Anandamide reduces infarct size in rat isolated hearts subjected to ischaemia—reperfusion by a novel cannabinoid mechanismNichola J Underdown, C Robin Hiley, and William R Ford
Br J Pharmacol. 2005 November
1. Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia—reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved.
2. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30min) and reperfusion (1h). Agonists were present from 5min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment.
3. In vehicle-treated hearts, 26±3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1μM anandamide (10±1%, n=7) or 1μM methanandamide (12±4%, n=6) but not 1μM HU210. Neither ACPA (1μM; CB1 receptor agonist) nor JWH133 (1μM; CB2 receptor agonist), individually or combined significantly affected infarct size.
4. Anandamide (1μM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1μM) or the CB2 receptor antagonist, SR144528 (1μM).
5. Despite sensitivity to CB1 and CB2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB2 receptors suggesting the involvement of a novel cannabinoid site of action.
Endocannabinoids have been indirectly implicated in the mechanisms of protection from cardiac ischaemia—reperfusion (IR) injury due to preconditioning induced by lipopolysaccharide (Lagneux & Lamontagne, 2001) or heat stress (Joyeux et al., 2002). In general, these studies suggest that the protection is mediated via activation of the CB2 receptor as the responses are sensitive to the selective CB2 receptor antagonist, SR144528.
Recently, direct evidence of the cardioprotective properties of endocannabinoids has been published using rat isolated hearts (LÃ©picier et al., 2003). In that study, protection from cardiac IR was observed with the endocannabinoids palmitoylethanolamide (PEA) and 2-arachidonoylglycerol (2-AG) but not anandamide. In addition, infarct size was limited by either a selective CB1 (arachidonoyl-2-chloroethylamide; ACEA) or a selective CB2 (JWH015) receptor agonist.
These results are somewhat confusing for a number of reasons. PEA is supposedly inactive at CB1 and CB2 receptors (Lambert et al., 1999), a fact supported by the finding that PEA was devoid of activity when administered to rat isolated hearts (Ford et al., 2002). In contrast, anandamide causes negative inotropy and coronary vasodilatation in rat isolated hearts (Ford et al., 2002). It is therefore interesting that anandamide failed to reduce IR injury whereas PEA and 2-AG were effective.
The pharmacology of 2-AG and anandamide is quite similar, in that both can bind to and stimulate CB1 and CB2 receptors (Sugiura et al., 2000; Steffens et al., 2005). Therefore, it is unlikely that the difference between 2-AG and anandamide in the study of LÃ©picier et al. (2003) is due to contrasting affinities or efficacies at cannabinoid receptors, particularly as cardioprotection could apparently be induced by agonists selective for either CB1 or CB2 receptors. It was suggested that anandamide might not have been effective because it was rapidly taken up and degraded. However, both 2-AG and anandamide are sensitive to uptake and metabolism and, furthermore, 2-AG is more labile than anandamide (Goparaju et al., 1998; Laine et al., 2002). It is therefore unlikely that the reason for the differences between 2-AG and anandamide are due to different rates of degradation.
Given the potential confusion about the effectiveness of endocannabinoids in mediating cardioprotection we decided to assess whether or not anandamide could alter infarct size induced by IR, measured directly using triphenyltetrazolium chloride staining, in rat isolated hearts and identify the pharmacological mechanism. A preliminary report of these findings has been communicated to the British Pharmacological Society (Underdown & Ford, 2004).
All animals were housed and treated in accordance with Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (Her Majesty's Stationary Office, U.K.). Male Wistar rats (300—400g), that had been fed ad libitum, were killed with an overdose of sodium pentobarbital (60mgkg−1 i.p, Sagatal; RhÃ´ne MÃ©rieux, Harlow, Essex, U.K.) and heparinised (100Ukg−1 i.p, CP Pharmaceuticals, Wrexham, Clwyd, U.K.). Hearts were rapidly excised and placed in ice-cooled Krebs—Henseleit solution.
After cannulating the aorta, constant pressure (80mmHg) Langendorff perfusion was commenced (Ford et al., 2001). The perfusate, a modified Krebs—Henseleit solution (NaCl 118mM, KCl 4.7mM, KH2PO4 1.2mM, MgSO4 1.2mM, 2.5mM CaCl2, 11mM glucose, 100mUl−1 insulin), was maintained at 37°C and continuously bubbled with a gas mixture of 95% O2/5% CO2. Hearts were immersed in a chamber containing perfusate at a constant 37°C. During periods of perfusion, hearts were electrically paced via bipolar platinum electrodes at a frequency of 5Hz (Stimulator 100; Palmer Bioscience, Sheerness, Kent, U.K.).
Left ventricular-developed pressure (LVDP) was measured by means of a pressurised (10—20mmHg) balloon inserted into the left ventricle and connected to a pressure transducer (Ohmeda, model P23XL-1). Coronary flow was measured by ultrasonic flow probes (Transonic H4X). The electrocardiogram (ECG) was continuously recorded by means of two stainless-steel needles inserted into the ventricular apex and base, connected to an ECG amplifier (Grass EKG/Tachograph model 7P4). Hearts that failed to respond to electrical pacing during aerobic periods of perfusion for more than 10min were excluded from all analyses. All parameters were continuously recorded using a PowerLab 800 (ADInstruments, Hastings, East Sussex, U.K.) and stored using a Macintosh PowerPC.
Hearts were randomly assigned to receive the cannabinoid vehicle, Tocrisolve 100 (a 1:4 soya oil:water mixture emulsified with poloxamer F188, Tocris Cookson Ltd, Avonmouth, Bristol, U.K.), or one of the following; 1μM anandamide, 1μM methanandamide, 1μM arachidonoylcyclopropylamide (ACPA), 1μM JWH133 or a combination of ACPA and JWH133 (1μM each).
Where used, antagonists or their vehicle dimethylsulfoxide (DMSO; 0.01% volvol−1, final concentration) were present in the perfusate throughout the protocol at a concentration of 1μM. Antagonists were prepared in DMSO as 1mM stock solutions before being added to the perfusate. Hearts were randomly assigned to one of six groups. In the first group, the DMSO was present throughout the protocol and hearts were infused with Tocrisolve 100. Hearts assigned to the second group were treated with DMSO and an infusion of anandamide (1μM final concentration). The third group were treated with 1μM rimonabant (SR141716A) and infused with Tocrisolve 100. In the fourth group, hearts were treated with 1μM rimonabant and an infusion of anandamide (1μM final concentration). The fifth group were treated with 1μM SR144528 and an infusion of Tocrisolve100. The sixth group received 1μM SR144528 and an anandamide infusion (1μM final concentration).
In all groups of hearts, cannabinoid agonists or Tocrisolve 100 vehicle were infused at a rate adjusted to be 10% of coronary flow. Drug or vehicle infusion was started 5min before ischaemia (Figure 1). Ischaemia, induced by clamping the aortic inflow line, was maintained for 30min. During ischaemia, drug or Tocrisolve 100 infusion and electrical pacing were stopped. After 30min of global, no-flow ischaemia, the clamps were removed and perfusate allowed to flow. Drug infusion and electrical pacing was restarted and continued throughout the 2h of reperfusion.
Diagrammatic representation of the ischaemia—reperfusion protocol. Hearts are aerobically perfused for the first 15min (open box). Baseline values are taken at 10min (a), pre-ischaemic values are taken just before ischaemia is (more ...)
Infarct size determination
At the end of reperfusion, hearts were frozen at −4°C and then cut into 3—4mm slices. The slices were immersed in a 1% solution of triphenyltetrazolium chloride (Sigma Chemical Co., Poole, Dorset, U.K.) in Krebs—Henseleit solution for 10—20min at room temperature. Stained hearts were stored overnight in 10% formalin solution (VWR, Loughborough, Leicestershire, U.K.) before infarct size was determined by an investigator blinded to the experimental treatment. The right ventricle and connective tissues were removed and the slices compressed between two plates with a spacing of 2.5mm. The total area of the left ventricle together with areas with an absence of red stain (infarct area) were traced onto acetate and scanned into a personal computer. The total area (in pixels) for each heart and the area of infarction was calculated using SigmaScan Pro 5 software (Systat Software U.K., Hounslow, Middlesex, U.K.). Infarct size for each heart is expressed as a percentage of the total left ventricular area.
Drug preparation and administration
The cannabinoids, anandamide, methanandamide, ACPA and JWH133 (Tocris Cookson Ltd, Avonmouth, Bristol, U.K.), obtained as solutions of 5mgml−1 dissolved in Tocrisolve 100 (Tocris Cookson Ltd, Avonmouth, Bristol, U.K.), were diluted in Krebs—Henseleit solution to a concentration of 10−5M such that when infused at a rate equal to 10% of coronary flow a final concentration of 1μM was obtained. The cannabinoid agonist vehicle, Tocrisolve 100 was diluted in Krebs bicarbonate to give a concentration of 0.07% solution that when infused at a rate controlled to be 10% of coronary flow a final concentration of 0.007% was obtained. Rimonabant (SR141716A) and SR144528 (gifts from Sanofi Synthelabo, France) were prepared in DMSO (Sigma Chemical Co., Poole, Dorset, U.K.) as 1mM stock solutions and added to the perfusate (final concentration was 1μM).
All data are expressed as mean±s.e.m. Multiple comparisons against a single control were made using analysis of variance (ANOVA) supported by Dunnet's post hoc test. Comparisons of time course data were analysed using ANOVA with repeated measures. Statistical significance was set at P<0.05.
Diastolic pressure, LVDP and coronary flow did not significantly vary among any of the experimental groups before ischaemia (Table 1).
Cardiac function and infarct size in hearts treated with cannabinoid agonists
In hearts treated with the cannabinoid agonist vehicle, Tocrisolve 100, diastolic pressure, markedly elevated at the beginning of reperfusion, fell steady throughout reperfusion remaining elevated compared to pre-ischaemic values by the end of reperfusion (Figure 2 and Table 1). LVDP recovered incompletely during reperfusion recovering to a maximum of 40±6% of preischaemic values after 50min of reperfusion. Thereafter, there was a steady decline in LVDP until the end of the reperfusion period when the values were 29±6% of the preischaemic values (Figure 2). Recovery of coronary flow during reperfusion was incomplete (Table 1) reaching a maximum (37±7% or pre-ischaemic values) after 20min of reperfusion then declining to be 24±6% of pre-ischaemic values by the end of reperfusion. Infarct size, measured at the end of the reperfusion was 26±3% of the left ventricle (Figure 3).
Diastolic pressure (panels a and b) and left ventricular developed pressure (LVDP, panels c and d) measured during periods of perfusion for hearts treated with vehicle (Tocrisolve 100), 1μM anandamide, 1μM methanandamide, (more ...)
Infarct sizes for individual hearts treated with Tocrisolve 100, anandamide, methanandamide, HU-210, ACPA, JWH133 and a combination of ACPA+JWH133. *=P<0.05 vs Control (ANOVA supported by Dunnett's post hoc test). (more ...)
None of the cannabinoid agonists used in the study had a significant effect on the recoveries of diastolic pressure, LVDP or coronary flow compared to Tocrisolve 100-treated hearts (Figure 2 and Table 1). However, infarct size was significantly reduced by the presence of anandamide (10±1% of the left ventricle) and methanandamide (12±4% of the left ventricle) but not HU-210 (19±3% of the left ventricle) compared to hearts that received
Tocrisolve100 (Figure 3). Infarct size in hearts treated with ACPA (26±5% of the left ventricle) or JWH133 (20±2% of the left ventricle) individually or in combination (27±4% of the left ventricle) were not significantly different from those obtained in hearts treated with Tocrisolve 100 (Figure 3).
Diastolic pressure, LVDP and coronary flow did not significantly vary among any of the experimental groups before ischaemia (Table 2).
Cardiac function and infarct size in hearts treated with cannabinoid agonists
In Tocrisolve 100-treated hearts with 0.01% volvol−1 DMSO present, diastolic pressure was markedly elevated at the beginning, falling steadily throughout but remaining significantly elevated at the end of the reperfusion period (Figure 4). Recovery of LVDP was incomplete reaching a maximum of 33±8% of pre-ischaemic values after 40min of reperfusion then falling to 18±4% of preischaemic values by the end of the reperfusion (Figure 4). Coronary flow was impaired during reperfusion (Table 2) recovering to a maximum of 33±7% of pre-ischaemic values after 20min of the start of reperfusion then falling to 2±1% of pre-ischaemic values by the end of the reperfusion period when 31±4% of the left ventricle was infarcted (Figure 4).
Diastolic pressure (panels a, c and e) and left ventricular developed pressure (LVDP, panels b, d and f) for hearts treated with Tocrisolve 100 or 1μM anandamide in the absence (panels a and b) or presence of 1μ (more ...)
Infusion of anandamide in the presence of 0.01 volvol−1 DMSO had no significant effect on the recovery of diastolic pressure, LVDP or coronary flow during reperfusion compared to vehicle control hearts (Figure 4 and Table 2). Infarct size, measured at the end of reperfusion, was significantly reduced to 12±2% of the left ventricle (Figure 5).
Infarct sizes for individual hearts treated with Tocrisolve 100 in the absence (control) and presence of 1μM rimonabant or 1μM SR144528, or 1μM anandamide in the absence or presence of rimonabant (more ...)
The recoveries of diastolic pressure, LVDP and coronary flow were not significantly different from Tocrisolve 100-treated hearts with 0.01% volvol−1 DMSO present to hearts where rimonabant (1μM) or SR144528 (1μM) was present (Figure 5). Infarcts obtained in hearts where rimonabant or SR144528 was present (Figure 5) were similar to those treated with Tocrisolve 100 in the presence of 0.01% volvol−1 DMSO.
The combination of either rimonabant or SR144528 with anandamide infusion had no significant effect on the recoveries of diastolic pressure, LVDP or coronary flow during reperfusion compared to hearts treated with rimonabant or SR144528 alone (Figure 5). However, the infarct size reduction observed when anandamide was infused alone was lost in the presence of either antagonist (Figure 5).
The main finding of this study is that exogenous addition of the endocannabinoid, anandamide, limits infarct size induced by IR. This infarct-limiting action was not mimicked by CB1 or CB2 receptor-selective agonists either used individually or in combination. Furthermore, the infarct-limiting action of anandamide was blocked by the presence of either rimonabant or SR144528, which are regarded as CB1 and CB2 receptor selective antagonists, respectively. These results therefore suggest that anandamide acts either by action at both CB1 and CB2 receptors or that it limits the cardiac infarction associated with IR by activation of one or more novel cannabinoid sites of action. However, since neither ACPA (a selective CB1 receptor agonist) nor JWH133 (which is selective for CB2 receptors), alone or in combination, affected infarct size the involvement of a novel site seems to be the most likely explanation.
The first indication that endocannabinoids might play a role in reducing cardiac IR injury came from indirect evidence concerning lipopolysaccharide- (Lagneux & Lamontagne, 2001) and heat stress-induced (Joyeux et al., 2002) preconditioning. Both of these types of preconditioning were sensitive to the selective CB2 receptor antagonist, SR144528, but not to rimonabant, leading the authors of both studies to conclude that the cardioprotection is mediated through activation of the CB2 receptor.
A more recent study has examined the direct effects of endocannabinoid administration on the severity of cardiac IR injury (LÃ©picier et al., 2003). In that study, the endocannabinoids PEA and 2-AG were shown to reduce infarction associated with low-flow ischaemia in rat isolated hearts. Although infarct size was not directly measured, 0.3μM anandamide, a relatively nonselective endocannabinoid (Glass & Northup, 1999), was ineffective at reducing leakage of creatine kinase or lactate dehydrogenase into the coronary effluent (biochemical markers of cellular damage). In our study, we have shown that anandamide infusion (final concentration 1μM) significantly reduced infarct size by more than 50% compared to vehicle-treated controls. Clearly this is not concordant with the previous investigation. However, there are a number of methodological differences between the two studies. One potentially critical difference is that the vehicles used to deliver anandamide differ. LÃ©picier et al. (2003) used propylene glycol (1:9 volvol−1 with Krebs—Henseleit buffer) to deliver anandamide whereas Tocrisolve 100 (from Tocris Cookson, Bristol, U.K.) was used here. Responses to anandamide in Tocrisolve 100 have previously been reported (Begg et al., 2002; Ford et al., 2002; Kwolek et al., 2005) whereas we are unaware of any reports where anandamide responses have been observed with propylene glycol used as the vehicle. Given the recent report that responses to anandamide can be lost depending upon the solvent/vehicle used (LÃ³pez-Miranda et al., 2004), this is a major consideration.
In their study, LÃ©picier et al. (2003) found that ACEA, a selective CB1 receptor agonist or JWH015, a selective CB2 receptor agonist, were able to reduce infarct size to a similar degree as either PEA or 2-AG. However, in the present study, neither ACPA, a selective CB1 receptor agonist (Hillard et al., 1999), nor JWH133, a selective CB2 receptor agonist (Huffman et al., 1999), reduced infarct size when used individually or in combination. At the present time, we are unable to account for this difference between the two studies. The differences in pharmacology between the two sets of selective CB1 and CB2 receptor agonists are not sufficient to explain the disparity in results.
Indeed the selective agonists in our study were used at a higher concentration than those used by LÃ©picier et al. (2003). However, the choice of vehicle for the selective agonists again differed in the two sets of experiments with LÃ©picier et al. (2003) using ethanol in contrast to the Tocrisolve 100 used here. Therefore, it could be argued that ACPA and JWH133 may be biologically inactive when delivered in Tocrisolve100 as these preparations have not been extensively tested as yet.
Cannabinoids are known to mediate responses via a number of different mechanisms, one of which is uptake and metabolism to form products of the arachidonic acid cascade (Ellis et al., 1995; Kozak et al., 2002). As methanandamide, a nonhydrolysable analogue of anandamide (Abadji et al., 1994), also reduced infarct size, it is unlikely that the mechanism of cannabinoid-induced cardioprotection involves uptake, metabolism and release of arachidonic acid metabolites.
Both anandamide (Smart et al., 2000) and methanandamide (Malinowska et al., 2001) are known to be able to activate vanilloid VR1 receptors. However, rimonabant does not seem to block VR1 receptor-mediated responses (del Carmen GarcÃa et al., 2003). As rimonabant was effective in blocking the reduction in infarct size induced by anandamide, it is unlikely that this process is mediated via activation of the vanilloid VR1 receptor.
Cannabinoid responses can also be mediated by two receptor subtypes that have been identified and cloned, namely the CB1 (Matsuda et al., 1990) and the CB2 receptors (Munro et al., 1993). Anandamide binds and activates both CB1 (Vogel et al., 1993) and CB2 receptors (Zoratti et al., 2003) although there are also examples in the literature where it does not activate CB2 receptors (Bayewitch et al., 1995; Facci et al., 1995). Methanandamide, on the other hand, is a relatively selective agonist for the CB1 receptor (Mechoulam et al., 1998). Therefore, it would appear that a likely candidate receptor for mediating the infarct size limiting actions of anandamide and methanandamide in our study is the CB1 receptor. However, the infarct-limiting response to anandamide is blocked by the selective CB2 receptor antagonist, SR144528 as well rimonabant, which is widely used as a selective CB1 receptor antagonist. There are two possible explanations for these data. Firstly, that the infarct-limiting action of anandamide requires costimulation of CB1 together with CB2 receptors such that if one or other is blocked the cardioprotective response is lost. Secondly, the response to anandamide is mediated by a receptor distinct from either the CB1 or CB2 receptor but which is sensitive to the two cannabinoid receptor antagonists.
We found that, individually, neither the selective CB1 agonist, ACPA (Hillard et al., 1999) nor the selective CB2 agonist JWH133 (Pertwee, 1999) reduced infarct size. This would be expected if the former hypothesis, that stimulation of both receptor subtypes is required in order to observe infarct size reduction, is true. However, when the two selective cannabinoid agonists are used in combination, the size of infarction should be reduced. Our data demonstrates that this is not the case, indicating that the mechanism of anandamide-induced infarct size limitation is not mediated by synergistic activation of CB1 and CB2 receptors, favouring the hypothesis that the response is mediated at a site distinct from these receptor subtypes.
The presence of a cannabinoid receptor which is distinct from either the CB1 or CB2 receptor and is located on endothelial cells has recently been proposed (Mo et al., 2004). Anandamide is thought to be able to activate this receptor and therefore activation of the putative endothelial cannabinoid receptor (JÃ¡rai et al., 1999) might play a role in the mechanism of anandamide infarct size limitation. However, the fact that the response to anandamide was abolished by SR144528 would indicate that the putative endothelial cannabinoid receptor is not involved as it is reportedly insensitive to this antagonist (Mo et al., 2004).
We have previously reported that anandamide mediates cardiac responses such as negative inotropy and coronary vasodilatation by interaction with a novel site distinct from any of the pathways already known to mediate cannabinoid responses (Ford et al., 2002). The pharmacological profile of the limitation in infarct size by anandamide observed in this study is the same as that we reported in our previous study (Ford et al., 2002). Therefore, it would appear that in addition to the previously reported cardiac responses, anandamide reduces infarct size by interaction with one or more novel mechanisms of cannabinoid signal transduction similar to those that mediate negative inotropy and coronary vasodilatation.
Although there is no direct evidence for the novel site of cannabinoid action in the heart being a receptor, it is the most likely explanation for our observations as responses to anandamide were mimicked by methanandamide, a nonhydrolysable analogue, and antagonised by rimonabant and SR144528 at concentrations below those reported to have nonspecific actions. The sensitivity of the novel cardiac cannabinoid site to SR144528 or rimonabant excludes the possibility that it shares identity with either the putative endothelial cannabinoid receptor (Mo et al., 2004) or the putative CB3 receptor identified in the CNS (Fride et al., 2003). The orphan, G-protein-coupled receptor, GPR55, can be activated by anandamide but is unlikely to be responsible for mediating the responses observed in the heart as rimonabant acts as a GPR55 agonist (Brown & Wise, 2001; Drmota et al., 2004; Brown et al., 2005) whereas responses to anandamide were antagonised in our study. Although the literature contains evidence supporting the existence of a number of cannabinoid receptors distinct from CB1, CB2 or VR1, responses to anandamide have a unique pharmacological profile in that they are sensitive to both rimonabant and SR144528.
In summary, anandamide and its stable analogue R-(+)-methanandamide, limit infarct size induced by IR in rat isolated hearts. The pharmacological profile of this response is the same as that previously reported for anandamide-induced cardiac responses (Ford et al., 2002) and fails to match with any of the previously known mechanisms of cannabinoid action. We therefore conclude that anandamide reduces infarct size in rat isolated hearts by interaction with one or more novel sites of cannabinoid action that might involve a new cannabinoid receptor subtype.
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