Although 40–60% of patients achieve the return of spontaneous circulation (ROSC) after cardiac arrest and cardiopulmonary resuscitation (CA-CPR), the postdischarge survival rate of these patients is less than one third (1–3). Myocardial injury after CA is one of the primary reasons for postarrest death despite successful resuscitation and ROSC (4). Therefore, the development of methods for reducing myocardial injury after CA is essential and of significant value.
“Mild hypothermia is an effective method currently in clinical use to improve human survival” (5). Unfortunately, recent reports have questioned the efficacy and safety of prehospital therapeutic hypothermia, and there is no clear evidence that mild hypothermia improves myocardial function (5). Therefore, the development of pharmacologic strategy to improve myocardial function after CA-CPR and survival is necessary. No pharmacologic interventions that provide cardioprotection for patients suffering from CA are currently available. Endocannabinoids play essential roles in several types of heart diseases by virtue of their hypothermic, vasorelaxant, and antioxidant properties and their ability to alter cardiometabolic risk factors (6). The endocannabinoid system comprises cannabinoid receptors; cannabinoid ligands, such as 2-arachidonoylglycerol (2-AG); and enzymes, such as monoacylglycerol lipase (MAGL) (7). The cannabinoid 1 agonist WIN 55, 212-2 has been shown to significantly improve cardiac function after resuscitation (6). However, the use of cannabinoid receptor agonists without organ tissue selectivity may result in cannabinoid-related complications that limit their use (7). Therefore, cannabinoid receptor activation by endogenously increasing cannabinoid ligands, such as 2-AG, may also be beneficial for resuscitation.
MAGL is an endogenous cannabinoid hydrolase that specifically hydrolyzes 2-AG to produce arachidonic acid (AA) (7–9). Although multiple studies have addressed liver, lung, and brain protection by means of MAGL inhibitors (10–13), to the best of our knowledge, no approach involving the regulation of endocannabinoid actions in interventions for improving outcomes and cardioprotection in CA-CPR have been reported.
In this study, we investigated whether blocking MAGL protects against postresuscitation myocardial injury and improves survival in a rat model of CA-CPR.
MATERIALS AND METHODS
Preparation of the Experimental Animals
Sprague-Dawley male rats weighing 450–550g at the age of 16–18 weeks were obtained from the Chengdu Dashuo Experimental Animal Center in Sichuan of China. All experimental procedures were conducted in accordance with the 2006 guidelines of the Ministry of Science and Technology of China on the Humane Care of Experimental Animals (14 , 15), and this study protocol was approved by the Animal Care and Use Committee of West China Hospital of Sichuan University (Protocol number 2016042). Rats had free access to food and water.
Asphyxial CA Model
We established the 8-minute asphyxia CA-CPR model based on the Katz L method with a slight modification (16). Briefly, each rat was anesthetized with pentobarbital sodium solution (50 mg/kg). When the rat showed no signs of sensation, a 14-G catheter was inserted into the trachea with oral assistance by a laryngoscope. During this process, the end-tidal CO2 (PETCO2) and rectal temperature (T°C) were monitored with a Multifunctional Monitor System (PHILIPS, Boblingen, Germany). Two PE-50 catheters (Smith Medical, Kent, United Kingdom) were inserted into the femoral artery and vein for blood pressure measurement and infusion of medicine. After the operation, a muscle relaxant (Vecuronium, 2 mg/kg) was given, and the ventilator was connected to offer mechanical ventilation (tidal volume 10 mL/kg, respiratory rate 60/min). Vecuronium (1 mg/kg) was given again 1 minute before asphyxia. During this procedure, the rectal temperature was maintained at 36.0–37.0°C by adjusting a heating blanket, and PETCO2 was maintained between 35 and 45 mm Hg. To maintain anesthesia, 10 mg/kg pentobarbital sodium was given by intraperitoneal injection every hour or when necessary. At the end of the 8-minute asphyxia procedure, CPR was initiated by unclamping the tracheal tube and reconnecting the ventilator to 100% oxygen for inhalation, injecting epinephrine (0.02 mg/kg) and sodium bicarbonate (1 mEq/kg), and applying external chest compressions (200 compressions/min). Successful ROSC was defined as an initial return of sinus electrocardiogram rhythm and blood pressure exceeding 60 mm Hg that lasted for at least 5 minutes. The clamp on the tracheal tube was released immediately after 8 minutes of asphyxia.
The flow diagram of the experimental groups is shown in Figure 1A. At 1 minute after ROSC, the rats were divided into the CPR or CPR + URB602 group by using a random number table. Rats in the CPR + URB602 group were injected with a loading dose of URB602 (5 mg/kg) (Cayman Chemical, Ann Arbor, MI) 1 minute after ROSC and a maintenance dose of 0.5 mg/kg at 24 hours after ROSC. However, the rats in the CPR group were injected with equivalent volumes of vehicle solution (dimethyl sulfoxide:Tween 80:saline = 1:1:18). The sham rats underwent the same procedures that were performed on rats in the CPR and CPR + URB602 group minus CA or asphyxia and were injected with equivalent volumes of vehicle solution immediately and 24 hours after all procedures were completed. All injections were administered intraperitoneally.
The present study included three parts (Fig. 1B). First, 44 rats that showed ROSC were randomly divided into the CPR and CPR + URB602 groups (22 rats in each group). The 7-day survival rates and hemodynamic variables were evaluated in these 44 experimental rats plus five sham rats. Next, additional surviving rats were assigned to the sham, CPR and CPR + URB602 groups (endpoint: six rats in each group) to determine the quantities of serum creatine kinase MB isoenzyme (CKMB); the levels of ligands, including anandamide, 2-AG, AA, prostaglandin (PG) E2, PGD2, and thromboxane (TX) B2 in the myocardial tissue; and the left ventricular (LV) ejection fraction (EF) and fractional shortening (FS) and to observe the structure and morphology of the myocardial and mitochondrial cells by light and electron microscopy at 6 hours after ROSC. Finally, myocardial cells were extracted from additional rats in the sham, CPR and CPR + URB602 groups (endpoint: six rats in each group) 15 minutes after ROSC, and mitochondria were extracted from myocardial cells to evaluate the opening of the mitochondrial permeability transition pore (mPTP).
Determination of Endocannabinoid and Eicosanoid Metabolism in Myocardial Tissues.
Heart samples collected 6 hours after ROSC were immediately frozen in liquid nitrogen and stored in a –80°C refrigerator until use. Then, 1 mL of 0.1% ice-cold formic acid was added as a homogenization buffer followed by an ice-cold spiking solution of deuterated endocannabinoids in acetonitrile and 3 mL of ice-cold ethyl acetate/hexane (9:1) as the extraction solvent. The mixture was homogenized by ultrasonication in an ice-bath and then centrifuged at 8,000 rpm for 15 minutes at 4°C. The upper organic phase was evaporated to dryness under nitrogen and reconstituted in 100 μL of acetonitrile. After centrifugation at 20,000 rpm for 15 minutes at 4°C, a 5-μL aliquot of the sample was injected into the LC–MS/MS system (Agilent 1260–6460, Santa Clara, CA) for analysis (17).
Hemodynamics and Survival Rate.
The mean arterial pressure (MAP) and heart rate (HR) of the rats in the sham, CPR and CPR + URB602 groups were recorded every 5 minutes from before resuscitation until 1 hour after ROSC. The survival times of the same rats were recorded at 6, 24, 48, 72, and 168 hours after ROSC.
Measurements of Myocardial Function and Serum CKMB.
The LVEF and LVFS were measured (18) at 6 hours after ROSC with a GE vivid ultrasound (GE VIVID 7, GE Healthcare, Milwaukee, WI) by two testers who were blinded to the experimental groups. At the same time, blood samples were collected from each rat for CKMB measurements. Blood was centrifuged for 10 minutes at 3,000 rpm; The supernatant was employed to measure CKMB activity with an automa tic biochemical analyzer (Mindray BS-120, Shenzhen, China).
Light and Electron Microscopy of Myocardial Tissue.
Six hours after ROSC, LV apical tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sliced and stained with hematoxylin and eosin for observation under a microscope. Simultaneously, apex tissue of 1 mm × 1 mm × 1 mm was immediately sampled, and the same tissue was prefixed with a mixed solution of 3% glutaraldehyde. Next, the LV tissue was post fixed in 1% osmium tetroxide, dehydrated in an acetone series, infiltrated with Epox 812 for an extended time, and embedded. The sample was cut into semithin sections, which were stained with methylene blue, cut into ultrathin sections with a diamond knife and stained with uranyl acetate and lead citrate. Sections were examined with a transmission electron microscope (H-600IV; HITACHI, Tokyo, Japan). Mitochondria were observed in each microscopic field and counted in five independent, randomly selected microscopic fields at 15,000× magnification in each specimen (n = 6). The morphological results were examined by an independent anatomist who was blinded to the grouping. The degree of mitochondrial damage was judged according to the semiquantitative score of FlaMeng (19).
Mitochondria were isolated from rat hearts 15 minutes after ROSC or the sham operation by differential centrifugation according to a previously reported method (20). mPTPs were measured by monitoring a decrease of absorbance at 540 nm, which is associated with mitochondrial swelling, as reported in the literature (21). The decrease of absorbance was monitored for 13 minutes at 37°C, with the reduction of absorbance detected with an Epoch12 microplate reader (Biotek, Winooski, VT) at 540 nm. We obtained the maximum rate of the decrease in absorbance by linear regression over a period of 3 minutes.
Data are presented as the mean ± SEM. Repeated-measures or simple analysis of variance, as appropriate, was performed to compare two or more groups. All pairwise comparisons were carried out with pairwise t tests. Survival curves were determined with the Kaplan-Meier method and compared with the log-rank test. Values of p less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA) and SPSS 21.0 software (SPSS, Chicago, IL).
Baseline and Resuscitation Characteristics Before CA in Rats
A total of 96 rats were prepared in this study (Fig. 1B), and 17 rats constituted the sham group. Of the animals that were successfully resuscitated (n = 72), four were excluded from the study (three did not survive to the 6 hr observation endpoint, and one of them could not be achieved due to ventilator issues). There were no significant differences in body weight, EF, FS, T°C, PETCO2, MAP, or HR before resuscitation in any group (p > 0.05).
URB602 Exerts Bidirectional Control Over Endocannabinoid and Eicosanoid Metabolism
The levels of anandamide, 2-AG, AA, PGE2, PGD2, and TXB2 in the rats of the CPR and CPR + URB602 groups were elevated respectively 6 hours after ROSC, and compared with the levels in the CPR group, the CPR + URB602 group exhibited higher levels of 2-AG and lower levels of AA, PGE2, PGD2, and TXB2 (p < 0.05) (Fig. 2).
URB602 Improves Survival and Hemodynamics Following CA-CPR
Administration of URB602 markedly improved survival after 6, 24, 48, 72, and 168 hours; the survival rates increased from 72.7%, 45.5%, 40.9%, 36.4%, and 31.8% to 95.4%, 77.3%, 72.7%, 68.2%, and 63.6%, respectively. All five rats in the sham group survived. The log-rank test showed that the differences were significant (p < 0.05) (Fig. 3A). Compared with basal values, the MAPs of the CPR and CPR + URB602 groups decreased significantly after resuscitation, and the most notable decreases were observed at 15–20 minutes after ROSC (p < 0.05); however, the reduction in blood pressure was lower in the CPR + URB602 group than in the CPR group (Fig. 3C). After resuscitation, the HR was decreased in both groups, with no significant difference between the two groups (p > 0.05), and returned to the baseline level 1 hour after ROSC (Fig. 3B).
URB602 Reduces Myocardial Injuries Following CA-CPR
Myocardial fibrinolysis and disorganization occurred in both the CPR + URB602 and CPR groups 6 hours after ROSC. However, in the CPR + URB602 group, the extent of myocardial fibrinolysis and disorganization was significantly decreased (Fig. 4A). The EF and FS of the CPR + URB602 and CPR groups were significantly higher than those of the sham group (p < 0.05). However, the EF and FS of the CPR + URB602 group were higher than those of the CPR group (p < 0.05) (Fig. 4 B–D). The CKMB concentrations of all resuscitated rats were increased compared with those of the sham group (p < 0.05), but the CKMB concentration in the CPR group was higher than that in the CPR + URB602 group (p < 0.05) (Fig. 4E).
URB602 Preserves Mitochondrial Morphology and Function Following CA-CPR
Mitochondrial morphology in both the CPR and CPR + URB602 groups was characterized by mitochondrial outer membrane injury, unclear intima, and increased electron density in some ridges. In the CPR + URB602 group, mitochondrial structural injury was significantly reduced, the outer membrane structure was relatively intact, and the electron density of the intima was obviously decreased (Fig. 5A).The FlaMeng semiquantitative score was significantly increased (p < 0.05) 6 hours after ROSC in both the CPR and the CPR + URB602 groups compared with that in the sham group, whereas the FlaMeng semiquantitative score in the CPR + URB602 group was significantly lower than that in the CPR group (p < 0.05) (Fig. 5B). Compared with the sham group, both the CPR and CPR + URB602 groups showed decreases in their mitochondrial absorbance values at 540 nm, and calcium-induced the opening of the mPTP; however, URB602 reduced both mitochondrial swelling and mPTP opening after resuscitation (p < 0.05) (Fig. 5, C and D).
It was demonstrated that the MAGL inhibitor URB602 significantly reduces myocardial injury and improves the survival of rats after CA-CPR in this study. Myocardial dysfunction after resuscitation was manifested as tachycardia, hypotension, and decreased LVEF and LVFS (22) were decreased. Early hemodynamic instability and severe LV dysfunction may be one of the leading causes of early mortality after ROSC. We also observed that MAP at the first hour and the LVEF and LVFS at 6 hours after ROSC were significantly reduced. Administration of URB602 markedly improved the MAP, EF, and FS values, indicating that URB602 can efficiently improve myocardial dysfunction. Additionally, we measured the levels of serum CKMB, a marker for the diagnosis of acute myocardial ischemia (23) was measured, and it was found that plasma CKMB levels decreased significantly after the administration of URB602 at 6 hours after ROSC. These results suggested that URB602 improves myocardial enzyme variables and reduces myocardial injury. The administration of URB602 can improve myocardial pathologic damage after resuscitation. Hence, we demonstrated that URB602 can protect against the development of myocardial dysfunction and injury.
Although the mechanism of myocardial damage caused by CA-CPR need to be further elaborated, evidence shows that the potential mechanisms include inflammatory factor activation and mitochondrial damage (24–26). Reports in the literature indicate that CA-CPR causes systemic inflammatory reactions and coagulation abnormalities (27 , 28). Our results showed that the 2-AG, anandamide, AA, TXB2, PGD2, and PGE2 content in the heart are increased after resuscitation, indicating that CA-CPR activates the endocannabinoid system and AA metabolic pathway. AA is an unsaturated fatty acid that is mainly present in cell membrane phospholipids (9). Phospholipase allows AA to produce PGs via the cyclooxygenase pathway and can cause inflammation and activation of the coagulation system (9). One major cause of the myocardial inflammatory response and blood hypercoagulation may be the activation of AA and PGs. On the one hand, URB602 mitigates myocardial damage by reducing the level of proinflammatory factors such as AA and its metabolites. On the other hand, URB602 may protect the myocardium by the reduction of the TXB2 content, the inhibition of platelet activation, and the decrease of myocardial hypercoagulation. TXB2 is a stable metabolite of TXA2 in the body and promotes tissue damage and inflammation by stimulating platelet aggregation (29). We speculated that MAGL inhibitors acts as anti-inflammatory and antiplatelet agents in a manner similar to other drugs, such as aspirin, but without the side effects of these drugs, such as gastric bleeding.
The current study shows (25 , 30–32) that the vicious cycle of mitochondrial damage is the leading cause of cardiac death after CPR. Similar to myocardial dysfunction and injury 6 hours after CA-CPR, mitochondrial injury was significantly mitigated by the administration of URB602. These results were reinforced by our finding that URB602 prevented the CA-CPR-induced acceleration of Ca2+-induced mitochondrial swelling, a measure of mPTP, 15 minutes after CA-CPR. These findings suggest that the cardioprotective effects of URB602 may be mediated via the inhibition of mitochondrial injury. Therefore, URB602 may also protect myocardial function by blocking the vicious cycle of mitochondrial damage during early resuscitation, thereby improving the survival of rats after CA-CPR.
There are still several limitations and shortcomings in the current research. First, the use of MAGL knockout mice is needed to further confirm the role of MAGL during CPR. Second, in this study, rats were used as experimental subjects, large animals such as dogs and pigs should be used for evaluating the protective effects of MAGL during CA-CPR.
The MAGL inhibitor URB602 can effectively reduce myocardial and mitochondrial damage and significantly improve survival in a rat model of CA-CPR.
1. Peatfield RC, Sillett RW, Taylor D, et al. Survival after cardiac arrest in hospital. Lancet 1977; 1:1223–1225
2. Sasson C, Rogers MA, Dahl J, et al. Predictors of survival from out-of-hospital cardiac arrest: A systematic review and meta-analysis. Circ Cardiovasc Qual Outcomes 2010; 3:63–81
3. Mozaffarian D, Benjamin EJ, Go AS, et al. Executive summary: Heart disease and stroke statistics–2016 update: A report from the american heart association. Circulation 2016; 133:447–454
4. Callaway CW, Donnino MW, Fink EL, et al. Part 8: Post-cardiac arrest care: 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation 2015; 132:S465–S482
5. Dankiewicz J, Schmidbauer S, Nielsen N, et al. Safety, feasibility, and outcomes of induced hypothermia therapy following in-hospital cardiac arrest-evaluation of a large prospective registry*. Crit Care Med 2014; 42:2537–2545
6. Montecucco F, Di Marzo V. At the heart of the matter: The endocannabinoid system in cardiovascular function and dysfunction. Trends Pharmacol Sci 2012; 33:331–340
7. Mechoulam R, Panikashvili D, Shohami E. Cannabinoids and brain injury: Therapeutic implications. Trends Mol Med 2002; 8:58–61
8. Ma L, Lu X, Xu J, et al. Improved cardiac and neurologic outcomes with postresuscitation infusion of cannabinoid receptor agonist WIN55, 212-2 depend on hypothermia in a rat model of cardiac arrest. Crit Care Med 2014; 42:e42–e48
9. Benyó Z, Ruisanchez É, Leszl-Ishiguro M, et al. Endocannabinoids in cerebrovascular regulation. Am J Physiol Heart Circ Physiol 2016; 310:H785–H801
10. Berardi A, Schelling G, Campolongo P. The endocannabinoid system and Post Traumatic Stress Disorder (PTSD): From preclinical findings to innovative therapeutic approaches in clinical Settings. Pharmacol Res 2016; 111:668–678
11. Grabner GF, Zimmermann R, Schicho R, et al. Monoglyceride lipase as a drug target: At the crossroads of arachidonic acid metabolism and endocannabinoid signaling. Pharmacol Ther 2017; 175:35–46
12. Carloni S, Alonso-Alconada D, Girelli S, et al. Pretreatment with the monoacylglycerol lipase
inhibitor URB602 protects from the long-term consequences of neonatal hypoxic-ischemic brain injury in rats. Pediatr Res 2012; 72:400–406
13. Mayeux J, Katz P, Edwards S, et al. Inhibition of endocannabinoid degradation improves outcomes from mild traumatic brain injury: A mechanistic role for synaptic hyperexcitability. J Neurotrauma 2017; 34:436–444
14. Cao Z, Mulvihill MM, Mukhopadhyay P, et al. Monoacylglycerol lipase
controls endocannabinoid and eicosanoid signaling and hepatic injury in mice. Gastroenterology 2013; 144:808–817.e15
15. Costola-de-Souza C, Ribeiro A, Ferraz-de-Paula V, et al. Monoacylglycerol lipase
(MAGL) inhibition attenuates acute lung injury in mice. PLoS One 2013; 8:1–15
16. National Society for Medical Research: Guide for the Care and Use of Laboratory Animals. 1996Washington, DC, National Academic Press.
17. Institute of Laboratory Animal Resources: Guide for the Care and Use of Laboratory Animals. 1985Atlanta, GA, National Institutes of Health.
18. Katz L, Ebmeyer U, Safar P, et al. Outcome model of asphyxial cardiac arrest in rats. J Cereb Blood Flow Metab 1995; 15:1032–1039
19. Lomazzo E, Bindila L, Remmers F, et al. Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain. Neuropsychopharmacology 2015; 40:488–501
20. Kaplan JA, Reich DL, Savino JS. Kaplan’s Cardiac Anesthesia: The Echo Era. 2011Sixth Edition. St. Louis, Elsevier Saunders.
21. Flameng W, Borgers M, Daenen W, et al. Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J Thorac Cardiovasc Surg 1980; 79:413–424
22. Marcu R, Neeley CK, Karamanlidis G, et al. Multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria. J Vis Exp 2012; 67:4131
23. Baines CP, Kaiser RA, Purcell NH, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 2005; 434:658–662
24. Stub D, Bernard S, Duffy SJ, et al. Post cardiac arrest syndrome: A review of therapeutic strategies. Circulation 2011; 123:1428–1435
25. Alper E, Arabul M. Serum myeloperoxidase, CPK, CK-MB, and cTnI levels in early diagnosis of myocardial ischemia during ERCP; once or repeated once? Turk J Gastroenterol 2014; 25:452–453
26. Varvarousis D, Varvarousi G, Iacovidou N, et al. The pathophysiologies of asphyxial vs dysrhythmic cardiac arrest: Implications for resuscitation and post-event management. Am J Emerg Med 2015; 33:1297–1304
27. Ertracht O, Malka A, Atar S, et al. The mitochondria as a target for cardioprotection in acute myocardial ischemia. Pharmacol Ther 2014; 142:33–40
28. Jacob CJ, Meshe DC, Cameron D. Myocardial dysfunction and shock after cardiac arrest. Biomed Res Int 2015; 11:1–14
29. Kern KB, Zuercher M, Cragun D, et al. Myocardial microcirculatory dysfunction after prolonged ventricular fibrillation and resuscitation. Crit Care Med 2008; 36:S418–S421
30. Wada T. Coagulofibrinolytic changes in patients with post-cardiac arrest syndrome. Front Med (Lausanne) 2017; 4:156
31. Hirakata H, Ushikubi F, Toda H, et al. Sevoflurane inhibits human platelet aggregation and thromboxane A2 formation, possibly by suppression of cyclooxygenase activity. Anesthesiology 1996; 85:1447–1453
32. Madungwe NB, Zilberstein NF, Feng Y, et al. Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic. Heart Am J Cardiovasc Dis 2016; 6:93–108