Author + information
- Received May 15, 2010
- Revision received July 5, 2010
- Accepted July 6, 2010
- Published online December 14, 2010.
- Mohanraj Rajesh, PhD⁎,
- Partha Mukhopadhyay, PhD⁎,
- Sándor Bátkai, MD, PhD⁎,
- Vivek Patel⁎,
- Keita Saito, PhD‡,
- Shingo Matsumoto, PhD‡,
- Yoshihiro Kashiwaya, MD, PhD†,
- Béla Horváth, MD, PhD⁎,
- Bani Mukhopadhyay, PhD⁎,
- Lauren Becker⁎,
- György Haskó, MD, PhD§,
- Lucas Liaudet, MD∥,
- David A. Wink, PhD‡,
- Aristidis Veves, MD¶,
- Raphael Mechoulam, PhD# and
- Pál Pacher, MD, PhD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Pál Pacher, Section on Oxidative Stress Tissue Injury, Laboratory of Physiological Studies, National Institutes of Health/NIAAA, 5625 Fishers Lane, MSC-9413, Bethesda, Maryland 20892-9413
Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose.
Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans.
Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry.
Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-κB and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38α) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-α, markers of fibrosis (transforming growth factor-β, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-κB activation, and cell death in primary human cardiomyocytes.
Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis.
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.
Animals and treatment
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 (Online Fig. 1). In another set of experiments, 8-week diabetic mice were treated with CBD or vehicle for 4 weeks (Online Fig. 2). 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.
Hemodynamic measurements in mice
Left ventricular performance was measured in mice anesthetized with 2% isoflurane as previously described (25,26).
Determination of superoxide dismutase activity, malondialdehyde, reduced glutathione, oxidized glutathione, 4-HNE, and protein carbonyl content
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.
Reverse transcription and real-time polymerase chain reaction
Determination of PARP, caspase 3/7 activities, chromatin fragmentation, TUNEL, and 3-NT content
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.
Western immunoblot analysis
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.
Cell culture studies
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.
Simultaneous determination of cytosolic and mitochondrial ROS generation and apoptosis by flow cytometry
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.
Blood glucose levels, pancreas insulin content, and body weights
Diabetic animals exhibited increased blood glucose levels (Online Figs. 1A and 2A) with the decrease in the body weight (Online Fig. 1D). Diabetic animals also had increased glycosylated hemoglobin (HbA1c) levels with concomitant decline in the pancreas insulin content (Online Fig. 1B and 2B). 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 (Online Figs. 1 and 2).
CBD treatment attenuates diabetes-induced hemodynamic alterations
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) (Fig. 1). The CBD treatment also improved the diabetes-induced myocardial dysfunction when it was given for 4 weeks in 8-week diabetic mice (Online Fig. 3).
CBD treatment attenuates diabetes-induced myocardial oxidative stress
There was increased accumulation of lipid peroxides (Figs. 2A and 2B), protein carbonyls (Fig. 2C), ROS generation (Fig. 2D), expression of messenger ribonucleic acid of various ROS-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (p22phox, p67phox, gp91phox) (Fig. 2E) with concordant decrease of reduced/oxidized glutathione ratio (Fig. 2F) and attenuated activity of the superoxide-eliminating enzyme, the SOD (Fig. 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 (Figs. 2A to 2G).
CBD treatment attenuates diabetes-induced myocardial nuclear factor-κB activation and inflammation
As shown in Figure 3A, 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 (Fig. 3B). Gel shift assay also confirmed the NF-κB activation in diabetic hearts (Fig. 3C). The CBD treatment of diabetic mice inhibited the IκB-α and subsequent p65NF-κB nuclear translocation (Figs. 3A to 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 (Figs. 3D and 3F) and pro-inflammatory cytokine tumor necrosis factor (TNF)-α (Figs. 3E and 3G), respectively, in the diabetic myocardial tissues.
CBD treatments attenuates diabetes-induced nitrative stress
There was significant increase in inducible nitric oxide synthase (iNOS) expression (Fig. 4A) and 3-NT accumulation (Figs. 4B to 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) (Figs. 4B to 4E).
CBD treatment attenuates diabetes-induced MAPK activation and apoptosis
There was marked increase in the p38MAPK (Fig. 5A) and c-Jun N-terminal kinase (JNK) (Fig. 5B) activation in the myocardial tissues of diabetic mice. In addition, there was marked activation p38αMAPK (Fig. 5C) and slightly diminished p38βMAPK (Fig. 5C) in the diabetic myocardium. There was also activation of MAPKAPK-2 in the diabetic heart (Fig. 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 (Fig. 5E). In diabetic myocardium, there was marked increase in caspase 3 cleavage, caspase 3/7 activity (Figs. 6A and 6B), chromatin fragmentation, and PARP activity (Figs. 6C and 6D), and enhanced apoptosis (Figs. 6E and 7); all these changes in diabetes were attenuated by CBD treatment.
CBD treatment attenuates diabetes-associated myocardial fibrosis
Real-time reverse transcriptase-polymerase chain reaction analysis revealed significant increases in the pro-fibrotic gene expressions (Fig. 8A) and in collagen deposition (Fig. 8B) in diabetic hearts, and these were attenuated by CBD (Fig. 8).
CBD post-treatment after the establishment of diabetic cardiomyopathy attenuates diabetes-induced myocardial oxidative/nitrative stress, cell death, and fibrosis
Remarkably, CBD 20 (mg/kg) treatment also attenuated the diabetes-induced increased myocardial nitrative stress, cell death (Online Fig. 4) and fibrosis (Online Fig. 5) when it was given for 4 weeks in 8-week diabetic mice.
CBD treatment attenuates high glucose-induced cytosolic and mitochondrial ROS generation and 3-NT formation in HCM
High glucose (HG) treatment of HCM for 48 h markedly increased cytosolic (Online Fig. 6A) and mitochondrial (Online Fig. 6B) 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 (Online Figs. 6A and 6B) and 3-NT accumulation in HCM (Online Fig. 6C).
CBD mitigates HG-induced NF-κB activation and apoptosis in HCM
The HG treatment induced NF-κB activation (Online Figs. 7A and 7B) and increased apoptosis and PARP-dependent cell death in cardiomyocytes (Online Figs. 8A and 8B); the antiapoptotic activity of CBD was mediated, at least in part, by its ability to modulate Akt activity (Online Fig. 8A).
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 (Online Fig. 1A.), we aimed to study if CBD treatment can prevent the development of characteristic alterations of type I diabetic cardiomyopathy; in the second treatment protocol (Online Fig. 2A), 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 p22phox, p67phox, gp91phox, 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 Fig. 1) was able to attenuate the oxidative-nitrative stress (decreased the myocardial ROS generation and expression of p22phox, p67phox, gp91phox, 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 (Online Figs. 2 to 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 (Online Figs. 6 to 8).
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
Drs. Rajesh and Partha Mukhopadhyay contributed equally to this article. This study was supported by the Intramural Research Program of the NIH/NIAAA (to Dr. Pacher) and NIDA Grant DA9789 (to Dr. Mechoulam). Dr. Horváth is a recipient of a Hungarian Research Council Scientific Research Fund Fellowship (NKTH-OTKA-EU, MB08-80238). Dr. Veves receives funding from Novartis for an investigator-initiated research grant, unrelated to this study. Dr. Mechoulam is a consultant for GW Pharmaceuticals, United Kingdom, which is not involved in this publication and is unaware of it. All other authors have reported that they have no relationships to disclose.
- Abbreviations and Acronyms
- adenosine diphosphate
- human cardiomyocytes
- high glucose
- intercellular adhesion molecule
- inhibitor of nuclear transcription factor nuclear factor-κB
- inducible nitric oxide synthase
- c-Jun N-terminal kinase
- mitogen-activated protein kinase
- matrix metalloproteinase
- nicotinamide adenine dinucleotide phosphate
- nuclear factor kappa B
- poly(ADP-ribose) polymerase
- reactive oxygen species
- superoxide dismutase
- delta 9-tetrahydrocannabinol
- tumor necrosis factor
- terminal deoxynucleotidyl transferase dUTP nick end labeling
- vascular cell adhesion molecule
- Received May 15, 2010.
- Revision received July 5, 2010.
- Accepted July 6, 2010.
- American College of Cardiology Foundation
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