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, 61 (3), 716-27

Cannabinoid 1 Receptor Promotes Cardiac Dysfunction, Oxidative Stress, Inflammation, and Fibrosis in Diabetic Cardiomyopathy

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Cannabinoid 1 Receptor Promotes Cardiac Dysfunction, Oxidative Stress, Inflammation, and Fibrosis in Diabetic Cardiomyopathy

Mohanraj Rajesh et al. Diabetes.

Abstract

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Figures

FIG. 1.
FIG. 1.
Enhanced CB1R expression and endocannabinoid (AEA) levels in diabetic hearts; improved diabetes-induced cardiac dysfunction in CB1−/− mice. A: After 12 weeks of established diabetes, mice were killed, heart left ventricles were excised, and protein samples were analyzed for CB1R expression by Western blot analysis. *P < 0.05 vs. control (CO). B: Levels of myocardial anandamide (AEA) were determined by liquid chromatography–mass spectrophotometry technique as described in research design and methods. *P < 0.05 vs. control. C: Twelve weeks of diabetes in CB1+/+ mice was characterized by decreased systolic (attenuated load-dependent [+dP/dt; ejection fraction, stroke work, cardiac output] and load-independent [D] [Emax, dP/dtmax–end-diastolic volume relation; preload-recruitable stroke work {PRSW}] indices of LV contractile function) and diastolic (decreased –dP/dt, prolonged time constants of LV pressure decay [τWeiss and τGlantz], increased LV end-diastolic pressure [LVEDP] [C], and decreased the slope of the end-diastolic pressure-volume relation [EDPVR; an index of LV stiffness {C, D}]) functions. The diabetes-induced cardiac dysfunction was less pronounced in CB1−/− mice than in CB1+/+ mice (C and D). *P < 0.05 vs. CB1+/+ control (CO), #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 6–9/group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Attenuation of diabetes-induced myocardial inflammation, oxidative/nitrative stress, β-MHC isozyme switch, and AT1R expression in CB1−/− mice. A: LV mRNA expressions of inflammatory cytokines and adhesion molecules. B: iNOS and COX2. C: α- and β-MHC. D: AT1R and p47phox NADPH isoform. E: Oxidative/nitrative stress was determined by measuring 4-HNE and 3-NT in the LV myocardial tissues. F: Protein of iNOS in the respective groups. G: SERCA2a. *P < 0.05 vs. WT/CB1+/+ control (CO); #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 8–9/group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
Attenuated diabetes-induced myocardial RAGE and AGE expression/accumulation, MAPK activation, and cell death in CB1−/− mice. A: LV mRNA expression of RAGE and accumulation of AGEs in the myocardium measured by ELISA. B: Representative Western immunoblot for the analysis of MAPKs (p38 and JNK) in the myocardial tissues. *P < 0.05 vs. CB1+/+ control (CO); #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 6/group. C and D: Markers of cell death (PARP and caspase 3 activities and chromatin fragmentation) in the LV myocardial tissues from the respective groups. *P < 0.05 vs. WT/CB1+/+ control (CO); #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 8/group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Attenuation of diabetes-induced myocardial fibrosis in CB1−/− mice. A: Representative formalin-fixed paraffin-embedded myocardial tissue sections stained with Sirius Red, indicating the marked interstitial fibrosis in the WT diabetic mice, which was attenuated in CB1−/− mice. *P < 0.05 vs. CB1+/+ control (CO); #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 6/group. B: mRNA expression of fibrosis markers in the myocardial tissues. *P < 0.05 vs. CB1+/+ control (CO); #P < 0.05 vs. CB1+/+ diabetes (Diab); n = 9/group. CTGF, connective tissue growth factor. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Attenuation of diabetes-induced cardiac dysfunction by CB1R inhibition. A: Twelve weeks of diabetes in control (CO) mice was characterized by decreased systolic (attenuated load-dependent [+dP/dt; ejection fraction, stroke work, cardiac output] and load-independent [B] [Emax, dP/dtmax–end-diastolic volume relation; preload-recruitable stroke work {PRSW}] indices of LV contractile function) and diastolic (decreased –dP/dt, prolonged time constants of LV pressure decay [τWeiss and τGlantz], increased LV end-diastolic pressure (LVEDP) (B), and decreased the slope of the end-diastolic pressure-volume relation (EDPVR; an index of LV stiffness [B]) functions. Eleven months of treatment with rimonabant (SR141716/SR1) attenuated the diabetes-induced cardiac dysfunction (A and B). *P < 0.05 vs. vehicle/SR1 control (CO); #P < 0.05 vs. diabetes (Diab); n = 6–9/group. B: Representative pressure-volume loops after vena cava inferior occlusions demonstrate attenuation of the contractile function and increased diastolic stiffness in diabetic hearts, which is attenuated by CB1R blockade with SR1. Treatment of 8-week diabetic mice for 4 weeks with SR1 attenuated the diabetes-induced functional alterations (Supplementary Fig. 6). *P < 0.05 vs. vehicle control (CO); #P < 0.05 vs. diabetes (Diab); n = 5–7/group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 6.
FIG. 6.
Attenuation of diabetes-induced myocardial inflammation, oxidative/nitrative stress, β-MHC isozyme switch, and AT1R expression by CB1R antagonists. A: LV mRNA expressions of inflammatory cytokines and adhesion molecules. B: iNOS and COX2. C: α- and β-MHC. D: AT1R, p47phox, gp91phox, and NADPH isoforms. E: SERCA2a. F: mRNA expression of RAGE and accumulation of AGE in the myocardium measured by ELISA. G: Oxidative/nitrative stress determined by measuring 4-HNE and 3-NT in the LV myocardial tissues in the respective groups as indicated. *P < 0.05 vs. vehicle control (CO); #P < 0.05 vs. diabetes (Diab), n = 8–9/group. AM/SR1 treatments alone in control mice had no significant effect on any of the markers studied (not shown) compared with vehicle-treated controls (CO). (A high-quality color representation of this figure is available in the online issue.)
FIG. 7.
FIG. 7.
Attenuation of diabetes-induced myocardial MAPK activation and apoptosis by CB1R antagonists. A: Representative immunoblot for the p38/JNK MAPKs in the myocardial tissues from the respective groups. *P < 0.05 vs. vehicle/AM/SR1 control (CO); #P < 0.05 vs. diabetes (Diab), n = 6/group. B and C: Cell death markers in the respective groups as indicated. *P < 0.05 vs. vehicle/AM/SR1 control (CO); #P < 0.05 vs. diabetes (Diab), n = 8/group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 8.
FIG. 8.
Attenuation of diabetes-induced myocardial fibrosis by CB1R antagonists. A: The representative formalin-fixed paraffin-embedded myocardial tissue sections stained with Sirius Red, indicating the marked fibrosis in the diabetic mice, which was attenuated by CB1R antagonists. *P < 0.05 vs. vehicle/AM/SR1 control (CO); #P < 0.05 vs. diabetes (Diab), n = 6/group. B: mRNA expression of fibrosis markers in the myocardial tissues. *P < 0.05 vs. vehicle/AM/SR1 control (CO); #P < 0.05 vs. diabetes (Diab), n = 9/group. (A high-quality digital representation of this figure is available in the online issue.)

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