Cannabinoid receptors may be found all over the cardiovascular system. The myocardium, human coronary artery, endothelial, and smooth muscle cells, as well as presynaptic sympathetic nerve terminals that innervate the cardiovascular system, all express the CB1 receptor. In addition to the myocardium, CB2 receptors have been discovered in human coronary endothelium and smooth muscle cells. Endocannabinoids are generated in endothelium and smooth muscle cells, as well as heart tissue, and their amounts in the blood may be measured. Despite this, the endocannabinoid system is unlikely to have a significant role in the control of cardiovascular function under normal circumstances [30]. JWH-018 is a SC with full agonist effect on both CB1 and CB2 receptors. It is known that the CB1 receptor affinity of SCs is 100 times higher than THC. Due to far more potent receptor activity, it is quite reasonable to expect the cardiovascular effects to be stronger and more problematic.

The negative effects of SCs on the cardiovascular system have been shown in previous studies [31–33]. According to our analyses of cardiac results, the HR response of JWH-018 was found as different due to dose and duration of application. While single high-dose administration resulted with reduced HR, chronic administration resulted with increased HR irrespective of JWH-018 dose. In the AHD group, reduced HR values were accompanying with reduced mean BP. In long-term, BP drop was continuing despite increased HR, especially in the rats are given prolonged high-dose JWH-018. In the related literature, the most frequently reported cardiovascular effect of SCs is a significant decrease in arterial BP, heart contraction and HR [30, 34, 35]. Despite the fact that numerous studies show that SCs related cardiovascular depression effects are mediated by CB1 receptors, they may also have vascular and cardiac effects that are independent of CB1 and CB2 receptors. The role of CB1 receptors in the vasodepressor response has been demonstrated by the relief of hypotension when a CB1 selective antagonist is used [36]. Hypotension caused by cannabinoid and the complete absence of bradycardia in CB1 receptor-deficient mice are the main evidence showing the effect of CB1 receptors on these cannabinoid-related effects [37]. In anaesthetized hypertensive mice, Batkai and colleagues discovered that CB1 receptor agonists reduce contractility and normalize BP [38]. Recently, it was shown that presynaptic CB1 receptor stimulation inhibits norepinephrine release both in vitro and in vivo [39, 40]. JWH-018 and HU-210 are both SCs that act as agonists at CB1 and CB2 receptors, but their differences in chemical structure, receptor binding affinity, and metabolism lead to distinct pharmacological and toxicological effects. JWH-018 contains an indole core with a nitrogen atom, distinguishing it from THC, which lacks nitrogen. HU-210, in contrast, is a classical cannabinoid structurally related to THC, but is fluorinated and contains a bicyclic structure instead of an indole ring. The presence of nitrogen in JWH-018’s indole ring system significantly alters its binding properties and metabolic stability, leading to more potent and toxic effects compared to THC and HU-210 [22]. Furthermore, the hypotensive response to HU-210, a SC, remained intact when sympathetic tone was reduced by ganglionic blockade and vascular tone was restored by vasopressin infusion, even if the bradycardic effect was gone [41]. Above-mentioned data indicate that cannabinoid-induced bradycardia is caused in the short term by inhibition of sympathetic tone to the heart; however, the hypotensive response is directly related to vasodilation [42]. According to our long-term results, increased HR response can be explained by reflex tachycardia for chronic reduced peripheral resistance and hypotension. Furthermore, cannabinoids have a strong ability to block acetylcholine release from heart.

In the current study, the effect of JWH-018 on cardiac structure and function was evaluated using transthoracic ECHO, although we did not recognize functional and structural changes in terms of ejection fraction or fractional shortening and any of diastolic function parameters. In addition, level of serum pro-BNP which is a sign of impaired cardiac functions was found to be increased in long-term high-dose JWH-018-treated group. It can be accepted that this situation is compatible with the cardio depressant effect of SCs as proven before. Pacher et al. highlighted that the hypotensive action of a SC, HU-210, is predominantly due to a decrease in ventricular contractility in pentobarbital-anesthetized mice in vivo, employing pressure–volume conductance [43]. In accordance with this finding, in another study, Wagner et al. reported decreased cardiac index and resulting BP by same SC using radiolabeled microsphere technique [41].

It has been reported in many clinical cases, SCs can cause cardiac arrhythmias and fatalities. CB1/CB2 effects appear to accumulate over time, altering cardiac function through multiple pathways: chronic CB1 activation leads to progressive downregulation of CB1 receptors, altering receptor sensitivity and cardiac autonomic regulation [53]. CB1-mediated inhibition of norepinephrine release may induce compensatory autonomic changes that contribute to progressive cardiac dysfunction [39]. Chronic CB2 activation has been linked to increased oxidative stress and inflammation in cardiac tissue, leading to electrophysiological changes that may prolong the QT interval [15]. In our preclinic study, cardiac arrhythmia frequency was found to be increased in all JWH-018 groups. It has been noticed that there is QT prolongation in rats in the group treated with SAHD JWH-018, unlike in other groups. Moreover, QT analysis, resembling arrhythmia risk, showed prolongation in long-term cannabinoid use, despite increased HR especially in the high-dose group. Al Kury et al. previously demonstrated that endogenous cannabis can produce arrhythmias in rat ventricular myocytes by blocking the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in the absence of CB1 and CB2 receptor activation [44]. In another study, Li et al. found that anandamide, an endocannabinoid, reduced L-type Ca²⁺ current in ventricular myocytes and delayed the length of action potential in cardiac tissues via CB1 but not CB2 receptors. Beside this, anandamide facilitated the inactivation of L-type Ca²⁺ current and inhibited its recovery from inactivation [45]. Recently, Yun et al. investigated the effect of JWH-30, a synthetic cannabinoid on duration of action potential and QT interval. They observed that inhibiting the human ether-a-go-go related gene (hERG) channels in rabbit Purkinje fibers shortened the duration of action potential, and that intravenous administration of JWH-030 (0.5 mg/kg) at the ECG measurement in rats lengthened the QT interval [46]. There is mounting evidence that using SCs increases the likelihood of a clinically significant lengthening of the rate-corrected QT interval of the ECG. Torsades de pointes is the main arrhythmia connected to delayed ventricular repolarization and, therefore, QT interval lengthening. This can progress to deadly ventricular fibrillation and is related to cellular origin of early-after depolarizations and enhanced repolarization dispersion. Therefore, QT prolongation caused by both prescription medications and illicit substances has some relevance [47]. In cases where SCs were detected as a result of toxicological studies conducted in the autopsy series, body fluids and tissue samples examined, the causes of death were usually due to cardiac problems. Cardiac problems identified include causes such as myocardial infarction, dilated cardiomyopathy, cardiomegaly, arrhythmias, and decontamination [48–50]. In the present study, during evaluation of QT interval and arrhythmia, we have also evaluated the ischemic ECG changes such as ST segment depression and T-wave negativity in all JWH-018-administered rats. Although we did not detect any ECG changes resembling ischemia, all JWH groups have more ischemic ECG findings despite prior reported coronary vasodilatory effect of cannabinoids [41]. This ECG changes may be result of decreased BP causing reduced coronary perfusion or increased HR causing supply demand mismatch. However, we did not find cardiac troponin-I elevations in serum as a myocardial injury biomarker in contrast to our histopathological observations which showed ischemic circumstance in the cardiac tissue. Histopathologically, we found morphological changes compatible with the first 4–12th h of ischemia in the SALD and SAHD groups. Although these ischemic changes were not seen on the ECG findings, we thought that they reflected the decreased BP result. Histopathological findings related to arrhythmia detected on ECG were not observed in the JWH-018 groups.

One more thing should be emphasized is the metabolic effect of JWH-018. The ECS regulates hunger and energy balance in the central nervous system, principally through managing both the homeostatic and hedonic components of food intake. By activating CB1 receptors in brain areas implicated in energy control, both endogenous and exogenous cannabis can promote food absorption, change the release of orexigenic and anorexic mediators, and boost hedonic valuation (i.e., the hypothalamus and mesocorticolimbic system) [51]. In contrast, agents with specific antagonistic effects for the CB1 receptor have been shown to suppress food intake and reduce body weight in laboratory animals [52]. Contrary to this knowledge, we found that all rats lost weight when long-term JWH-018 was used, regardless of dose. Cooper reported that among the common side effects of SCs of moderate severity, there may be a decrease in body weight due to loss of appetite [12]. Dalton et al. reported that weight loss was linked to a dose-dependent downregulation of CB1 receptors that lasted throughout chronic exposure [53]. We also found that triglyceride levels in blood lipids were decreased, possibly related to weight loss of rats.

Previously, it is well shown that metabolic enzymes are involved in the biotransformation of SCs. The main ring involved in the molecular structure of SCs is metabolized, especially by the CYP1A enzyme [54]. In some studies, CYP2C9 and CYP1A2 enzymes have been responsible for the metabolism of JWH-018 [55]. The elimination half-life of JWH-018 in animals is approximately 2 h, meaning it is largely cleared within 8–10 h [58]. However, some hydroxylated and carboxylated metabolites have longer half-lives and persist in tissues for 24 h or more, depending on metabolic clearance [55]. This suggests that while JWH-018 itself exerts strong immediate effects, its metabolites could contribute to prolonged pharmacological and toxicological consequences, including cardiovascular stress. The persistence of JWH-018 metabolites in the SAHD group despite lower JWH-018 levels suggests that these metabolites might sustain the toxic effects even after the parent compound has been cleared. Given that SC metabolites can have partial or full agonist activity, their continued presence could extend receptor activation, leading to prolonged hypotension, tachycardia, and arrhythmias [57]. At least nine mono-hydroxylated metabolites of JWH-018 have been found. It has been shown in studies that these metabolites bind to the CB1 and CB2 receptors [56, 57]. It was found that the heart value was between 0.16 and 1.63 ng/mg in the rats in the AHD group. In addition, it was estimated that the heart value was between 0.02 and 0.08 ng/mg in the SALD group. It is detected at lower levels than the rats in the ALD group. One of the possible explanations of these results is that it is oxidized by CYP2C9 and CYP1A2 cytochrome P450 isoforms in drug metabolism and it was thought that the increase in metabolites and the storage of substance metabolites in the organs were due to the effect of UGT2B7 found in hepatic tissue and UGT1A3 main function isoforms found in extraheptic tissue in the conjugation step [11]. The HRs of the rats increased statistically significantly in the groups in which JWH-018 was administered subacutely, compared to the groups in which it was administered acutely. This circumstance may lead to increase renal perfusion and increased renal excretion of JWH-018 and its metabolites. JWH-018 tests on animals have indicated a half-life of some 2 h [58, 59]. Considering that it takes approximately 4–5 half-lives for a drug to be completely removed from the body, the levels of the drug and its metabolites may have been low in SA JWH-018 administered groups due to increased renal clearance with increased HR.

In addition, it was determined that the heart value was between 0.02 and 0.08 ng/mg in the SAHD group. Based on this relationship, in a clinical study, two volunteers received 100 and 150 mg SCs containing 2.9% JWH-018. JWH-018 serum concentration peaked 5 min after inhalation, reaching 8.1 mcg/L and 10.2 mcg/L. However, it is reported that the concentration decreases rapidly after 1 h and cannot be detected in the 24th h [60]. Compared to the AHD group, there was a higher blood level and substance-metabolite accumulation in the tissues. The reason for this situation was thought to be the decrease in the metabolic rate of the drug in excess and/or the toxic effect of the drug on the tissues. At lower doses, metabolic enzymes may process the drug more efficiently, preventing significant accumulation in the heart. Increased HR in the SALD group might have further accelerated drug clearance by enhancing renal and hepatic blood flow, leading to faster elimination. Low-dose JWH-018 might preferentially accumulate in the brain or fat tissue rather than the heart, making it undetectable in cardiac tissue samples. Previous studies show that SCs have a high affinity for lipid-rich tissues, which could explain the lack of detection in the heart at low doses [59].

JWH-018 and metabolite determinations are mostly measured in tissues such as serum, blood, oral fluid, urine, brain, kidney, lung, liver and spleen. Although JWH-018 has been determined in heart tissue before, the study was in mice and only the level of JWH-018 was assayed [61]. On the other hand, we aimed to examine the JWH-018 level in the heart tissue along with the other 5 metabolites, and we determined the levels of 4 metabolites that can be separated from each other correctly. In addition, unlike the other study, the fact that we looked at 4 separable metabolites that we reached as consumables, rather than a few metabolites in serum and heart tissue, constitutes another important uniqueness of our study.