Opioid δ1 and δ2 Receptor Agonist Attenuate Myocardial Injury Via mPTP in Rats With Acute Hemorrhagic Shock

Materials and Methods Forty-eight adult male SD rats were adopted 60-min hemorrhagic shock through removing 30% (5 mL) of the total blood volume, and followed by 2-h resuscitation with shed blood and L-lactated Ringer's solution. At the end of shock and prior to resuscitation, NS, δ 1 -OR agonist TAN-67 (10 mg/kg) and antagonist BNTX (3 mg/kg), and BNTX+TAN-67, DMSO, δ 2 -OR agonist Deltorphin II (1 mg/kg) and antagonist NTB (2 mg/kg), and NTB+Deltorphin II in 0.5 mL were administrated. Left ventricular function parameters were measured during the whole experimental period. Myocardial mitochondria were isolated to determine opening of mitochondrial permeability transition pore (mPTP). Morphologic changes in myocardium and mitochondria were observed by electron microscope. Results The hemodynamic indexes in group TAN-67 and group Deltorphin II were higher than control group at each time point during resuscitation, respectively ( P < 0.05). TAN-67 and Deltorphin II decrease but their antagonists BNTX and NTB increase the opening of mPTP ( P < 0.05). Myocardial and mitochondrial damage were attenuated in group TAN-67 and group Deltorphin II. Conclusions δ 1 -OR agonist TAN-67 and δ 2 -OR agonist Deltorphin II protect the heart by targeting the mPTP in rats with acute hemorrhagic shock. Key Words hemorrhagic shock delta OR hemodynamics mitochondrial permeability transition pore Introduction Acute hemorrhagic shock as a common urgent and serious condition mainly occurs in serious trauma and operations, which can cause life-threatening hypotension, multiple organ failure, and even death. Cellular dysfunction following hemorrhagic shock also occurs in many organs, including cardiovascular system. Although myocardium has strong tolerance to ischemia, its function still declines significantly due to hypoperfusion and damage of cytokine during acute hemorrhagic shock [1] . Currently, the primary measures for treatment of hemorrhagic shock include fluid resuscitation and administration of vasoactive drugs. However, reducing myocardial injury induced by shock may still be difficult and myocardial suppression may eventually occur even though above treatments have been carried out. Therefore, it is very important to lessen reperfusion damage and enhance myocardial function during early of resuscitation. It has been well documented that endogenous δ opioid peptide and receptor have a higher expression in heart compared with other organs [2] . The regulative roles of δ opioid peptide and receptor on myocardial contractile function under physiologic or pathologic conditions also have been demonstrated [3] . Many studies over the past decade have been conducted that exogenous opioid receptor (OR) agonists reduced ischemia/reperfusion injury when administered prior to ischemia or at the time of reperfusion [4] . Although δ-OR have been shown to confer ischemic preconditioning [5] or postconditioning [6] to the myocardium, their roles in providing extended protection to myocardium in hemorrhagic shock have not been well explored [7] . Recently, Summers et al. [8] described that DADLE, a nonselective δ OR agonist, appeared to increase survival rate during hemorrhagic shock. There are two subtypes (δ 1 , δ 2 ) of δ OR in brain and peripheral circulatory system. The two subtypes of δ receptors may serve distinctly different roles in the regulation of myocardial ischemia/reperfusion injury during hemorrhagic shock. High affinity and selectivity for δ 1 OR agonist TAN-67 could reduce myocardial infarct size and enhance myocardial function through opening K + ATP channel [9, 10] . In addition, studies found that δ 2 OR agonist could facilitate MAP recovery and prolong survival time after severe hemorrhagic shock [11–13] . However, the precise mechanisms of different δ OR agonist in hemorrhagic shock should be further investigated. Mitochondrial permeability transition pore (mPTP), as a nonspecific channel, remains closed during the ischemic period and only opens in the first few min of reperfusion. The open of mPTP can lead to mitochondrial swelling and efflux of cytochrome c , which induces apoptosis in the setting of ischemia/reperfusion injury [14] . Could δ OR agonists close mPTP to reduce myocardial injury during hemorrhagic shock when administered before resuscitation? In this study, acute hemorrhagic shock animal model was established according to a modification of Wigger's method [15] . The myocardial systolic function, myocardial morphologic structure, mitochondrial damage score, and changes of absorbance at 540 nm (A 540 ) of myocardial mitochondria were observed to explore protective effects of different δ OR agonist on myocardial injury induced by acute hemorrhagic shock, and investigate the role of myocardial mPTP in the protection induced by δ OR activation. Materials and Methods All animal procedures used were in strict accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals. The approval of the ethical committee of the Fourth Military Medical University (Xi'an, P.R. China) was obtained. Animals and Grouping Forty-eight adult male Sprague Dawley rats weighing 250–300 g were obtained from Experimental Animal Center of the Fourth Military Medical University. The rats were randomized into eight subgroups ( n = 6) of two groups. Group 1: control group: NS (normal saline), group δ 1 OR agonist: TAN-67 (2-methyl-4aa-(3-hydroxyphenyl)-1, 2, 3, 4, 4a, 5, 12, 12a α -octahydroquinolino[2, 3, 3-g]isoquinoline dihydrobromide, Tocris, Bristol, Britain) (10 mg/kg i.v.); group δ 1 -OR antagonist: BNTX (7-benzylidenenaltrexone maleate, Tocris) (3 mg/kg i.v.); group BT (BNTX+TAN-67). TAN-67 and BNTX were dissolved in 0.5 mL 0.9 % normal saline. Shultz et al. [16] demonstrated that pretreatment with TAN-67 (10 mg/kg) significantly reduced infarct size in the ischemic rat heart. The cardioprotection conferred by TAN-67 was subsequently reversed with the use of the BNTX (3 mg/kg). Therefore, we chose the same dose of TAN-67 and BNTX in our experiment and we found that the dose were appropriate after preliminary experiment. Group II: control group: DMSO (Sigma, St. Louis, MO, USA), group δ 2 -OR agonist: deltorphin II ([D-Ala 2 ]-Deltorphin II, Tocris, Britain) (1 mg/kg i.v.); group δ 2 -OR antagonist: NTB (17-(cyclopropylmethyl)-6, 7-didehydro-3, 14β-dihydroxy-4, 5 α -epoxy-6, 7-2,3'-benzo furanomorphinan mesylate; Tocris) (2 mg/kg i.v.) and group ND (NTB+Deltorphin II). According to product information and related references [17] , Deltorphin II was dissolved in 6 % DMSO to 0.5 mL. We found that 1 mg/kg Deltorphin II was the minimum effective dosage during preliminary experiment in which 0.5 mg/kg, 1 mg/kg, and 2 mg/kg Deltorphin II were used in the rats with hemorrhagic shock, it could effectively improve myocardial function and recovery of MAP; 2 mg/kg NTB could effectively reverse the role of Deltorphin II and have a lower fatality rate. Hemorrhagic Shock Animal Model Animal model of hemorrhagic shock was established by a modification of Wigger's method [18] . Briefly, rats were anesthetized with intraperitoneal injection of 30 mg/kg sodium pentobarbital. After tracheotomy and placement of an endotracheal tube, spontaneous breathing was preserved. The left femoral artery was catheterized for bleeding and infusion of shed blood. The right femoral artery was catheterized for measurement blood pressure. The right carotid artery was isolated, and a catheter was inserted into the left ventricle for hemodynamic measurement. The left jugular vein was catheterized for injection drugs and fluid. Esophageal temperature was maintained at 38–39 °C with a heating pad during experiment. After that, rat was injected with 3 mg/kg heparin and stabilized for 15 min. Then rats were bled for 10 min until the mean arterial pressure (MAP) of ∼40 mm Hg was obtained and maintained for 60 min by withdrawal further blood or infusion of shed blood. Approximately 30% (5 mL) of the total blood volume (TBV) was removed [19] . At the end of hemorrhagic shock, rats were given corresponding drugs at the rate of 1 mL/min. Resuscitation was begun by giving L-lactated Ringer's solution (21 mL/kg) and returning shed blood. MAP, left ventricular pressure (LVP), and positive and negative LVdp/dt were monitored continuously by a multichannel physiologic monitor RM-6200 (Chengdu Instrument Plant, Chengdu, China) for analysis. After 120 min resuscitation period, rats were euthanized for electron microscopy examination and detection of mPTP. The experimental procedure and group are diagrammed in Fig 1 . Electron Microscopy Examination and Quantified Score At the end of the experiment, a segment of myocardial tissue (1 mm × 1 mm × 1 mm) was taken from the root of the anterior papillary muscle of left ventricle. Myocardial ultrathin sections were elaborated according to standard procedures including fixing, incubation, rinse, gradient dehydration, embedding, ultrathin section. Changes in the ultrastructure of the myocardial tissues were observed under a JEM-2000EX transmission electron microscope (JEOL, Kyoto, Japan). Flameng's semi-quantitative analysis was used to analyze electron microscope specimens [20] . Five visions on each electron microscope sample and 20 mitochondrial on each vision were randomly selected to observe the myocardial damage. Score was the average of scores on all specimens in each group. Detection of mPTP At the end of experiment, myocardium was homogenated and gradient was centrifugated at low temperature (4 °C); myocardial mitochondria were isolated using Mitochondria Isolation Kit (Genmed Scientifics Inc., U.S.A, Boston, MA, USA) and identified by Janus green (Sigma). Changes in absorbance at 540 nm were measured by spectrometer (Molecular Devices, Sunnyvale, CA, USA) using Colorimetry mPTP Detection Kit (Genmed Scientifics Inc.). Opening of mPTP was determined by Ca 2+ induced mitochondrial swelling. The decrease in light scattering was closely paralleled with the percentage of mitochondrial population undergoing permeability transition. Statistical Analysis Data were expressed as a mean value ± SEM, and data were analyzed using SPSS software with P < 0.05 considered significant. HR, MAP, cardiac function values, were analyzed using repeated measures analysis of variance (ANOVA) to test for significant differences within and between groups. Further comparisons to evaluate mean differences at individual time points were made using an ANOVA with Student-Newman-Keuls (SNK-q ) analysis. Mortality and mitochondria quantified score were analyzed using one-way ANOVA to test for significant differences between groups. Log-rank analysis was used for further comparison of survival rate in each group. Results General Condition Body weight and total bleeding volume had no significant difference among groups ( P > 0.05 ) ( Table 1 ). All rats survived after 120 min of resuscitation. Changes in Hemodynamics There was no significant difference in HR, MAP and cardiac function among groups before bleeding ( P > 0.05 ). Hemorrhage produced significant slowing of the HR and decreasing of MAP, LVP, and ±LVdp/dt max in comparison with the corresponding pre-bleeding ( P < 0.05 ). Hemodynamics was improved in all groups after resuscitation. MAP, LVP and ±LVdp/dt max in group TAN-67 and group Deltorphin II were higher than in group NS and DMSO, respectively, at each time point during resuscitation, but HR was not significant difference in group TAN-67 and group Deltorphin II compared with other groups. These indexes in group BNTX and group NTB were not significantly difference compared with group NS and group DMSO, respectively ( P > 0.05 ), but MAP in group NTB recovered better than group DMSO within the last 30 min ( P < 0.05 ). ( Figs. 2 and 3 ) Changes in Myocardial Ultrastructure Quantified Score Myocardial ultrastructure of various groups showed varying degrees of hypoxic injury at the end of experiment. The myofilaments and mitochondria were normal in group TAN-67, but they were seriously damaged in Groups BNTX and group BT, which were similar to group NS ( Fig. 4 ). Structure of myofilaments and mitochondria were generally normal in group deltorphin II, but serious damages were found in groups NTB, ND, and DMSO. The myocardial damage was more serious using NTB, and there were a lot of vacuoles ( Fig. 5 ). Myocardial damage in each group was analyzed using Flameng's semiquantitative analysis. The higher scores indicated more serious damage. The score of mitochondria in group TAN-67 was less than group NS ( P < 0.05 ). Score was also significant decreased compared group deltorphin II with other groups ( P < 0.05 ). Nucleus damage score of group TAN-67 was less than group NS ( P < 0.05 ), and it was decreased in group Deltorphin II compared with group DMSO. Myofilament damage score of group TAN-67 was less than group NS. Score was also significant decreased compared group Deltorphin II with other groups ( P < 0.05 ) ( Table 2 ). Changes in Absorbance at 540 nm (maxA 540 -minA 540 ) After myocardial mitochondria were isolated and verified by Janus green, A 540 of isolated mitochondria was measured by spectrometer. The optical density decreased in each group after adding in CaCl 2. Changes of A 540 (maxA 540 -minA 540 ) of group TAN-67 and group Deltorphin II were significantly less compared with group NS and group DMSO, respectively ( P < 0.05 ). But, BNTX and NTB significantly increased changes of A 540 compared with group NS and group DMSO, respectively ( P< 0.05 ) ( Figs. 6 and 7 ). It indicated that TAN-67 and Deltorphin II decrease but their antagonists BNTX and NTB increase the opening of mPTP. Discussion This study demonstrates that both δ 1 OR agonist TAN-67 and δ 2 OR agonist Deltorphin II administered at the beginning of resuscitation can promote hemodynamic recovery and protect integrity of myocardial ultrastructure and lessen myocardial ischemia/reperfusion injury in rat with hemorrhagic shock. These protective effects of TAN-67 and Deltorphin II can be eliminated by selective OR antagonists, BNTX and NTB. The inhibition of the opening of mPTP may be one of the important protective mechanisms of δ OR agonist in rat with hemorrhagic shock. Several studies have documented the beneficial roles of δ OR agonist for hemorrhagic shock. Summers et al. [8] demonstrated that the nonselective δ OR agonist DADLE (D-Ala 2 -D-Leu 5 -enkephalin) improved hemodynamic stability and attenuated myocardial injury during hemorrhagic shock in rat. Oeltgen et al. [12] found that pretreatment with δ OR agonist Delt-D var for 24 h decreased plasma lactate levels and improved hemodynamic stability and survival during hemorrhagic shock. The novel δ 2 OR agonist deltorphin E was found to facilitate the recovery of hemodynamic indices without accompanying fluid resuscitation, even after the onset of severe hemorrhagic shock [10] . Moreover, studies showed that another δ opioid agonist pentazocine, acting via δ receptors and K ATP + channels, improved outcome and post-resuscitation myocardial performance in a rat hemorrhagic shock model [21] . However, the myocardial protection roles and the mechanisms of hemodynamic stability during resuscitation of high selective δ 1 and δ 2 OR agonist in hemorrhagic shock should be further explored. In addition, the effects of δ 1 and δ 2 opioid receptor agonist on the opening of mPTP have not been investigated. TAN-67 has high affinity for δ OR in rat with 2070-fold lower affinity at the μ OR and 1600-fold lower affinity at the κ OR. The activity of TAN-67 is blocked by the putative δ 1 OR subtype selective antagonist BNTX but not by δ 2 OR antagonist naltriben. Therefore, TAN-67 is defined as a selective nonpeptide δ 1 receptor agonist. It also implied that there is a lack of cross-activity between δ 1 and δ 2 OR agonist [22] . Deltorphin II has high affinity for the δ 2 OR, whereas its binding to the μ OR is weak and extremely low for the κ OR, μ/δ, and κ/δ, selectivity was about 900 and 1000, respectively [23] . Our experimental results are consistent with these previous studies and indicate that both δ 1 and δ 2 OR agonist could promote hemodynamic recovery and protective integrity of myocardial ultrastructure. In addition, our study has more clinical potential because TAN-67 and Deltorphin II were administered at the onset of resuscitation. The cardioprotective effects of δ OR agonist have been consistently demonstrated in different models of ischemia/reperfusion injury in vivo as well as in vitro [5, 23, 24] . Most studies indicated that mechanism of cardioprotection of δ OR agonist was similar to ischemic preconditioning, in which brief periods of coronary artery occlusion before a prolonged ischemic insult reduced the degree of irreversible tissue damage and slowed the rate of adenosine triphosphate depletion [2, 25] . We found that myocardial tissues of various groups showed varying degrees of hypoxic injury at the end of experiment. The myofilaments and mitochondria were nearly normal in group TAN-67 and group Deltorphin II, but serious ischemic damages were found in others groups. Meanwhile, semiquantitative analysis of myocardium also found injuries that were significantly less in group TAN-67 and group Deltorphin II than in other groups. These changes in myocardial ultrastructure indicated that preservation of myocardial ultrastructure integrity was also the key to recovery of myocardial function. Mitochondrium as an arbitrator of cell death can not only provide energy for cells, but also can trigger apoptosis or death [25] . Thus the protection of TAN-67 and Deltorphin II to mitochondrial structure and function directly improve myocardial function. Studies showed that mPTP located on mitochondria was the key factor that elicited ischemia/reperfusion injury [26] . mPTP as a nonspecific channel, remains close during the ischemic period and only opens in the first few minutes of reperfusion. The opening of mPTP could lead to mitochondrial swelling and efflux of cytochrome c , which induced apoptosis in the setting of ischemia/reperfusion injury. To further clarify cellular mechanisms of cardioprotective effect by activation of δ OR, we studied the role of mPTP in mediating this effect in hemorrhagic shock rats. Our present results showed that both δ 1 and δ 2 OR agonist diminished opening of mPTP, protected structure of myocardium, and improved function of mitochondria. Argaud [26] demonstrated that ischemic postconditioning enhanced the tolerance of myocardium to Ca 2+ overload and decreased intracellular Ca 2+ concentration and opened mPTP to lessen myocardial injury. The above experimental results were consistent with our previous supposition that improvement of myocardial function and preservation of myocardial ultrastructure integrity was probably mediated by inhibiting opening of mPTP. However, the mechanism through which δ OR agonist prevent opening of mPTP at the time of myocardial reperfusion is currently unclear. Jang et al. [27] examined the effects of OR activation on the mPTP in isolated rat hearts. They found that the protective effects produced by morphine given at reperfusion reversed the δ OR antagonists naltrindole and BNTX, implying that δ ORs are involved in the action of morphine. Besides, they found the protective effects was also reversed by L-NAME, the guanylyl cyclase inhibitor ODQ, and the PKG inhibitor KT5823, suggesting that the action of morphine was mediated by the NO/cGMP/PKG pathway. The effect of morphine was further abrogated by the mPTP opener atractyloside, indicating that OR activation may protect the heart by modulating the mPTP opening. In cardiomyocytes, morphine prevented H 2 O 2 – induced loss of mitochondrial transmembrane potential (ΔΨm), which was reversed by L-NAME, suggesting that morphine inhibits mPTP opening via a NO-dependent mechanism. Further studies indicated that glycogen synthase kinase 3β (GSK-3β) was translocated to mitochondria by morphine and interacts with the voltage-dependent anion channel (VDAC), a major component of the mPTP. Therefore, it seems that δ OR activation prevents reperfusion injury by inhibiting mPTP opening via a NO-dependent mechanism. Translocated GSK- 3β may interact with VDAC leading to inhibition of mPTP opening. We noted that administration of δ OR agonist after hemorrhage led to a rapid, short latency recovery of MAP, returning MAP to baseline levels. Accompanying the increase in MAP was an increase in HR and ± dp/dt max , suggesting that the recovery in MAP might be due to increased cardiac function. All of these responses demonstrated that δ OR agonist could improve myocardial contractile function. It seems that there were better effects on pressure recovery of Deltorphin II compared with TAN-67 after 30 min recovery, but we could not directly compare the effects of Deltorphin II and TAN-67 on hemodynamics due to different solvents used for Deltorphin II and TAN-67. Meanwhile, we also found that TAN-67 had revealed better myocardial ultrastructure and mitochondria protection compared with Deltorphin II. Although we could not conclude that δ 1 and δ 2 OR agonist have distinct roles during resuscitation of hemorrhagic shock, it is reasonable that they have different roles on myocardial protection at hemorrhagic shock. Fryer et al. [28] demonstrated that the protective effect of DADLE on reducing infarct size was completely abolished by the selective δ 1 but not the δ 2 OR antagonist. In both rat and rabbit isolated heart preparations, Schultz et al. [5] and Bolling et al. [29] independently demonstrated that activation of δ 1 OR reduced infarct size but did not improve postischemic myocardial function. However, a selective δ 2 antagonist completely abolished the improved postischemic myocardial mechanical function that followed opioid activation [9, 30] . These studies supported the concept that δ 1 OR mainly attenuated myocardial structure injury, and δ 2 OR was mainly conducive to the recovery of hemodynamics. In summary, the results of our study are the first to demonstrate that two subtypes of δ OR agonist produced a reduction in ischemia/reperfusion injury in rats with hemorrhagic shock, and mPTP played an important role in myocardial protection. These findings have important clinical potential, since synthetic opioid derivatives will not only alleviate pain postoperatively but also provide a cardioprotective effect for patients suffering from hemorrhagic shock or receiving cardiac surgical interventions. However, the exact mechanism of TAN-67 and Deltorphin II against hemorrhagic shock and the suitable dose are still unclear. Subsequent studies are required to determine the exact nature of the interaction between the δ 1 and δ 2 OR subtypes in hemodynamic regulation after hemorrhage. 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