Coronary occlusion studies

Male Sprague-Dawley rats (300–400 g), were bred and housed in the University of Aberdeen Medical Research Facility. Animals were maintained at a temperature of 21 ± 2°C, with a 12 h light/dark cycle and with free access to food and tap water. Animals were obtained on a daily basis and allowed to acclimatize before commencing the study. All studies were performed under an appropriate Project License authorized under the UK Animals (Scientific Procedures) Act 1986.

Studies to investigate the pharmacological mechanism of CBD

In a separate group of rats we aimed to elucidate the type of receptors CBD acts on in the anaesthetized rat. Animals were anaesthetized and cannulated as previously described. MABP was measured via the carotid cannula and heart rate (HR) was calculated from the ECG. After surgery, rats were allowed to stabilize for 15 min before drug/vehicle administration. Post stabilization, animals were administered a bolus dose of vehicle followed subsequently (at regular intervals) by increasing doses of the proposed GPR55 agonist, O-1602 (5–100 ng·kg⁻¹; n = 3–8), firstly in the absence then presence of CBD (50 µg·kg⁻¹). To investigate the role of CBD at the CB₁receptor, the haemodynamic effects of the CB₁ agonist, arachidonyl-2¢-chloroethylamide (ACEA; 3 mg·kg⁻¹; n = 4), were investigated in the absence and then presence of CBD (50 µg·kg⁻¹). To compare the effects of CBD on the ACEA-mediated vascular response with a known fatty acid amide hydrolase (FAAH) inhibitor, URB597 (1 mg·kg⁻¹; n = 4) was administered to rats prior to the administration of a bolus dose of ACEA (3 mg·kg⁻¹), as ACEA is thought to be susceptible to hydrolysis by FAAH.

Statistical analyses

For the haemodynamic data (expressed as mean ± SEM) Student’s two-tailedt-test was used to compare pre-injection and post-injection MABP/HR values. One-way analysis of variance (anova) and Dunnett’s post hoc test was used to compare pre-occlusion and post-occlusion MABP/HR values. Post-occlusion MABP/HR comparisons between the control and CBD-treated groups were made using a two-way anova and Bonferroni post hoc test. Ventricular and reperfusion arrhythmias were determined from the ECG trace and classified according to the Lambeth Conventions (Walker et al., 1988). The effect of CBD on the number of ventricular ectopic beats [VEBs; reported as singles, salvos, ventricular tachycardia (VT) and total VEB count and values expressed as mean ± SEM] was analysed using a one-way anovaand Dunnett’s post hoc test. The effect of CBD on the incidence of VT, reversible and irreversible ventricular fibrillation (VF) and on mortality were analysed using Fisher’s exact test. The effect of CBD treatment on both PR and QT intervals at various time points was investigated using a two-wayanova and Bonferroni post hoc test. The effects of CBD on infarct size, ex vivoand in vitro platelet aggregation, mast cell degranulation, and the effects of both CBD and URB597 on ACEA-induced vascular responses were analysed using Student’s t-test or a one-way anova and Dunnett’s post hoc test, where appropriate.

Effects of CBD on haemodynamic variables

The effects of CBD administration prior to and during CAO on MABP and HR are summarized in Table 1. Administration of 50 µg·kg⁻¹, but not 10 µg·kg⁻¹, CBD prior to CAO induced a significant but transient fall in MABP that reached a nadir 5 min post administration (P < 0.05). Treatment with CBD (50 µg·kg⁻¹), 10 min prior to reperfusion, similarly induced a significant but transient fall in MABP, which reached a nadir 11 min post administration (P < 0.01). All groups exhibited the characteristic fall in MABP upon occlusion of the coronary artery (P < 0.001). Administration of CBD (50 µg·kg⁻¹) prior to reperfusion had no effect on the recovery of MABP post occlusion when compared with control animals. None of the CBD administration regimens had any significant effects on HR at any time point (Table 1).

Table 1

Summary of MABP and HR in rats given saline or CBD either prior to (time −10 min) ischaemia (performed at time 0 min), or prior to reperfusion (at +30 min)

Effect of CBD on I/R-induced ventricular arrhythmias

Induction of myocardial ischaemia resulted in the generation of a significant number of ventricular arrhythmias in the control group (Figure 1A). In most cases arrhythmias commenced 9–10 min post coronary occlusion in all groups (data not shown) and the majority occurred as VT (Figure 1A). Treatment with 50 µg·kg⁻¹, but not 10 µg·kg⁻¹, CBD prior to coronary occlusion significantly reduced the incidence of ischaemia-induced VEBs occurring as VT, and consequently the total number of VEBs compared with vehicle-treated animals (both P < 0.001; Figure 1A). Although CBD (50 µg·kg⁻¹) administration prior to coronary occlusion tended to reduce the incidence of reversible and total VF this did not achieve statistical significance (Figure 1B). The lower dose of CBD (10 µg·kg⁻¹) did not alter any type of VF compared with vehicle-treated animals. Further examination of the ECG revealed that myocardial ischaemia prolonged the QT interval in all groups but this was not significantly affected by CBD (50 µg·kg⁻¹) treatment (Figure 2A). Neither CAO nor CBD administration significantly altered the duration of the PR interval in any of the groups examined (Figure 2B). Reperfusion of the myocardium resulted in the generation of ventricular arrhythmias, the majority occurring as VT. Treatment with CBD (50 µg·kg⁻¹) immediately prior to reperfusion did not affect the occurrence of VEBs when compared with the vehicle control [77 ± 50 vs. 92 ± 45 (total VEBs); not significant]; neither control nor CBD (50 µg·kg⁻¹)-treated groups experienced any VF during reperfusion.

Figure 1

Effect of pre-ischaemic administration of CBD on (A) ischaemia-induced arrhythmias and (B) the incidence of VF. The incidence of each type of arrhythmia was recorded and the data expressed as mean ± SEM (n = 4–14). *P < 0.05, ** …

Figure 2

Effect of CBD administration on (A) PR and (B) QT intervals. Both PR and QT intervals were measured from the ECG in milliseconds (ms) and the data expressed as mean ± SEM (n = 7–8). ***P < 0.001 versus pre-ischaemic values. CBD, …

Effect of CBD on infarct size

Figure 3 illustrates the effects of the higher dose of CBD (50 µg·kg⁻¹) on both area at risk (percentage of left ventricular area) and infarct size (percentage of area at risk). Area at risk was similar across all groups. Administration of CBD (50 µg·kg⁻¹) prior to coronary occlusion significantly reduced infarct size, as did its administration immediately prior to reperfusion, when compared with vehicle-treated control rats (both P < 0.001; Figure 3).

Figure 3

Effect of CBD administered both prior to ischaemia and prior to reperfusion, on area at risk and infarct size. Area at risk was measured as a percentage of total left ventricular area and infarct size was measured as a percentage of area at risk. Both …

Effect of CBD on platelet aggregation

In time-matched sham-operated rats CBD (50 µg·kg⁻¹) significantly reduced collagen-induced platelet aggregation ex vivo compared with vehicle-treated sham-operated rats (P < 0.05; Figure 4A). Administration of CBD (50 µg·kg⁻¹) prior to ischaemia similarly attenuated collagen-induced platelet aggregation measured ex vivo (P < 0.05; Figure 4A). Interestingly, when CBD (50 µg·kg⁻¹) was administered immediately prior to reperfusion it did not significantly affect platelet aggregation when compared with the control. In a series of experiments to investigate the in vitro effects of CBD on agonist-induced platelet aggregation only the highest concentration of CBD investigated (1 mM) significantly attenuated collagen-induced platelet aggregation compared with the vehicle (P < 0.05; Figure 4B).

Figure 4

Effect of CBD (50 µg·kg⁻¹) treatment on (A)ex vivo and (B) in vitro platelet aggregation in response to collagen (5 µg·mL⁻¹). Platelet aggregation was expressed in terms of ohms (Ω) and expressed …

Effect of CBD on I/R-induced cardiac mast cell degranulation

Figure 5 summarizes the effects of CBD on cardiac mast cell degranulation. In vehicle-treated sham-operated animals, approximately 44% of cardiac mast cells were degranulated and similar numbers were found in sham-operated rats given CBD (50 µg·kg⁻¹). Myocardial I/R induced significant (P< 0.001) mast cell degranulation in vehicle-treated control rats, when compared with the vehicle sham-operated group; and administration of CBD (50 µg·kg⁻¹) either prior to or post CAO did not alter the extent of mast cell degranulation induced by I/R alone.

Figure 5

Effects of CBD (50 µg·kg⁻¹) and I/R on the percentage of mast cells degranulated in the rat myocardium. Mast cell degranulation was measured as the percentage of the total number of mast cells present that had undergone degranulation …

Receptor-mediated effects of CBD

The haemodynamic effects of a range of doses (5–100 ng·kg⁻¹) of O-1602 (GPR55 agonist) were examined; however, no reproducible measurable depressor response was obtained over the dose range tested (data not shown). Administration of the CB₁ receptor agonist, ACEA (3 mg·kg⁻¹), induced a depressor response that was unaffected by pretreatment with CBD (50 µg·kg⁻¹; Figure 6), a proposed CB₁ antagonist. Furthermore, a similar ACEA-induced depressor response, to that observed in the presence of CBD, was demonstrated when ACEA was administered in the presence of the selective FAAH inhibitor, URB597 (1 mg·kg⁻¹; Figure 6).

Figure 6

Receptor-mediated effects of (-)-cannabidiol (CBD). The role of CBD as either a CB₁antagonist or potential fatty acid amide hydrolase (FAAH) inhibitor was investigated by comparing the effects of CBD (50 µg·kg⁻¹) and the selective …

Anti-arrhythmic effects of CBD

There are several explanations for the mechanisms underlying the anti-arrhythmic effect of CBD, one of which could be a direct electrophysiological effect. CBD has been reported to inhibit the slow component of the delayed rectifying potassium channel (IKs) in ventricular myocytes (Mamas and Terrar, 1998). IKs blockers prolong cardiac action potential duration and QT interval and suppress electrically induced arrhythmias in the presence of myocardial ischaemia (Tamargo et al., 2004). However ECG analysis revealed that CBD did not prolong QT interval before ischaemia, nor did it further enhance the ischaemia-induced QT prolongation, suggesting that this is an unlikely explanation for CBD’s anti-arrhythmic effects.

The finding that CBD inhibits collagen-induced platelet aggregation ex vivosuggests an alternative mechanism for its anti-arrhythmic effect, as numerous studies have shown that anti-platelet agents are anti-arrhythmic by virtue of their ability to prevent release of arrhythmogenic substances such as thromboxane A2 and 5-hydroxytryptamine (Wainwright et al., 1988; Barnes and Coker, 1995). What is interesting, however, is that CBD only inhibited platelet aggregation ex vivo when given to sham-operated animals or prior to ischaemia, but not when given prior to reperfusion. While this finding supports the notion that an effect on platelets may be responsible for its anti-arrhythmic effect during ischaemia but not following reperfusion, it cannot explain the ability of CBD to preserve tissue from cell death. Moreover, what this observation may also suggest is that, the mechanism by which CBD inhibits collagen-induced platelet aggregation when administered under physiological conditions (i.e. in sham-operated and pre-ischaemia) is somehow absent or abrogated under ischaemic conditions (i.e. administered prior to reperfusion). In addition, as data from the in vitro studies demonstrated that CBD (in micromolar concentrations) did not affect platelet aggregation, this may further support the idea that CBD only modulates platelet aggregation through interference with an endogenous system and not directly.

While there is no immediate explanation for this, recent studies have demonstrated that platelets express both CB₁ and CB₂ receptors (Deusch et al., 2004) and that the endocannabinoids anandamide (Maccarrone et al., 1999) and 2-arachidonoylglycerol (2-AG; Baldassarri et al., 2008) both induce platelet activation/aggregation, although whether or not through CB₁and/or CB₂ receptor activation remains controversial. Studies have demonstrated that levels of 2-AG are increased in the ischaemically preconditioned heart (Wagner et al., 2006), thus ischaemia-induced elevated levels of 2-AG may contribute to platelet activation by abrogating the anti-platelet effects of CBD via competition for the same receptors. However, further studies to investigate the various effects of endocannabinoids within the ischaemic myocardium are clearly required.

A third explanation for the anti-arrhythmic effect of CBD is through an action on mast cells, as previous studies have demonstrated that CBD induces mucosal mast cell degranulation (Giudice et al., 2007). Treatment with mast cell degranulating agents prior to ischaemia has been shown to elicit a profound anti-arrhythmic effect via the depletion of mast cell-derived cytotoxic compounds (Parikh and Singh, 1997; Walsh et al., 2009a). However, in the present study it was demonstrated that CBD does not induce cardiac mast cell degranulation, as shown by the lack of effect in hearts from rats subjected to sham treatment. Moreover, CBD did not prevent ischaemia-induced mast cell degranulation, a strategy that has also been demonstrated to be cardioprotective (Humphreys et al., 1998; Walsh et al., 2009b). Taken together this evidence does not support the involvement of a cardiac mast cell-dependent pathway in the anti-arrhythmic effects of CBD.

Although we did not investigate this in the current study, CBD may also mediate its anti-arrhythmic effects through modulation of one or more endogenous cardioprotective agents that have demonstrated anti-arrhythmic effects, including anandamide (Ugdyzhekova et al., 2001; Krylatov et al., 2002; Hajrasouliha et al., 2008). In a previous study, CBD (10–20 µM) was shown to inhibit both the anandamide membrane transporter (thus preventing cellular uptake) and FAAH (thus preventing hydrolysis of anandamide) (Bisogno et al., 2001), both of which would elevate endogenous anandamide levels. In the present study, we attempted to determine whether or not CBD behaved in a similar way to the selective FAAH inhibitor, URB597, by assessing their ability to enhance the vascular response to ACEA, which has been shown to be susceptible to FAAH hydrolysis. However, neither URB597 nor CBD augmented the response to ACEA, therefore the question as to whether or not CBD is acting via inhibition of endocannabinoid breakdown remains to be answered. Moreover, whether or not the estimated low plasma concentration of CBD (~2 µM) achieved in the myocardial I/R study was sufficient to increase endogenous anandamide levels also remains to be determined.

Infarct sparing effect of CBD

In relation to the infarct sparing effect, CBD has previously been shown to protect against both cerebral (Mishima et al., 2005; Hayakawa et al., 2007) and myocardial I/R injury (Durst et al., 2007) and evidence points to this being achieved through a direct anti-inflammatory effect (Weiss et al., 2008) mediated by CB₂ receptors (Hajrasouliha et al., 2008). Our data agree with the findings of Durst et al. (2007) in that CBD significantly reduces tissue injury; however, our study significantly extends their observations in two ways. First, the study by Durst’s group involved both prolonged (7 day) CBD administration and a much later time point for assessment of tissue injury (i.e. at a time when the key pathological events are inflammation and scar formation), whereas we have assessed tissue injury at a time when immediate lethal injury has occurred (within 2 h of reperfusion) but delayed injury has not yet begun. Thus our data show that CBD can undoubtedly reduce the initial injury that is associated with rapid events such as oxidative stress and activation of death signalling pathways (Logue et al., 2005). Second, we have also shown that CBD can do this when given just before restoration of blood flow, implying a potentially valuable clinical application in patients undergoing clinical reperfusion.

Quite how CBD exerts cardioprotection against immediate lethal injury has yet to be fully explored. One suggestion is that CBD may act as a peroxisome proliferator-activated receptor gamma agonist (O’Sullivan et al., 2009), activation of that has previously been shown to reduce infarct size in a murine model of myocardial I/R via a profound anti-inflammatory effect (Honda et al., 2008). In addition, CBD may confer tissue protection by acting as a CB₁ receptor antagonist resulting in preferential activation of CB₂receptors by endocannabinoids, as the bulk of evidence points to endocannabinoids reducing infarct size via activation of CB₂ rather than CB₁receptors (Hajrasouliha et al., 2008; Lim et al., 2009). This is in contrast to the effects of synthetic CB₁ or CB₂ receptor agonists, neither of which reduce infarct size (Underdown et al., 2005), suggesting that endocannabinoid-induced protection may be mediated by receptors other than the typical CB₁/CB₂ receptors. In support of the latter, our own study demonstrated that CBD does not prevent ACEA-induced hypotension, suggesting that under the present conditions, CBD (at a dose of 50 µg·kg⁻¹) does not act as a CB₁receptor antagonist. It could act as an antagonist at the orphan receptor GPR55, which has been proposed as a third cannabinoid receptor (Ryberg et al., 2007), through inhibition of a detrimental effect of anandamide action at this receptor. However, data from the present study suggest that GPR55 receptors are not present on the rat vasculature (due to a lack of observed haemodynamic effects of the GPR55 agonist, O-1602), although this does not rule out the presence of these receptors in the myocardium. To date there are no studies that have explored the role of GPR55 in the setting of acute myocardial I/R, although this clearly would be of value.

Rather than acting through a receptor, CBD may induce a tissue sparing effect through a direct action on ion channels. A very recent study (Ryan et al., 2009) has shown that the neuroprotective effect of CBD may be a result of restoration of intracellular Ca²⁺ homeostasis at the level of the mitochondria; using hippocampal slices this group found that under normal physiological conditions CBD had minimal effects on mitochondrial calcium mobilization, while under conditions of high extracellular K⁺ it significantly reduced cytosolic Ca²⁺ concentration. This effect was abolished by inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (NCX), but not via an inhibitor of the mitochondrial permeability transition pore (mPTP), suggesting that, under pathophysiological conditions, CBD improves intracellular Ca²⁺homeostasis through modulation of NCX activity. A similar effect on the cardiomyocyte mitochondria would therefore be expected to help prevent calcium overload, one of the key mechanisms of immediate lethal injury following reperfusion. In addition, anandamide has recently been shown to reduce inositol-1,4,5,-trisphosphate receptor (IP₃R)-mediated nuclear Ca²⁺release in cardiomyocyte nuclear envelopes expressing both CB₁ and CB₂receptors (Currie et al., 2008). This effect was significantly attenuated by both CB₁ and CB₂ receptor antagonists, providing the first evidence for a nuclear receptor site of action for cannabinoids in cardiomyocytes. Further study of a cardioprotective role for CBD, mediated at either the mitochondrial or nuclear level, is therefore clearly warranted.

In summary, to our knowledge this is the first study to demonstrate an anti-arrhythmic effect of CBD following myocardial I/R. This study is also the first to demonstrate that acute administration of a single dose of CBD is sufficient to reduce myocardial tissue injury irrespective of whether it is administered prior to or post coronary occlusion. While further detailed studies are required to elucidate the mechanism by which CBD preserves tissue in I/R, these data expand on the currently very limited literature detailing the role of CBD in the cardiovascular system and firmly establishes its potential as a cardioprotective agent.

Abbreviations:

2-AG

2-arachidonoylglycerol

5-HT_1A

5-hydroxytryptamine_1A receptor

Δ⁹-THC

(-)-Δ⁹-tetrahydrocannabinol

ACEA

arachidonyl-2¢-chloroethylamide

CAO

coronary artery occlusion

CB₁

cannabinoid receptor 1

CB₂

cannabinoid receptor 2

CBD

(-)-cannabidiol

FAAH

fatty acid amide hydrolase

GPR55

G-protein-coupled receptor 55

H₂O₂

hydrogen peroxide

I/R

ischaemia/reperfusion

IKs

delayed rectifying potassium channel

IP₃R

inositol-1,4,5,-trisphosphate receptor

mPTP

mitochondrial permeability transition pore

NCX

Na⁺/Ca²⁺ exchanger

TRPV1

transient receptor potential vanilloid type 1 receptor

VEB

ventricular ectopic beat

VF

ventricular fibrillation

VT

ventricular tachycardia

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