Previous studies have indicated that body mass index and heart weight are significantly higher in MA abusers than in non-MA users(Abdullah et al., 2020). In addition, compared with deaths caused by multiple drug toxicities, those from MA toxicity show heavier hearts(Darke et al., 2018). In the current study, we demonstrated that rat body weight increased significantly after 4 weeks of MA administration (Fig. 2A). These results are similar to previous findings showing an increasing trend in body weight in mice receiving MA (2 mg/kg, IP, 10 days), followed by withdrawal (7 days)(Garcia-Carmona et al., 2018), but differ from other research showing no significant differences in body weight in mice receiving MA (0–6 mg/kg) via subcutaneous injection 5 days a week for 4 weeks(Abdullah et al., 2020). Heart weight was higher in the MA group than in the control group, but not significantly. Relative heart weight, calculated as the heart-to-body weight ratio, increased significantly in the MA group compared to the control group (Fig. 2C), consistent with that found in mice (i.e., increased 4–5 mg/week for 8 weeks at 35 mg/kg MA and 2 mg/week after 20 weeks at 40 mg/kg)(Marcinko et al., 2019). In contrast, other research has reported no significant differences in relative heart weight in rats after an acute dose of 50 mg/kg MA or in mice after 10 days of exposure to 2 mg/kg MA(Islam et al., 2009, Garcia-Carmona et al., 2018). These discrepancies could be explained by the different concentrations and treatment times of MA administration, animal species tested, or specific experimental protocols used. The current study is also the first to report on the protective effects of CBD (80 mg/kg) against MA-induced cardiomegaly in rats.
Various autopsy reports have revealed that cardiomyopathy, coronary artery stenosis, valvular heart disease, and inflammatory heart disease are involved in many MA-toxicity deaths, with myocyte hypertrophy, myocarditis, endocarditis, pericarditis, perivascular and interstitial fibrosis, fiber necrosis, collagen deposition, and subendocardial myocardial infarction found via microscopic examination(Nishida et al., 2003, Darke et al., 2017, Darke et al., 2018, Abdullah et al., 2020). The non-specific cardiac histopathology observed in our study following MA exposure is similar with that reported in autopsy studies and animal models(Islam et al., 2009, Marcinko et al., 2019, Abdullah et al., 2020). Notably, inflammatory response, fibrosis, and necrosis of myocardial tissue were confirmed by H&E staining, ELISA analysis, Masson’s trichrome staining, and western blotting. Compared to the MA group, however, CBD pretreatment alleviated these lesions to a certain extent.
Long-term MA administration in ApoE−/− mice can lead to a significant increase in the levels of plasma C-reactive protein, inflammatory cytokines (ICAM-1, VCAM-1, TNF-α), and neuropeptide Y in the aortic root and myocardial tissue, which promote inflammation and atherosclerosis(Gao et al., 2015). Furthermore, acute exposure to MA in mice (30 mg/kg for 6 h) results in a significant increase in serum IL-6, TNF-α, and IL-10, with a further increase under MA exposure and water-restraint stress(Tomita et al., 2011). Both the Nfkbiz gene (a regulator of NF-κB) and the Nr4a1 gene (a transcription factor) are up-regulated by NF-κB signaling activation, which is associated with inflammatory response(Yamamoto et al., 2004, Shinone et al., 2010). Similarly, mRNA expression of Nfkbiz and Nr4a1 in the heart and TNF-α, IL-1β, and IL-6 levels in serum are significantly induced in mice after MA exposure (30 mg/kg), with further increases in TNF-α and IL-6 when the mice are restrained after MA administration(Shinone et al., 2010). These studies indicate that inflammation plays a key role in myocardial damage induced by MA, which can be aggravated by additional environmental stimuli. In the present study, IL-6 increased and IL-10 decreased after MA administration, but these changes were reversed by CBD (40 or 80 mg/kg), suggesting that CBD may have an anti-inflammatory protective effect on myocardial damage induced by MA (Fig. 4A and 4B). Previous research has indicated that CBD treatment (2 µg/µl) can inhibit the increase in IL-1β mRNA expression in the prefrontal cortex of rats following MA exposure(Karimi-Haghighi et al., 2020). Furthermore, CBD treatment (1.5 mg/kg, IP, 10 weeks) can significantly decrease pro-inflammatory cytokine IL-23, its receptor, CXCL-9, and CXCL-11 in mice with spinal cord injury, but not IL-6 or INF-γ(Li et al., 2018). In rats with myocardial ischemic reperfusion injury, CBD (5 mg/kg, IP, 7 days) can reduce infract size, myocardial inflammation, and serum IL-6(Durst et al., 2007). Myocarditis, focal and diffuse myocardial fibrosis, and myocardial dysfunction are reported in patients with pheochromocytoma, indicating that catecholamine toxicity may lead to myocarditis and myocardial fibrosis(Ferreira et al., 2016). This is supported by our study, whereby MA induced a cardiac inflammatory response and myocardial fibrosis, but these effects were attenuated by CBD in a dose-dependent manner (Fig. 3 and Fig. 4). Thus, CBD exhibited considerable preventive and therapeutic effects against cardiac damage induced by MA exposure, which may be mediated by a reduced inflammatory response.
To evaluate the extent of myocardial necrosis caused by MA, we detected cTnI levels in the left ventricle using western blot analysis, as shown in Fig. 4C. Creatine kinase myocardial band (CK-MB) is a key biomarker of myocardial infarction. Autopsy studies have shown high cTnI expression in fatal MA abusers, with CK-MB levels also increased in cardiac and peripheral blood(Zhu et al., 2007) and pericardial and cerebrospinal fluids of MA abusers(Wang et al., 2011). Elevated levels of cTnI and CK-MB are indicative of increased myocardial necrosis, as found in our study following MA administration. However, we also found that CBD (80 mg/kg) pretreatment decreased cTnI levels compared to the MA group, indicating that high-dose CBD may have a protective effect on cardiac damage. Similar findings have been reported in rabbits with acute myocardial infraction, with CBD administration (100 µg/kg) significantly decreasing plasma levels of cTnI and reducing ischemic injury in the myocardium(Feng et al., 2015).
The distribution of MA in the major organs of MA-sensitized rats is reported to be higher in the brain and heart than in the kidney, blood, and abdominal muscle, and delayed efflux of MA in the heart may be associated with cardiac toxicity(Nakagawa et al., 2003). The brain corticotrophin releasing factor system, which is associated with cardiac sympathetic control, is activated by chronic MA administration and withdrawal in mice, which further activates the sympathetic pathways in the heart with increased levels of phospho-tyrosine hydroxylase (p-TH) and p-heat shock protein 27 (p-HSP 27), which may be the mechanism of cardiovascular risk related to MA abuse(Garcia-Carmona et al., 2018). Although various pathological mechanisms have been investigated, the mechanism underlying myocardial injury caused by MA remains unclear. Our data showed that the PKA/CREB signaling pathway was activated and p-PKA and p-CREB increased in rats under chronic MA administration. These findings suggest that the PKA/CREB pathway participated in MA-induced myocardial inflammation and myocardial pathology. Increased cellular cAMP promotes the dissociation of PKA, the catalytic subunit of which migrates to the nucleus and phosphorylates CREB at a single phospho-acceptor site (ser 133), with p-CREB then promoting further transcription(Mayr and Montminy, 2001). PKA is the key kinase for CREB phosphorylation(Meyer et al., 2000), and CREB plays an important role in drug addiction(Zhou and Zhu, 2006). PKA, p-PKA, CREB, and p-CREB are highly expressed in different brain regions of MA-induced conditioned place preference (CPP) rats and in SH-SY5Y cells, but can be inhibited by gastrodin(Yang et al., 2020b). The cAMP/PKA/CREB pathway is also involved in the apoptosis of cortical neurons induced by MA, but can be regulated by the neuroprotective effects of gastrodin(Ma et al., 2020). In this study, the expression levels of PKA, p-PKA, CREB, and p-CREB decreased following CBD pretreatment, indicating that CBD may attenuate myocardial inflammation and cardiac pathology by mediating the PKA/CREB signaling pathway. Similarly, CBD has shown potential therapeutic effects on MA-induced CPP in rats via the PI3K/AKT/GSK-3β/CREB signaling pathway(Yang et al., 2020a).
To the best of our knowledge, this study is the first to report on the protective effects of CBD on cardiac pathology elicited by chronic MA exposure in rats, with inhibition of cardiomegaly and reversal of histopathology, inflammatory response, and necrosis. Results showed that CBD at 40 and 80 mg/kg had a protective effect on MA-induced cardiac damage, although the effect was stronger at the higher concentration (80 mg/kg). These findings are similar with previous study showing that CBD at 80 mg/kg, but not 40 mg/kg, can reduce motivation of self-administered MA and drug-seeking behavior after extinction(Hay et al., 2018). CBD may exhibit cardioprotective effects by modulating the expression of crucial components of the cAMP/PKA/CREB signaling pathway. Our study highlights the potential clinical application of CBD in MA-induced cardiac pathology.
Our study demonstrated that chronic MA administration induced cardiomegaly and cardiac pathology in rats, with a notable increase in inflammatory response and myocardial necrosis. Interestingly, CBD pretreatment significantly and dose-dependently reduced the inflammatory response and myocardial necrosis via regulation of the PKA/CREB signaling pathway. These results indicate that CBD may have potential clinical application for the treatment of MA-induced cardiotoxicity. However, the specific molecular mechanism of MA-induced cardiotoxicity and the protective effects of CBD need to be further investigated.