Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy

Abstract

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.

Fig.1. Cannabidiol attenuates diabetes-induced left ventricular dysfunction

A) Representative pressure–volume (P–V) loops at different preloads after inferior vena cava occlusion, showing differences in the end-systolic and end-diastolic P–V relations (ESPVR and EDPVR) in control and diabetic mice treated with vehicle or cannabidiol (CBD). The shift of P-V loops right and changed slope of ESPVR and EDPVR in diabetic mice indicates decreased systolic and diastolic functions, which were less pronounced in diabetic mice treated with CBD (20 mg/kg/day) for 11 weeks. B) 12 weeks of diabetes was associated with decrease in left ventricular systolic pressure (LVSP), maximum first derivative of ventricular pressure with respect to time (+dP/dt), stroke work, ejection fraction, cardiac output, and load-independent indexes of contractility (preload-recruitable stroke work (PRSW), dP/dt–end-diastolic volume relation (dP/dt-EDV), and end-systolic pressure–volume relation (Emax), respectively), and an increase in left ventricular end-diastolic pressure (LVEDP) and prolongation of relaxation time constants (τ Weiss and Glantz), which were largely attenuated by CBD treatment (20 mg/kg/day I.P.) for 11 weeks. Results are mean±SEM of 8-11/group. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D].

Fig.2. CBD attenuates diabetes-induced myocardial oxidative stress

Oxidative stress in the myocardial tissues were determined by measuring (A) MDA, (B) 4-HNE, (C) protein carbonyls content and (D) ROS levels by EPR as described in the Methods section and the (E) NADPH oxidase subunits mRNA expression by real time RT-PCR (F) endogenous antioxidants (GSH) content and (G) SOD activity. *P<0.05 vs. vehicle control/CBD alone, #P<0.05 vs. diabetes [D], n=6-9/group.

Fig.3. CBD attenuates diabetes-induced myocardial NF-κB activation

(A) Western blot analysis demonstrates IκB-α expression and its phosphorylation in the cytosolic fraction and (B) the nuclear translocation of p65NF-κB in the nuclear fraction of the heart tissue homogenates. (C) Shows the gel shift assay demonstrating NF-κB activation. (D) Shows the mRNA expression of ICAM-1/VCAM-1 (E) TNF-α in the respective groups as indicated (F) Shows the western blot analysis for the protein expression of ICAM-1/VCAM-1 and (G) TNF-α protein in the myocardial tissues. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n=6-9/group.

Fig.4. CBD inhibits diabetes-induced myocardial, iNOS expression and 3-NT accumulation

(A) iNOS expression was determined by Western immunoblot in the heart tissues (B) 3-NT levels in the heart samples were quantitatively determined by ELISA with indicated CBD concentration (mg/kg body weight) respectively. (C) Representative gel indicating the nitrated proteins analyzed by immunoprecipitation with 3-NT specific antibody (D) shows the representative images for the histochemical staining for 3-NT levels in the formalin-fixed myocardial tissues. (E) Depicts the immunofluorescence staining for 3-NT from frozen sections as described in methods. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n= 6-8/group.

Fig.4. CBD inhibits diabetes-induced myocardial, iNOS expression and 3-NT accumulation

(A) iNOS expression was determined by Western immunoblot in the heart tissues (B) 3-NT levels in the heart samples were quantitatively determined by ELISA with indicated CBD concentration (mg/kg body weight) respectively. (C) Representative gel indicating the nitrated proteins analyzed by immunoprecipitation with 3-NT specific antibody (D) shows the representative images for the histochemical staining for 3-NT levels in the formalin-fixed myocardial tissues. (E) Depicts the immunofluorescence staining for 3-NT from frozen sections as described in methods. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n= 6-8/group.

Fig.5. CBD mitigates diabetes-induced myocardial activation of MAPKs and augments Akt activation

Western blot analysis shows the (A) p38MAPK (B) JNK (C) p38α/βMAPK (D) MAPKAPK-2 and (E) Akt activation in the myocardial tissues. P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n=6/group.

Fig.6. CBD mitigates diabetes-induced myocardial apoptosis and cell death

(A) Western blot analysis for the caspase 3 and (B) caspase 3/7 activity (C) chromatin fragmentation and (D) PARP activation and (E) quantitative TUNEL assay was performed as described in Methods. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n=6-9/group.

Fig.7. CBD mitigates apoptosis in the diabetic myocardium

Shown are the representative TUNEL images in the diabetic myocardium and mice that were treated with CBD for 11 weeks. For details see the supplemental methods.

Fig. 8. CBD attenuates diabetes-induced cardiac fibrosis

(A) Shows the mRNA expression of the pro-fibrotic genes in the myocardial tissues. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n=9/group. (B) Depicts the Sirius red staining indicating collagen deposition and implying and the extent of cardiac fibrosis. Images shown are representative from 4 independent experiments. *P<0.05 vs. vehicle control/CBD alone; #P<0.05 vs. diabetes [D], n= 4-6/group.