprotective effects of remifentanil preconditioning remain largely unclear. Thus, the present study further investigated the influences of remifentanil preconditioning in both experimental rat models and hypoxia/reoxygenation-challenged H9c2 cardiomyocytes. Herein, rat MI/RI models were successfully established via 30 minutes' ligation of left anterior descending coronary artery and subsequent two hours' blood flow restoration. It is well acknowledged that heart damage is usually featured by myocardial infarction and cytoplasmic LDH release [31,32]. Moreover, it is widely acknowledged that oxidative stress and cardiomyocyte apoptosis are crucial biological events during the process of MI/RI development [33][34][35][36]. Based on rat model experiments, it was found that remifentanil preconditioning remarkably alleviated the increase in myocardial infarction size and cytoplasmic LDH release induced by I/R treatment. Furthermore, remifentanil preconditioning led to a remarkable increase in HIF-1 level the cardiac tissues. Notably, recent studies have revealed that elevated HIF-1 could protect the organs from I/R-induced damage through potentiating antioxidant activity and facilitating cell survival [37][38][39]. Besides, cell model experiments showed that remifentanil preconditioning significantly reversed the increase in intracellular ROS accumulation and apoptosis of H9c2 cardiomyocytes triggered by hypoxia/reoxygenation exposure. Taken together, our in vivo and in vitro experiments manifested the cardioprotective effects of remifentanil preconditioning.
It is well documented that Notch signaling pathway is a highly evolutionally conserved intercellular communication mechanism, which plays important roles not only in embryonic development but also in inflammation, cell differentiation, cell fate decision and cell apoptosis [40,41]. Furthermore, accumulating researches have implied that activation of Notch signaling pathway would facilitate organ protection and improve heart function via repressing oxidative stress and enhancing cell survival [42][43][44][45]. In order to illustrate the potential molecular mechanisms by which remifentanil preconditioning exerted its cardioprotective effects, we assessed the Collectively, the current study may expand our knowledge regarding the potential mechanisms underlying the cardioprotective effects of remifentanil preconditioning, thereby facilitating a better understanding for MI/RI treatment.

Declaration of conflict of interest
No conflict of interest exists in the current study.

Data availability statement
The data generated or analyzed during this study are available from the corresponding author on rational request.