Cardiovascular complications are the leading cause of morbidity and mortality in diabetic patients. Diabetic cardiomyopathy characterized by myocardial left ventricular dysfunction (both diastolic and later systolic), independent of atherosclerosis and coronary artery disease, has been well documented in both humans and animals (1- 3). The mechanism of diabetic cardiac dysfunction is complex and involves increased oxidative/nitrative stress (4- 7), activation of various downstream transcription factors, pro-inflammatory and cell death pathways such as nuclear factor (NF)-κB (8- 9), poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) (10), and mitogen-activated protein kinase (MAPK) (11- 12), inactivation of pro-survival pathways such as Akt (13), eventually culminating in cell death (14) and changes in the composition of extracellular matrix with enhanced cardiac fibrosis and increased inflammation (15).

Various components of the Cannabis sativa (marijuana) plant, termed cannabinoids (e.g., the most characterized active ingredient, the delta 9-tetrahydrocannabinol [THC]), exert potent analgesic effects through the activation of classic CB₁ receptors located in the central nervous system and anti-inflammatory properties through the activation of CB₂ cannabinoid receptors on immune cells (16). However, the major limitation of the therapeutic utility of THC is the development of centrally mediated CB₁-dependent psychoactive effects (16). Furthermore, the CB₁ receptor activation in the cardiovascular system by endocannabinoids may also contribute to the pathophysiology of multiple cardiovascular diseases, including heart failure and atherosclerosis (17). In contrast to THC, cannabidiol (CBD), the most abundant cannabinoid of Cannabis sativa, which has been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans since 2005 in Canada (18), does not bind to these receptors (19); therefore, it is devoid of psychoactive properties and has no potential to cause adverse cardiac toxicity (20). Importantly, CBD is well tolerated without side effects when chronically administered to humans (21- 22).

A previous study has demonstrated cardiac protection by CBD in myocardial ischemic reperfusion injury (23); therefore, we have investigated the potential protective effects of CBD in diabetic hearts and in primary human cardiomyocytes exposed to high glucose. Our findings underscore the potential of CBD for the prevention/treatment of diabetic complications.

All the animal protocols conformed to the National Institutes of Health (NIH) guidelines and were approved by the Institutional Animal Care and Use Committee of National Institute on Alcohol Abuse and Alcoholism (NIAAA)-NIH. Diabetes mellitus was induced in male C57/BL6J mice 8 to 12 weeks old, weighing 23 to 25 g (Jackson Laboratories, Bar Harbor, Maine) by intraperitoneal injection of streptozotocin (Sigma, St. Louis, Missouri) at the dose of 50 mg/kg dissolved in 100 mM citrate buffer pH 4.5 for 5 consecutive days. After 1 week, blood glucose levels were measured using Ascensia Coutour Glucometer (Bayer HealthCare, Tarrytown, New York) by mandibular vein puncture blood sampling. Mice that had blood sugar values >250 mg/dl were used for the study. In the first set of experiments 1-week diabetic mice were treated with CBD (1, 10, or 20 mg/kg intraperitoneally) or vehicle for 11 weeks (5). In another set of experiments, 8-week diabetic mice were treated with CBD or vehicle for 4 weeks (5). The CBD was isolated as described earlier (24). The corresponding control groups were treated with either vehicle or CBD alone for the same duration. All the animals were provided with food and water ad libitum.

Left ventricular performance was measured in mice anesthetized with 2% isoflurane as previously described (25- 26).

The superoxide dismutase (SOD) activities, and reduced glutathione and oxidized glutathione, malondialdehyde, 4-HNE, and protein carbonyl levels in the myocardial tissues were determined as described in the Online Appendix.

Determination of myocardial reactive oxygen species (ROS) by electron paramagnetic resonance spectrometer is described in the Online Appendix.

Preparation of samples and reverse transcription and real-time polymerase chain reaction (PCR) experiments from heart tissues and the primers are described in the Online Appendix and (5).

The PARP and caspase 3/7 activities, chromatin fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and 3-nitrotyrosine (3-NT) content in the heart homogenates and/or human cardiomyocyte extracts are described in the Online Appendix.

Sample preparations, Western immunoblot analysis, and sources of antibodies are described in the Online Appendix.

The immunohistochemistry/staining from frozen or formalin-fixed myocardial tissues (nitrotyrosine, TUNEL, Sirius red) is described in the Online Appendix.

Human cardiomyocytes (HCM) along with the culture medium were purchased from ScienCell Research Laboratories (Carlsbad, California) and were maintained and treated as described in the Online Appendix.

Mitochondrial superoxide/ROS generation and cell death were determined as described (27) and are detailed in the Online Appendix.

Results are expressed as mean ± SEM. Statistical comparisons were made by 1-way analysis of variance followed by Newman-Keuls post-hoc analysis using GraphPad Prism 5 software (San Diego, California). When heterogeneity of variance was present, analysis of variance was performed after logarithmic transformation of the data. Probability values of p < 0.05 were considered significant.

Diabetic animals exhibited increased blood glucose levels (5) with the decrease in the body weight (5). Diabetic animals also had increased glycosylated hemoglobin (HbA_1c) levels with concomitant decline in the pancreas insulin content (5). The CBD or vehicle treatment (1, 10, or 20 mg/kg intraperitoneally) for 11 or 4 weeks did not significantly alter the body weight, blood glucose level, or pancreas insulin content in either control or diabetic animals (5).

Twelve weeks of established diabetes was associated with impaired diastolic and systolic left ventricular function, which was largely attenuated by the treatment with CBD for 11 weeks (starting 1 week after the establishment of diabetes) (Figure 1). The CBD treatment also improved the diabetes-induced myocardial dysfunction when it was given for 4 weeks in 8-week diabetic mice (5).

There was increased accumulation of lipid peroxides ((Figure 2)A and Figure 2B), protein carbonyls (Figure 2C), ROS generation (Figure 2D), expression of messenger ribonucleic acid of various ROS-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (p22^phox, p67^phox, gp91^phox) (Figure 2E) with concordant decrease of reduced/oxidized glutathione ratio (Figure 2F) and attenuated activity of the superoxide-eliminating enzyme, the SOD (Figure 2G), in hearts of diabetic mice. These changes were attenuated when mice were treated with CBD for 11 weeks during the course of the diabetes ((Figure 2)A to Figure 2G).

As shown in (Figure 3)A, there was a marked inhibitor of nuclear transcription factor NF-κB (IκB-α) degradation in the cytosol of diabetic hearts, with increased phosphorylation of IκB-α leading to release of active p65 NF-κB, which subsequently translocates to the nucleus to induce the inflammatory and apoptotic gene expressions (Figure 3B). Gel shift assay also confirmed the NF-κB activation in diabetic hearts (Figure 3C). The CBD treatment of diabetic mice inhibited the IκB-α and subsequent p65NF-κB nuclear translocation ((Figure 3)A to Figure 3C). The CBD treatment also inhibited the NF-κB–dependent mRNA and/or protein expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 ((Figure 3)D and Figure 3F) and pro-inflammatory cytokine tumor necrosis factor (TNF)-α ((Figure 3)E and Figure 3G), respectively, in the diabetic myocardial tissues.

There was significant increase in inducible nitric oxide synthase (iNOS) expression (Figure 4A) and 3-NT accumulation ((Figure 4)B to Figure 4E) in hearts of diabetic mice compared to vehicle or CBD alone treated mice. The CBD treatment attenuated the diabetes-induced iNOS expression and 3-NT accumulation (marker of nitrative stress) ((Figure 4)B to Figure 4E).

There was marked increase in the p38MAPK (Figure 5A) and c-Jun N-terminal kinase (JNK) (Figure 5B) activation in the myocardial tissues of diabetic mice. In addition, there was marked activation p38αMAPK (Figure 5C) and slightly diminished p38βMAPK (Figure 5C) in the diabetic myocardium. There was also activation of MAPKAPK-2 in the diabetic heart (Figure 5D). CBD treatment for 11 weeks significantly mitigated p38MAPK, JNK, p38αMAPK, MAPKAPK-2 activation, while it was not effective in restoring the p38βMAPK levels. In addition, Akt activation was also significantly hampered in the diabetic myocardium, which was attenuated with CBD treatment (Figure 5E). In diabetic myocardium, there was marked increase in caspase 3 cleavage, caspase 3/7 activity ((Figure 6)A and Figure 6B), chromatin fragmentation, and PARP activity ((Figure 6)C and Figure 6D), and enhanced apoptosis ((Figure 6)E and Figure 7); all these changes in diabetes were attenuated by CBD treatment.

Real-time reverse transcriptase-polymerase chain reaction analysis revealed significant increases in the pro-fibrotic gene expressions (Figure 8A) and in collagen deposition (Figure 8B) in diabetic hearts, and these were attenuated by CBD (Figure 8).

Remarkably, CBD 20 (mg/kg) treatment also attenuated the diabetes-induced increased myocardial nitrative stress, cell death (5) and fibrosis (5) when it was given for 4 weeks in 8-week diabetic mice.

High glucose (HG) treatment of HCM for 48 h markedly increased cytosolic (5) and mitochondrial (5) ROS/superoxide generation compared with cells treated with either D-glucose 5 mM, L-glucose 30 mM, or CBD (4 μM) alone for the same duration. The CBD markedly attenuated the HG-induced increased ROS generation (5) and 3-NT accumulation in HCM (5).

The HG treatment induced NF-κB activation (5) and increased apoptosis and PARP-dependent cell death in cardiomyocytes (5); the antiapoptotic activity of CBD was mediated, at least in part, by its ability to modulate Akt activity (5).

Accumulating evidence suggests that increased oxidative/nitrative stress coupled with activation of various downstream pro-inflammatory and cell death pathways play pivotal roles in the development of complex biochemical, mechanical, and structural alterations associated with diabetic cardiomyopathy (3- 4,6,11- 12,14- 15). However, in spite of the accumulating knowledge obtained during the past decades, the treatment of diabetic cardiomyopathy still remains poor and largely symptomatic (1- 2).

Cannabidiol, a nonpsychoactive component of marijuana, has been shown to exert anti-inflammatory and antioxidant effects both in vitro and in various preclinical models of neurodegeneration and inflammatory disorders, independent from classical CB₁ and CB₂ receptors (20). Furthermore, CBD has recently been reported to lower the incidence of diabetes among nonobese diabetic mice (28) and to preserve the blood-retinal barrier in experimental diabetes (29).

In the present study, we have evaluated the effects of CBD treatment (for 11 weeks administered after the destruction of pancreatic beta cells and development of frank type 1 diabetes mellitus, as well as in 8-week diabetic animals for 4 weeks) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy or primary human cardiomyocytes exposed to HG. Because significant cardiac dysfunction in this model starts to develop from 4 weeks of established diabetes (4,10), with gradually increasing fibrosis thereafter (12,15) (peaking around 8 weeks of established diabetes), in the first treatment protocol (5.), we aimed to study if CBD treatment can prevent the development of characteristic alterations of type I diabetic cardiomyopathy; in the second treatment protocol (5), we sought to determine if it is able to reverse these changes once they have already developed.

Consistent with previous reports, diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with enhanced myocardial expression of NADPH oxidase isoforms p22^phox, p67^phox, gp91^phox, attenuated antioxidant defense (decreased glutathione content and SOD activity) coupled with increased myocardial ROS generation and lipid peroxidation (4,6,10,12,15). The HG-induced ROS generation in addition to inducing lipid peroxidation may also initiate activation of various stress signaling pathways (e.g., jun N-terminal kinase and p38MAPK). Our results are also in agreement with previous studies demonstrating enhanced activation of p38MAPK and its downstream effector (p38MAPKAPK-2) in diabetic cardiomyopathy models and demonstrating that pharmacological inhibition of p38MAPK signaling attenuates the expression of cardiac inflammatory markers, such as TNF-α, interleukin-1β, and interleukin-6, and collagen content associated with diabetic cardiomyopathy (11- 12). Likewise, with recent evidence supporting an emerging role of p38α activation in diabetic cardiomyopathy (12), in addition to its already established role in mediating cell death during myocardial ischemic-reperfusion injury. The HG-induced ROS generation also impairs important pro-survival signaling pathways such as Akt in diabetic hearts (13), activates pro-inflammatory and cell death pathways such as NF-κB (8- 9) and nuclear enzyme poly(ADP)-ribose polymerase 1 (10), which in turn regulate expression of important pro-inflammatory cytokines, cell adhesion molecules, and iNOS. The latter results in increased nitrosative/nitrative stress, which is also implicated in cardiovascular complications of diabetes (5). A recent study has also suggested that the NF-κB activation may induce increased oxidative stress and contributes to mitochondrial and cardiac dysfunction in type II diabetes (9). Importantly, the oxidative-nitrative stress, stress signaling, and inflammatory pathways in diabetic cardiomyopathy are closely interrelated, eventually promoting the development of myocardial fibrosis (3,9,11,15,30).

Treatment with CBD (Supplemental Figure 1) was able to attenuate the oxidative-nitrative stress (decreased the myocardial ROS generation and expression of p22^phox, p67^phox, gp91^phox, restored glutathione content and SOD activity, decreased 3-NT formation) and alterations of the pro-survival (Akt) and stress signaling (p38, p38α, JNK) pathways in diabetic hearts. It also attenuated the NF-κB activation, expression of iNOS, TNFα, and ICAM-1, cell death, and fibrosis in diabetic myocardium, and improved the associated characteristic functional alterations. Importantly, CBD treatment was able to attenuate/reverse (although to a lesser extent) some of the discussed diabetes-induced myocardial biochemical and functional changes after the establishment of diabetic cardiomyopathy with fibrosis (5). The CBD treatment also attenuated the HG-induced increased reactive oxygen and nitrogen species generation, NF-κB activation, and cell death in primary human cardiomyocytes (5).

The discussed beneficial effects of CBD could be explained in part by its potent antioxidant properties, which was first suggested by the Nobel Prize winner Dr. Julius Axelrod (31). In the Axelrod study, CBD was more protective against glutamate-induced neurotoxicity than any of the well-know antioxidants (e.g., ascorbate or α-tocopherol), indicating additional cytoprotective effects of CBD beyond its potent antioxidant properties (31). Indeed, our recent results suggest that CBD may exert potent effects on key pro-inflammatory pathways such as NF-κB and on pro-survival signaling such as Akt in vivo, which is most likely not related to its antioxidant effect. This is also supported by observations that CBD decreases inflammation in models in which conventional antioxidants are not very effective (e.g., in arthritis [20,32]), as well as by recent studies demonstrating that CBD is a potent inhibitor of bacterial lipopolysaccharide-activated NF-κB proinflammatory pathway in microglia cells (33). These results are also in support of the emerging role of the inflammation in the development and progression of diabetic cardiomyopathy (9,11,15,30).

Collectively, our results strongly suggest that CBD may have tremendous therapeutic potential in the treatment of diabetic cardiovascular and other complications by attenuating diabetes-induced oxidative/nitrative stress, inflammation, cell death, and fibrotic pathways.

The authors are indebted to Dr. Murali C. Krishna for generously providing his resources and expertise with electron paramagnetic resonance spectrometer measurements, to Drs. Sergey Dikalov and Kathy K. Griendling for sending the electron paramagnetic resonance spectrometer probe during the time when it was not commercially available, and to Dr. George Kunos for providing key resources and support. Dr. Pacher dedicates this study to the 80th birthday of Professor Raphael Mechoulam and to Dr. Julius Axelrod.

For a detailed discussion of the Methods, supplemental references, table, and figures, please see the online version of this article.

Cannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress, Fibrosis, Inflammatory and Cell Death Signaling Pathways in Diabetic Cardiomyopathy