Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

INTRODUCTION Doxorubicin (DOX) is one of the most commonly used broad-spectrum chemotherapeutic drugs; however, its clinical application is limited because of its dose-dependent cardiotoxicity, which may lead to the development of irreversible cardiomyopathy and/or heart failure (1). The mechanism of DOX’s cardiotoxicity is complex and may involve oxidative (2,3), nitrosative and nitrative stress (4,5), mitochondrial dysfunction/ toxicity (1,6–8), dysregulation of various metabolic (9) and lipid signaling pathways (10–12), activation of various stress kinases and cell death mechanisms (both apoptotic and necrotic) (13), triggering of secondary inflammation and remodeling (14), eventually culminating in cardiac dysfunction and heart failure (1,15). Cannabidiol (CBD) is the most abundant nonpsychoactive constituent of marCannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

Cannabidiol (CBD) is the most abundant nonpsychoactive constituent of mar-ijuana (Cannabis Sativa), which was considered initially to be biologically inactive (16,17). It has a negligible effect on conventional cannabinoid 1 and 2 receptors (CB1/2) in vivo (16,17) and is very safe in humans (18). A pioneering study by J Axelrod and D Wink in 1998 (19) demonstrated that CBD was a potent neuroprotective antioxidant. They found that it was more protective against glutamateinduced neurotoxicity than either ascorbate or α-tocopherol, suggesting that it has potential therapeutic utility in neurodegenerative disorders associated with oxidative stress (19). Later, large numbers of preclinical studies also confirmed potent antioxidant and antiinflammatory effects of CBD in preclinical models of colitis, ischemic reperfusion injury, various neurodegenerative and cardiovascular disorders, diabetes and diabetic complications (20), among others (17,21). An oromucosal spray containing 50% CBD (Sativex) is approved in the United Kingdom, Canada and various other European countries to alleviate pain and spasticity associated with multiple sclerosis (22), and CBD recently received orphan drug approval by FDA for the treatment of refractory childhood epilepsy.
In this study, we aimed to explore the effects of CBD in a well-established, clinically relevant mouse model of DOX-induced cardiomyopathy (4,5,23), particularly focusing on oxidative and nitrative stress and mitochondrial dysfunction/ biogenesis. Our results may also provide a novel mechanistic insight on the protective effects of CBD in various models of tissue injury and a promising tool for the prevention of devastating cardiovascular complications of DOX chemotherapy.

Animals/Drugs
All protocols involving the use of animals were approved by the Institutional Animal Care and Use Committees and were performed in line with the Guidelines for the Care and Use of Laboratory Animals adopted by the National Institutes of Health (NIH) (24). Male C57BL/6J mice weighing 22-30 g were acutely administered with single high dose (20 mg/kg) of DOX (Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally (IP) freshly dissolved in physiological saline. CBD obtained from Tocris (Ellisville, MO, USA) or isolated as described earlier (25) was dissolved in vehicle solution (one drop of Tween-80 in 3 mL 2.5% DMSO in saline). Mice were treated with CBD 10 mg/kg IP, or vehicle started 1.5 h before the DOX injection and once every day. Mice were subjected to hemodynamic measurements at the end of study (5 d). Hearts were excised and snap frozen in liquid nitrogen for biochemical measurements or fixed for histological evaluation as described previously (10,11).

Hemodynamic Measurements in Mice
Echocardiography was performed by using the VisualSonics Vevo0770 system (VisualSonics Inc., Toronto, ON,Canada), which is equipped with a 30 MHz mechanical scan probe which can obtain high resolution 2-dimensional images. Animals were anesthetized with isoflurane (2%) inhalation anesthesia. B mode images were obtained in the plane containing aortic and mitral valves. M mode images were obtained from the parasternal short-axis view at the level of papillary muscles. LV end-diastolic diameters, ejection fraction and fractional shortening were calculated by using Vevo Analysis software as described previously. The investigators performing echocardiography were blinded to the treatment status.

Serum Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) Levels
The blood samples were collected and the serum was removed immediately for CK and LDH measurements using a clinical chemistry analyzer system (VetTest 8008, IDEXX Laboratories, Westbrook, ME, USA).

Myocardial DNA Fragmentation ELISA
The quantitative determinations of cytoplasmic histone-associated-DNAfragmentation (mono and oligonucleosomes) due to in vivo cell death were measured using ELISA kit (Roche Diagnostics GmbH, Indianapolis, IN, USA) (12).

Determination of Myocardial Glutathione (GSH) Content
Myocardial GSH content was determined using kits from Trevigen according to the manufacturer's protocols, as previously reported (12).

Histological Examination of Heart Sections
Heart samples were fixed in 4% buffered formalin. After embedding and cutting 5 μm slices, all sections were stained with nitrotyrosine (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's protocol and/or as described (20). Nitrotyrosinestained sections were counterstained with hematoxylin. The specific staining was visualized and images were acquired using BX-41 microscope and U-TV1 × 2 camera (Olympus, Tokyo, Japan) with 200 × fold magnification.

Real-Time PCR Analyses of mRNA
Total RNA was isolated from heart homogenate using TRIzol reagents (Invitro-gen [Thermo Fisher Scientific Inc., Waltham, MA, USA]) according to manufacturer's instructions. The isolated RNA was treated with RNase-free DNaseI (Ambion [Thermo Fisher Scientific]) to remove traces of genomic DNA contamination. Each RNA sample (1 μg) was mixed with 2 μL of 5× Genomic DNA Elimination Buffer to remove any residual genomic DNA as supplied with the kit and made up to a final volume of 10 μL. RT cocktail (prepared according to the manufacturer's instruction) was added to RNA containing Genomic DNA Elimination Mixture and incubated at 42° C for 15 min, followed by heating at 95° C for 5 min. Each 20 μL of cDNA Synthesis Reaction received an addition of 91 μL of sterile water. Real-time PCR was performed using Oxidative Stress and Antioxidant Defense RT Profiler PCR array or specific primer sets (SAB Bioscience, Frederick, MD, USA). Relative quantification was calculated using the comparative CT method. The normalizer used for each cDNA sample was housekeeping gene β-actin as described (11,12,20,27).

PARP Activity
Myocardial PARP activity was assayed using a colorimetric kit according to the manufacturer's protocol (Trevigen) as described (12).

Myocardial 4-Hydroxy-2-Nonenal (4-HNE) Content
Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form more complex and reactive compounds such as 4-HNE, which has been shown to be capable of binding to proteins and forming stable HNE adducts. HNE in myocardial tissues was determined using a kit (Cell Biolabs). In brief, BSA or Myocardial tissue extracts were adsorbed onto a 96-well plate for overnight at 4° C. HNE adducts present in the sample or standard were probed with anti-HNE antibody, followed by an HRP-conjugated secondary antibody. The HNE protein adduct content in an unknown sample was determined by comparing with a standard curve (12).

Myocardial Glutathione Peroxidase Assay
Glutathione peroxidase enzyme activity was assayed using a SpectraMax spectrophotometer (Trevigen) according to the manufacturer's instructions.

Determination of Myocardial Mitochondrial Complex Activity
Microplate assay kits were obtained from MitoSciences (Eugene, OR, USA) and used to determine the activity of mitochondrial complex I, complex II and complex IV according to the manufacturer's instructions. The complex enzymes were immunocaptured in the microplate and activities were determined in Spectramax M3 (Molecular Devices [Thermo Fisher Scientific]). Complex activities were expressed as percentage activity compared with the heart samples of the vehicle-treated mice similar to that described previously for liver (27).

Mitochondrial DNA Content
Mitochondrial DNA (mtDNA) copy/content determination was performed as described previously (27).

Analyses of Data
Results are expressed as means ± SEM. Statistical significance among groups was determined by paired student t test or ANOVA followed by Tukey post hoc test for multiple comparisons using GraphPad Prism 6 software (San Diego, CA, USA). Probability values of P < 0.05 were considered significant.

CBD Attenuates DOX-Induced Cardiac Injury and Cardiac Dysfunction
First we investigated if CBD attenuates the DOX-induced increased serum LDH and CK levels (markers of tissue/ cardiac injury) and cardiac dysfunction (evaluated by measurement of ejection fraction [EF] and fractional shortening [FS] using echocardiography). DOX-markedly increased serum levels of LDH and CK ( Figure 1A) and reduced EF and FS (Figures 1B, C). The DOX-induced tissue injury and cardiac dysfunction were markedly attenuated by CBD treatment (Figures 1A, B).

CBD Attenuates DOX-Induced Myocardial Oxidative Stress
Next we explored the effects of CBD on myocardial oxidative stress. DOX induced marked increase in myocardial HNE content (Figure 2A), reduced total  Figure 2B), attenuated glutathione peroxidase ( Figure 2C) and mitochondrial complex I and II (but not IV) activities ( Figures 2D, E) (the unchanged complex IV activity is not shown). DOX also induced a marked increase in the myocardial expression of reactive oxygen species generation NADPH oxidase isoform NOX2 (Figure 2F). All markers of DOX-induced oxidative stress were attenuated by CBD treatment (see Figures 2A-F).

CBD Enhances DOX-Induced Impaired Mitochondrial Biogenesis
Since DOX-induced cardiotoxicity is known to be associated with mitochondrial injury and dysfunction (see also Figure 2), we further explored the effects of CBD on mitochondrial copy number and various markers of mitochondrial biogenesis. DOX decreased the myocardial mitochondrial copy number and attenuated expression of mRNA of various markers of mitochondrial biogenesis such as peroxi-some proliferator-activated receptor γ coactivator 1-α (PGC1A), peroxisome proliferator-activated receptor α (PPARA), estrogen-related receptor α (ERRA), uncoupling protein 2 and 3 (UCP2, UCP3) and medium-chain acyl-CoA dehydrogenase (MCAD) (Figures 5 A-G). CBD largely prevented the above-mentioned changes (see Figures 5A-G), suggesting that it enhanced mitochondrial biogenesis in damaged hearts.

CBD Attenuates DOX-Induced Myocardial Cell Death and Matrix Metalloproteinases 2 and 9 (MMP2 and MMP9) Activation
Cell death of cardiomyocytes and endothelial cells, as well as activation of proteolytic MMP enzymes, is a consequence of increased oxidative and nitrative stress and mitochondrial injury. We found that DOX-induced  marked increases in myocardial apoptotic and PARP-dependent cell death in the myocardium and in mRNA expression of MMP2 and MMP9, which all were largely attenuated by CBD treatment (Figures 6A-E).

DISCUSSION
DOX-induced cardiomyopathy/ heart failure is a serious complication of chemotherapy with very limited therapeutic options, mostly including supportive treatment or heart transplantation (1). The currently available preventive strategies are not satisfactory and there is an urgent need for development of novel approaches to prevent this devastating complication of DOX chemotherapy. CBD is a nonpsychoactive constituent of marijuana with potent antioxidant and antiinflammatory effects in preclinical disease models which are independent from classical G-protein coupled cannabinoid 1 and 2 receptors (16,17).
Herein, we show that CBD exerts protective effects against DOX-induced cardiotoxicity and cardiac dysfunction by (i) attenuating oxidative and nitrative stress, (ii) improving mitochondrial function, (iii) enhancing mitochondrial biogenesis, (iv) decreasing cell death and expression of MMPs and (v) decreasing myocardial inflammation.
Consistent with numerous previous reports (1-8) we found that DOX treatment markedly increased oxidative stress and impaired antioxidant defense in the heart. DOX also decreased myocardial mitochondrial complex I and II activities, as well as the activity of the glutathione peroxidase (GPX). This is consistent with studies suggesting a key role for mitochondrial dysfunction and reactive oxygen species generation in triggering the deleterious cascade of the DOX-induced cardiotoxicity (4,5,28,29). In addition to mitochondrial, NADPH oxidase-dependent ROS generation (particularly NOX2dependent) also may contribute to DOX-induced cardiotoxicity (4,30). We also found that DOX increased myocardial expression of iNOS and enhanced 3-nitrotyrosine (3-NT) formation. 3-NT is a marker of peroxynitrite formation, or, more broadly, of nitrative stress (26). Peroxynitrite formed from the diffusion limited reaction of superoxide anion and NO recently was implicated as the key downstream effector of promoting DOX-induced cardiotoxicity (26). Peroxynitrite may induce lipid peroxidation, nitration of key contractile proteins and/or sarco/endoplasmic reticulum Ca 2+ pump, trigger activation of mitogen-activated protein kinases promoting apoptotic cell death and impair mitochondrial function favoring increased reactive oxygen species generation, eventually culminating in cardiac dysfunction (26). In addition, peroxynitrite also can diffuse to the nucleus, inducing DNA breaks and consequent overactivation of the nuclear enzyme PARP-1, which, in turn, leads to energetic crisis and cell necrosis (26). Indeed, both neutralization of peroxynitrite by peroxynitrite decomposition catalysts or inhibition of iNOS or PARP is protective against DOX-induced cardiotoxicity in murine models of DOX-induced cardiomyopathies. (4,5,13). DOX via increased generation of reactive oxygen and nitrogen species also leads to activation of MMP enzymes (31), which in turn promotes pathological remodeling (4,14,32,33).
CBD attenuated DOX-induced myocardial lipid peroxidation and the decline in antioxidant glutathione level and in the activity of glutathione peroxidase, which could be the consequence of attenuated formation or enhanced inactivation of reactive oxygen and nitrogen species. CBD also decreased the DOX-induced increased iNOS expression in the myocardium, most likely contributing to the attenuation of DOXinduced myocardial nitrotyrosine formation as a consequence of decreased NO availability for peroxynitrite forma-tion from NO and superoxide anion. In agreement with this, CBD also attenuated nuclear factor-κB (NF-κB) and/or iNOS expression in a model of liver ischemia-reperfusion injury (34), cisplatin-induced nephropathy (35), and in diabetic cardiomyopathy (20,36). In these models, CBD also attenuated oxidative stress by attenuating mitochondrial dysfunction and reactive oxygen species generation or by decreasing the expression of various reactive oxygen species generating NADPH oxidase isoforms (20,35,36). CBD also attenuated the DOX-induced decreased myocardial mitochondrial complex I activity, in agreement with its effect in a liver ischemia-reperfusion injury model (34). Furthermore, CBD also reduced the DOX-induced decline in myocardial mitochondrial copy number and enhanced mitochondrial biogenesis in injured hearts. Consistent with previous studies, DOX also induced both apoptotic and PARP-dependent cell death in hearts (5,12,23,37), which could be largely attenuated by CBD. CBD also attenuated the DOX-induced increased myocardial inflammation. These results are in agreement with the attenuation of proinflammatory response by CBD in diabetic hearts (20) or in human cardiomyocytes (20) or coronary artery endothelial cells (36) exposed to high glucose, likewise with beneficial effects of CBD in rodent models of myocardial infarction, primary diabetes and diabetic complications (17,(38)(39)(40).
So far, over 50 metabolites of CBD were identified in the urine, showing considerable variations between different species (including humans), however their biological activity was not evaluated in detail (41,42). Substantial evidence from in vitro or in vivo studies suggests that CBD by itself exerts potent antiinflammatory and antioxidant effects, independently from cannabinoid receptors (for example in liver ischemia/ reperfusion injury model CBD exerted comparable antiinflammatory effects in both cannabinoid 2 receptor knockout mice and their wild- type littermates). Nevertheless, the potential protective effect of certain CBD metabolites in vivo cannot be excluded either.
Another issue which deserves some discussion is whether the abovementioned antioxidant and antiinflammatory effects of CBD would interfere with the antitumor activity of DOX. The answer is that it is not likely to do so. For example, mitochondrial-targeted antioxidants appear to have synergistic effects with various chemotherapeutic agents in tumor cell killing (43). Activation of MMPs may be involved in tumor progression and metastasis formation, which is inhibited by CBD, likewise the NF-κB signaling. Furthermore, CBD by itself has been reported to have antitumor effects in a large variety of cancer cell lines, as well as in some explanted tumor models (44)(45)(46), which would rather predict a synergistic effect with antineoplastic drugs. In fact, Insys Therapeutics just received FDA orphan drug designation for CBD as a potential treatment for glioblastoma multiforme in humans.

CONCLUSION
In summary, our results indicate that CBD may represent a novel promising approach for the prevention of DOXinduced cardiomyopathy/heart failure by attenuating oxidative/nitrative stress, mitochondrial dysfunction, cell death and inflammation, and by promoting mitochondrial biogenesis. These results, coupled with the excellent safety of CBD in humans, its protective effects against cisplatin-induced nephropathy, its recently reported antineoplastic properties in various malignancies and its orphan drug approval for glioblastoma multiforme and childhood epilepsy, are particularly encouraging from a therapeutic point of view.

ACKNOWLEDGMENTS
This study was supported by the Intramural Research Program of National Institutes of Health/NIAAA. P Pacher is grateful to George Kunos, Scientific Director of NIAAA, for continuous support, and dedicates this study to collaborator and friend, Itai Bab. The study also was supported by the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism (to P Pacher).

DISCLOSURES
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.  Real-time PCR shows significant increase of (A) TNFA, (B) IL1B, (C) MCP-1 mRNA level. Pretreatment with CBD significantly attenuates DOX-induced increased proinflammatory chemokine levels. *P < 0.05 versus vehicle group; # P < 0.05 versus DOX group; n = 4-6/group.