We conducted a systematic search and review following the international guidelines of PRISMA and CAMARADES. The protocol itself was previously uploaded at PROSPERO as recommended per guidelines. Preclinical and clinical research studies were sought in English and/or Spanish through the PUBMED, Web of Science, and Scopus database search engine from its inception to April 2022, using the following keywords and boolean operators: CBD OR Endocannabinoids OR Cannabinoids OR Cardiovascular Disease OR Myocardial Ischemia OR Coronary Disease OR Myocardial Infarction OR I/R OR Oxidative Stress OR Heart Disease, in the title or abstract, finally, results were manually conditioned as follows [(CBD AND Cardiovascular Disease), (CBD AND Myocardial Ischemia), ( CBD AND Coronary Disease), (CBD AND Myocardial Infarction), (CBD AND I/R), (CBD AND Oxidative Stress), (CBD AND Heart Disease)].

During this phase, we found there are no randomized clinical trials that utilize CBD as an experimental intervention in the context of CS, thus we continued looking for preclinical evidence.

Two certified physicians from the cardiology department of our medical center, independently analyzed the titles and abstracts of preclinical studies that contained the pre-specified terms and criteria for inclusion and exclusion. Inclusion criteria were: studies with in-vivo experimental animal models of myocardial infarction, myocardial I/R, coronary disease, myocardial ischemia and/or oxidative stress, that use CBD as a pharmacological intervention in control groups against experimental groups and that report primary and secondary objectives related to CS. Subsequently, the two physicians again independently, reviewed the full text of the studies choosen in the previous phase and select those that met the inclusion criteria. The differences in the review and choice made for each study were agreed upon by V.G as the third reviewer. The electronic platform developed by CAMARADES was used for the compilation and review of studies.

Pre-established data was included on a verification sheet by 3 researchers independently during the review of the selected works, these were: the study design and its methodology, the number of animals, the intervention, the outcomes, the results and finally miscellaneous data (funding source, key conclusions of the study authors). Numerical data from tables, graphs, text and/or figures were used to generate a table that describes the data obtained and synthesize the information. CAMARADES format was used to handle these data. Summaries of the interventions for each study were made by calculating RR (dichotomous variables) and/or SD (continuous variables), using dedicated statistical data management software. During data management, it was prematurely concluded that meta-analysis would not be possible due to the limited number of included studies and the very heterogeneity of the experimental models.

The qualitative information was treated and reported through a narrative textual synthesis, structured around the type of intervention (CBD), the characteristics of the animals, the types of outcomes and the clinical value of the intervention.

Included studies were evaluated for publication bias utilizing the tools suggested by Cochrane and SYRCLE group [38] , strict adherence to international regulations on the management of animal models and previously ethical approval on all studies was mandatory.

In 2007, Durst et al. [39] carried out the first study with CBD in experimental models of myocardial infarction. CBD 10mg/kg intraperitoneal was administered 1 hour prior to the obstruction (30 minutes) of the left anterior descending artery (LAD) and subsequently the same dose of CBD for 7 days in Sprague Dawley rats. Finally, the ex vivo hearts were analyzed; the infarct size (IS) was smaller in the CBD group vs control (9.6% ± 3.9% vs 28% ± 7%, p < 0.001) the cellular inflammatory infiltrate was also smaller in the CBD group, finally, interleukin-6 was also found to be significantly lower (CBD 264 ± 254 pg/ml vs control 2812 ± 500 pg/ml).

Feng et al. in 2015 [40] reproduced myocardial infarction and reperfusion models in medium-sized animals using rabbits, with the aim of standardizing the use of in-vivo magnetic resonance imaging (IVMRI) to assess the effect of intervention with CBD in experimental models. 100 um/kg of CBD was intravenously administered 10 minutes prior to the obstruction of the circumflex artery (90 minutes) and 10 minutes before reperfusion. The rabbits had 24 hours to recover, to later undergo IVMRI and finally blood extraction and ex vivo analysis. Serum troponin I levels were significantly lower in the CBD group vs the control group (3.99 ng/ml ± 1.955 vs 6.39 ng/ml ± 2.93, p = 0.02). The IS was also lower in the CBD group vs control (60.51% ± 12.94% vs 72.66%, p. 0.05). Other reported outcomes in IVMRI such as parietal thickening of the lateral wall was better in the CBD group vs control (34.84% ± 8.515% vs 23.35% ± 9.89%, p < 0.05), microvascular obstruction as well (7.25% ± 2.3% vs. 14.91% ± 4.17%).

The most recent work on CBD in models of myocardial infarction/reperfusion is that of Vadillo et al. [41] , who in 2021 reproduced the experimental model of Durst [39] , but by modifying the intervention with CBD, giving 5mg/kg intraperitoneal for 10 days in rats, prior to LAD obstruction (45 minutes) and 3 control groups. In turn, they documented new mechanisms of action not previously proposed, by directly studying the molecular expression of angiotensin 1 and 2 receptors (AT-1 and AT-2) and the function of the “RISK” (Reperfusion Injury Salvage Kinase) celullar pathway studying the expression of AKT/PKB and ERK proteins. No means were reported for the IS, but rather a 20.55% reduction in the CBD group vs. the control group. AT-2 elevation was found in the models with CBD vs control (p 0.0002). All forms of AKT and ERK were found to be increased in the CBD group vs controls (p 0.0001).

In 2010, Walsh et al. [42] carried out the first experiment on arrhythmias by I/R in rats; different doses of CBD (10 and 50 um/kg intravenous) and placebo were administered in 4 experimental groups, 10 minutes before the obstruction of the LAD and 10 minutes before reperfusion. At the same time, they sought to determine its effect on IS and mechanisms related to platelet aggregation and degranulation of mast cells. CBD treatment was able to decrease IS in the group receiving 50 µm /kg when administered before ischemia and before reperfusion vs control (p < 0.001). Treatment before ischemia also significantly reduced the incidence of ventricular tachycardia and ventricular ectopic beats. On the other hand, the same dose of 50 um/kg was able to decrease ex vivo collagen-induced platelet aggregation in a sham-operated experimental group (p < 0.05). There were no significant differences when studying mast cell degranulation in any group.

Gonca et al. in 2015 [43] analyzed the work of Walsh [42] , deciding to reproduce the same but with a shorter ischemia time. Walsh produced 30 minutes of ischemia and observed a greater number of ventricular arrhythmias. Gonca performed small periods of I/R every 6 minutes in 4 experimental groups. Walsh had previously proposed that adenosine and its receptors might be involved in the antiarrhythmic effects of CBD, so he included a group treated with the selective adenosine type 1 receptor antagonist (DPCPX). CBD 50 µg/kg intravenously was administered 10 minutes before obstruction, and CBD + DPCPX 10 and 15 minutes before obstruction, respectively. Treatment with CBD decreased the incidence of ventricular tachycardia (CBD 2.2 ± 0.4 vs Control 4 ± 0.4) and its duration vs control (CBD 21 sec ± 5 vs 80 sec ± 22). Concomitant administration of DPCPX abrogated these effects.

Some studies that were included in the initial full-text analysis were excluded after their complete reading due to their experimental model. Also, during the review of these models, there were differences that could not be agreed upon by the researchers, so it was ultimately decided to exclude studies that simulated states similar to metabolic syndrome for their mention in this section.

The studies reported a significant reduction in IS in the groups receiving CBD compared to controls, using 2,3,5-triphenyltetrazolium chloride (TTC) stained histopathology. Within the pathophysiology of the I/R, key moments are recognized to establish proper treatment, under this concept, we could not determine the ideal moment to administer the CBD, since the 5 models provided CBD at different moments, however, Feng et al. used IVMRI results and compared them ex vivo with TTC staining, presenting an adequate correlation with a decrease in IS. IS is an important factor in the prognosis of patients with an AMI, the cardioprotective mechanisms suggested in these models are probably related to multiple forms of attenuation of cardiomyocyte death.

Durst et al. demonstrated a reduction in IL-6, perhaps regulating cell necroptosis and pyroptosis, since this form of cell death promotes different proinflammatory pathways. Feng et al., documented a decrease in serum troponin values in the CBD models, also proving a lower IS.

There is also scientific information regarding the CBD anti-inflammatory propierties, the studies presented here suggests that administration previous to myocardial reperfusion and during reperfusion can lead to reduce IS; thus, we beleive that CBD might provide cardioprotection in multiple scenarios of myocardial ischemia, not only during ongoing ischemia like ACS, but also in chronic scenarios where inflamattion is perennial.

The physiological aspects of ischemic conditioning have been proposed as effective cardioprotective strategies, but with difficult technical reproducibility in clinical settings, particularly due to their inconclusive results. The intracellular signaling cascades involved in ischemic conditioning have 3 well-documented components: an activation pathway through G protein-coupled receptors and type A, C, and G protein kinases; the RISK signaling pathway, which involves inhibitory G-protein receptors, RACa serine/threonine protein kinase (AKT), and extracellular regulatory kinase (ERK); and the last one, known as survival activation factor enhancement (SAFE), mobilized by activation of JAK and STAT-3 and 5 cytokine receptors. Valdillo demonstrated for the first time the activation of the RISK signaling pathway in their animal models, as well as increased expression of AT-2 receptors. Their results suggest that one of the cardioprotective mechanisms of CBD could be the modification of this signaling pathway.

Arrhythmias caused by myocardial I/R are related to a worse clinical prognosis. Two studies were grouped in this scenario due to the similarity of the intervention with CBD and the reporting of outcomes.

The 2 models that used CBD in I/R-induced arrhythmias, differed in the time at which the coronary artery was ligated. One study ligated the artery for 30 minutes and evaluated a dose of 50 µg/kg of CBD prior to obstruction and reperfusion respectively, demonstrating a notable decrease in ventricular arrhythmias, particularly ventricular ectopic beats. The second study, with a coronary obstruction time of 6 minutes, demonstrated a decrease in post-reperfusion arrhythmias and correlated these effects to the levels of adenosine and its receptors. The beneficial effects of adenosine have not been reproducible in clinical trials with patients suffering from ST-elevation ACS. However, CBD has been shown to alter adenosine metabolism, so we also suggest that CBD has cardioprotective effects upon activation of adenosine receptors.

Two studies with CBD in models of provoked diabetes excluded for data extraction demonstrated positive effects by reducing oxidative stress, inflammation, cell death and fibrosis. Both models described cellular signaling pathways that allow the mechanism of action of CBD to be correlated within the inflammatory substrate of coronary syndromes.

Rajesh et al. [45] used treatment with CBD for 11 weeks, managing to reduce the levels of proinflammatory cytokines by modifying the NF-κB signaling pathway, CBD also modified the overactivation of activated mitotic kinases (MAPK) documented in diabetic versus CBD models. They were also able to demonstrate increases in AKT activation, which, as we had commented in the Castillo et al. models, is related to cardioprotection through RISK. For their part, Rahman et al. [46] used a synthetic analogue of CBD, called abnormal cannabidiol, which has previously show cardioprotective effects by GPR18 receptor activation and been pointed out as responsible for the positive effects observed on ventricular function of the heart in a previous experimental model and its relationship with adiponectin, a protein involved in the metabolism of fatty acids and glucose. The abnormal CBD compared to the placebo-managed to improve the expression of the GPR18 receptor as well as the levels of adiponectin, in turn, by means of molecular analysis of the AKT proteins, an ERK 1/2 increase in their expression was observed in comparison with the rats not treated with CBD. The effects studied in this model were abolished after the administration of the antagonist (O-1918) of the GPR18 receptor.

One of the limitations of this review is the few publications found and the heterogeneity presented in the studies included. However, this is the first systematic review of preclinical evidence to objectively synthesize the effect of CBD intervention in the context of I/R models. The decrease in IS is a very important prognostic variable for the justification of experimental drugs that seek to offer cardioprotection; one study was even able to quantify this result by means of IVMRI, giving high translational value to the findings.