Wagner, Jens A. MD*; Abesser, Marco*; Harvey-White, Judith†; Ertl, Georg MD*
Endogenous cannabinoids exert physiologic responses through activation of specific receptors, 2 of which have been cloned and named CB11 and CB2.2 Among potentially protective signal transduction pathways are stimulation of nitric oxide (NO) release3 and inhibition of noradrenaline release.4 Both endogenous cannabinoids, arachidonylethanolamide (anandamide) and 2-arachidonylglycerol (2-AG), have been implicated in neuroprotection after brain injury5 and during transient cerebral ischemia.6 Cell protective effects are not limited to the brain. In the heart, Lagneux and Lamontagne were the first to suggest an involvement of endocannabinoids in the cardioprotection against myocardial ischemia triggered by lipopolysaccharide.7 From the same group followed a study showing that 2-AG, but not anandamide, improved myocardial recovery in isolated rat hearts subjected to low-flow ischemia and reperfusion.8 This cardioprotective effect could be completely blocked by the CB2 receptor antagonist SR144528 and partially blocked by the CB1 receptor antagonist SR141716A. They concluded that the effect is mediated mainly through CB2 receptors, although the selective CB1 tagonist arachidonyl-2-chloroethylamide also reduced myocardial infarction.8 Further work described an involvement of endocannabinoids in the cardioprotection against myocardial ischemia conferred by heat stress with evidence for involvement of either CB2 cannabinoid receptors and/or NO synthase (NOS) activation in delayed cardioprotection.9 However, there is evidence for CB110,11 but not CB212 receptors in the rat heart, which precludes any molecular basis for CB2 receptors in mediating endocannabinoid effects in the heart.
The late phase of preconditioning (PC) which protects against myocardial infarction and stunning lasts 3 to 4 days and may have important clinical relevance, for review see Ref. 13. The cardioprotective effects of late PC can be triggered by heat stress or exercise, but may also be reproduced pharmacologically with, for example, opioids or NO donors. Because this approach might be used for therapeutic purposes, the cascade of signaling events and possible involvement of cannabinoids is of interest.
Therefore, we aimed to find out whether endocannabinoids are involved in delayed PC using a rat model of transdermal nitroglycerin (NTG) application followed by global, no-flow ischemia in isolated Langendorff-perfused hearts and whether cannabinoid receptor blockade reduces infarct size.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
PC via Transdermal NTG and Experimental Protocol
The study was carried out in 2 parts. In the first part, male Wistar rats (250 to 330 g) were submitted to either transdermal NTG treatment or sham procedure. In the second part, a global ischemia (20 min)-reperfusion (120 min) protocol was performed in isolated Langendorff hearts.
Rats were lightly anesthetized in an ether chamber to ease the following procedure. After shaving, a patch releasing 0.15 mg/h/kg NTG continuously for a period of 24 hours were applied on the back skin of the rat and fastened with 4 stitches. The patch was cut according to the weight of the rat. After removal of the patch, a 2-day interval without treatment was allowed before rat hearts were removed for the Langendorff preparation.
Stock solutions of AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxa-mide] and AM-630 (6-iodopravadoline, all from Tocris, Bristol, UK) were dissolved in dimethylsulfoxide. The final concentration of dimethylsulfoxide in the perfusate was <0.1% and this vehicle concentration was without own effects. Perfusion with either vehicle, 0.3 μM AM-251 or 0.3 μM AM-630, was initiated 30 minutes before global ischemia and continued throughout the 120 minutes of reperfusion. We used AM-251 (0.3 μM) rather than SR141716A because there is evidence that receptors distinct from CB1 and CB2 cannabinoid receptors are inhibited by SR141716A but not by AM-251.14,15 Further, AM-251 has a better selectivity (Ki: 7.5 nM at CB1 receptors, 306-fold selective over CB2 receptors16) than SR141716A (Ki: 11.5 nM at CB1 receptors, 143-fold selective over CB2 receptors16).
For CB2 antagonism we took AM-630 (Ki: 31.2 nM at CB2 receptors, 165-fold selective over CB1 receptors17) rather than SR144528 (Ki: 0.3 nM at CB2 receptors and 400 nM at CB1 receptors18), because AM-630 has a lower inverse efficacy than SR144528 at CB2 receptors.17
In another set of experiments, the cannabinoid receptor agonists 2-AG or its metabolically stable derivative noladinether (both from Tocris) were given to native Langendorff rat hearts in concentrations of 1 or 0.1 μM, respectively, starting 30 minutes before global ischemia and continued throughout the 120 minutes of reperfusion.
Rats were deeply anesthetized with sodium pentobarbital (40 mg/kg i.p.) and heparinized (500 IU/kg i.p.). After rapid excision and removal of the pericardium in 4°C Krebs-Henseleit solution, the hearts were mounted on a Langendorff perfusion apparatus by cannulating the aorta within 90 seconds of excision. Perfusion of the heart was started at constant pressure (100 mm Hg) at 37°C with Krebs-Henseleit buffer preequilibrated with 5% CO2 in O2. Heart rate (HR) and left ventricular pressure were monitored continuously with a latex balloon inserted into the left ventricle and a cannula connected to a pressure transducer (Gould Instruments, Oxnard, CA, USA). Preparations were stabilized for 30 minutes with the end diastolic pressure (LVEDP) set at 8 to 10 mm Hg via inflation of the balloon. Coronary flow (CF) was constantly monitored using an electromagnetic flow probe (Statham P23Db, Gould Statham Instruments, Hato Rey, Puerto Rico). HR and left ventricular developed pressure (LVDP, difference between left ventricular systolic pressure and LVEDP) were continuously recorded on a 4-channel recorder (Graphtec Corp., Tokyo, Japan).
Tissue Levels of Endocannabinoids
Control and NTG-treated rat hearts which were not used for infarct size measurement were analyzed with and without ischemia/reperfusion. Vital left ventricular muscle without scar tissue was homogenized and deproteinated with 10 volumes of ice-cold acetone and extracted 3 times with 2 volumes of chloroform/methanol (2:1) containing 7 nM of 2H4-anandamide as internal standard. The extract was dried under nitrogen and reconstituted in 50 μL methanol for analysis by liquid chromatography/in line mass spectrometry, using an Agilent 1100 series LC-MSD, equipped with a thermostated autosampler and column compartment. Anandamide and 2-AG in cellular extracts were quantified by LC/MS, as described.19 The mass sensitive detector (model LS) was set for atmospheric pressure chemical ionization, positive polarity, and selected-ion-monitoring to monitor ions m/z 348 for anandamide, 352 for 2H4-anandamide, and 379 for 2-AG. The amounts of anandamide and 2-AG were determined using inverse linear regression of standard curves, produced using synthetic anandamide and 2-AG.
Infarct Size Measurement
At the end of the reperfusion period, hearts were perfused with triphenyl tetrazolium chloride for 10 minutes and 6 transverse myocardial slices from each heart were used to identify necrotic tissue. Infarct size was calculated for each section by dividing the sum of the planimetered necrotic endocardial and epicardial circumferences by the sum of the total epicardial and endocardial circumferences of the left ventricle using a calibrated digitizer (Numonics Digitizer 2200).
Results are expressed as mean±SEM. As appropriate, Student's t test (when only 2 groups were compared) or one-way ANOVA followed by Dunnett test (more than 2 experimental groups) were used. Statistical significance was set at P<0.05.
Antagonists and Hemodynamics
Hemodynamic data are summarized in Table 1. PC with NTG did not alter baseline values for CF (mL/min), HR (b/min), the maximum of the first derivative of left ventricular pressure (dp/dt, mm Hg/s), and LVDP (mm Hg). During early reperfusion, transdermal NTG pretreatment increased CF, +dp/dt, and LVDP in otherwise untreated rats. After ischemia, recovery of CF, +dp/dt, and LVDP was incomplete in all groups. AM-630 had significantly protective effects on +dp/dt and LVDP during early reperfusion; AM-251 had protective effects on +dp/dt and LVDP during late reperfusion (Table 1).
Infarct Size After CB1 and CB2 Antagonism
The extent of myocardial infarction after ischemia reperfusion in different treatment groups is presented in Figure 1. Transdermal pretreatment with NTG reduced the myocardial scar from 40.9±3.9% to 27.5±3.8% (P<0.05). The CB1 receptor antagonist AM-251 (0.3 μM) had no infarct-limiting effects in sham-treated rats, but completely abolished the cardioprotective effect of NTG pretreatment. Interestingly, the selective CB2 receptor antagonist AM-630 (0.3 μM), which is a weak inverse agonist at CB1 receptors,27 reduced rather than increased infarct size in rats without PC and did not prevent the infarct-size–reducing effect of NTG.
As detected by liquid chromatography and mass spectrometry, PC with NTG significantly increased the content of viable left ventricular tissue of 2-AG but not of anandamide. Ischemia/reperfusion alone did not increase the 2-AG or anandamide content versus controls (Table 2).
Hemodynamics and Infarct Size After Treatment With 2-AG or Noladinether
The presence of 1 μM 2-AG (n=5) or 0.1 μM of the metabolically stable 2-AG derivative noladinether (n=5) given 30 minutes before ischemia (20 min) and throughout the reperfusion time (120 min) significantly reduced infarct size from 41±4% in controls to 30.5±6.1% in 2-AG–treated hearts (P<0.05) and to 21.1±3.8% in noladinether-treated hearts (P<0.01, Fig. 2). 2-AG and noladinether treatment significantly reduced baseline CF. More important, after ischemia and reperfusion, both 2-AG and noladinether promoted the recovery of the hearts as detected by significantly increased values for +dp/dt and LVDP (Table 3).
In this study we could show that endocannabinoids are involved in delayed PC using a rat model of transdermal NTG application followed by global, no-flow ischemia in isolated Langendorff-perfused hearts. Previous studies of others suggested involvement of CB2 cannabinoid receptors.8,9 In our model, CB1 but not CB2 cannabinoid receptor blockade reduced the cardioprotective effects of NTG pretreatment. Delayed PC through transdermal NTG application significantly increased 2-AG, but not anandamide tissue levels of the left ventricle. Finally, 2-AG or its metabolically stable derivative noladinether, given before ischemia/reperfusion in unpreconditioned hearts, mimicked the cardioprotective effects of PC and reduced infarct size.
The early phase of PC lasts only hours and protects against infarction but not against stunning. Clinically more relevant, the late phase of PC lasts 3 to 4 days and protects against both stunning and infarction and seems to be an universial response of the heart to stress in general (for review see Ref. 13). A complex cascade of signaling events can be triggered by exercise, heat stress, endotoxin, opioids, and NO donors. The latter substances may be used for therapeutic purposes which makes them specifically interesting.
Similarities to Cannabinoid-mediated Protection of the Brain
Recently, cannabinoid receptor activation has been implicated in cell protective mechanisms in the brain and heart. In our ischemic heart model, PC increased the left ventricular tissue content of 2-AG from 4.58±1.00 nmol/g to 10.88±1.39 nmol/g (P<0.05). The first group studying traumatic brain injury induced by closed head injury in mice found significantly increased 2-AG levels and synthetic 2-AG administered to brain-injured mice reduced brain edema and infarct volume. The beneficial effects of 2-AG were attenuated by the CB1 receptor antagonist SR141716A.5 Importantly, in a follow-up study of the same group, it could be shown that CB1 receptor knock out mice showed minor spontaneous recovery after closed head injury compared to wild-type mice. Moreover, 2-AG administration did not improve edema formation and neurologic function in CB1−/− mice. Instead, 2-AG abolished the increase of nuclear factor kappa B (NF-kappa B) only in wild-type mice which led the authors propose that in the brain, 2-AG exerts neuroprotection after traumatic injury via CB1 receptor-mediated mechanisms that involve inhibition of intracellular inflammatory signaling pathways.20 Other laboratories confirmed the above results in showing that CB1 receptor knock-out mice with ischemic stroke show enhanced mortality, infarct size, and neurologic deficits.21
Infarct-size Reducing Effects of Cannabinoids in the Heart
In the heart, endocannabinoid tissue levels in viable left ventricular muscle after PC have not been measured so far. Therefore, our results proposing 2-AG-mediated cardioprotection through CB1 receptor activation are in line with the data available for traumatic brain injury but cannot be compared with cannabinoid tissue levels in other PC models used in the rat heart.
Lagneux and Lamontagne were the first to implicate endocannabinoids in cardioprotection. Bacterial lipopolysaccharides are known triggers of protective mechanisms against ischemia-reperfusion injury.7 They could show that the cardioprotective effects of lipopolysaccharides treatment in Langendorff rat hearts, in terms of infarct size reduction and functional recovery, were abolished by the CB2 receptor antagonist SR144528 (1 μM) and the NOS inhibitor N-nitro-L-arginine. The CB1 receptor antagonist SR141716A (1 μM) showed only a nonsignificant trend for infarct size reduction.7 In a follow-up study they extended their findings to rat hearts preconditioned by heat stress. Again, they found that NOS inhibition or CB2 receptor blockade by SR144528 (1 μM), but not by the CB1 receptor antagonist SR141716A (1 μM), were able to abolish the infarct-size–reducing effects of PC.9
Besides the fact that heat stress is no clinically relevant trigger of PC, the studies are remarkable because so far molecular studies failed to identify mRNA for CB2 receptors in the heart.12 CB2 receptors are mainly located on immune cells. One may speculate that during PC CB2 receptor activation on macrophages may trigger the release of various cytokines resulting in cardioprotection. However, all 3 studies suggesting cardioprotective effects through CB2 receptors were using buffer-perfused hearts and not whole blood perfusion or cell suspensions,7–9 which excludes possible effects through CB2 receptors on macrophages, neutrophils, or platelets.
On the contrary, CB1 receptors were identified in rat hearts on mRNA22 and protein level.10,11 In our study, we found that delayed PC through transdermal NTG application increases the production of 2-AG which elicits protective effects against myocardial infarction via CB1 cannabinoid receptors, because the CB1 antagonist AM-251 (0.3 μM), but not the CB2 antagonist AM-630 (0.3 μM), abolished the NTG effects. It is noteworthy that the effective concentration of the CB1 antagonist used in our study is 3.3-fold lower than the concentrations used in the studies showing antagonism by CB2 receptor blockade (1 μM). As SR144528 has Ki values of 0.3 and 437 nM in cell lines expressing human CB2 or CB1 receptors,18 respectively, partial inhibition of CB1 receptors using 1 μM of SR144528 is likely.
Interestingly, Le`picier et al8 could show that 2-AG, but not anandamide, reduced infarct size in rat isolated hearts exposed to low-flow ischemia and reperfusion. This effect was mimicked by either the selective CB1 receptor agonist arachidonyl-2-chloroethylamide or the selective CB2 receptor agonist JWH015, both used at a concentration of 50 nM.8 Either the CB2 antagonist SR144528 or the CB1 antagonist SR141716A completely or partially, respectively, blocked the cardioprotective effects of 2-AG.8 As a possible reason for the noneffectiveness of anandamide compared to 2-AG, the authors discussed a rapid uptake and degradation of anandamide. In our hands, PC increased heart tissue levels of 2-AG, but not of anandamide. However, a recent study using rat isolated perfused hearts subjected to global, no-flow ischemia (30 min) and reperfusion (1 h) found no protective effects of the CB1 agonist ACPA, the CB2 agonist JWH133, or the potent agonist on both CB1 or CB2 receptors, HU-210. Instead, anandamide and the metabolically stable derivative methanandamide (1 μM each) reduced infarct size.23 Unfortunately, 2-AG was not used in that study. As anandamide did not reduce infarct size in the presence of SR141716A (1 μM) or SR144528 (1 μM), the authors concluded that a novel cannabinoid site of action is involved.23
Endocannabinoids and Cardioprotection
There is no doubt that endocannabinoids are able to protect the heart from ischemic damage,7–9,23 which is confirmed in our study. The mechanism remains controversial. Our results favor a CB1 receptor-mediated mechanism for 2 reasons: (1) AM-251, but not AM-630, used in selective concentrations of 0.3 μM reversed the infarct-limiting effect of PC through transdermal NTG application and (2) there is molecular proof of CB110,11,22 but not CB2 receptors in the heart.12 It is possible that stimulation of CB2 receptors in noncardiac tissue may ultimately result in a beneficial effect on the heart. However, we found CB2 antagonism rather than CB2 stimulation associated with reduced infarct size in sham hearts which makes the former statement unlikely.
CB1 and CB2 receptor antagonists have not been used in specific concentrations in earlier studies.7–9,23 As CB1 antagonists may partially block effects mediated by “CB3” or “anandamide” receptors,24,25 the involvement of a novel cannabinoid site of action, as proposed by Underdown and coworkers,23 cannot be ruled out. Importantly, we were able to detect a markable increase of 2-AG, but not anandamide, after PC. That may explain results of Le`picier et al,8 who did not see protective effects by anandamide but 2-AG. In our study, both 2-AG (1 μM) and its metabolically stable derivative noladinether (0.1 μM), significantly improved hemodynamics and reduced infarct size after ischemia/reperfusion, thus mimicking the cardioprotective effects of PC. It is noteworthy that lower concentrations of noladinether elicited even more pronounced effects on infarct size, suggesting that metabolism of 2-AG may partially prevent its cardioprotective effects.
In summary, we show that endocannabinoid activation may be one of several mechanisms contributing to the infarct-limiting effects of delayed PC. We propose that this effect is mainly mediated through 2-AG acting on CB1 receptors which resembles 2-AG–mediated neuroprotection after closed head injury5,20 or ischemic stroke.21 Additionally, we successfully introduced transdermal NTG application in a rat model of PC. So far, the use of this technique has been limited to rabbits and is not affected by the development of nitrate tolerance.26
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