| Background:Atrial fibrillation(AF)is the most common arrhythmia with a high incidence and is a risk factor for many diseases such as ischemic stroke,heart failure,and sudden cardiac death.The pathogenesis of AF is complicated.The current treatment of AF includes medical therapy and surgical therapy,and both of them have limitations.Atrial remodeling,including electrical remodeling and structural remodeling,is the major pathophysiological mechanism of AF initiation and maintenance.Studies have shown that oxidative stress is potentially related to atrial remodeling,which is one of the main mechanisms involved in the development of AF.Ca2+/calmodulin-dependent kinase II(CaMKII)is a multifunctional serine/threonine kinase.When oxidative stress occurs,CaMKII can be activated by oxidation of methionine 281/282(Met281/282)to form oxidized CaMKII(ox-CaMKII)to participate in cellular activities.The overexpression of ox-CaMKII was observed in patients with arrhythmias.Besides,the absence of Met281/282 in the CaMKII was found to reduce angiotensin Ⅱ(Ang Ⅱ)-induced susceptibility to AF in mice.CaMKII may serve as a target of antioxidative stress and become a valuable approach to developing new treatments for AF.Endocannabinoid system(ECS)is a widely conserved lipid signaling system that consists of endocannabinoids,their metabolic enzymes and the cannabinoid receptors.The ECS is involved in a variety of physiological and pathological processes,including embryo implantation,cognition,cell differentiation and mechanisms regulating inflammation,nociception,ion homeostasis,and energy balance.It has been demonstrated that cannabinoid receptor 1(CB1R)and cannabinoid receptor 2(CB2R)are both expressed in the cardiovascular system,and the activation of CB2R provides cardioprotective effects in animal models of atherosclerosis,myocardial ischemia and drug-induced myocardial injury.However,the role of CB2R in AF remains unclear.Besides,it has been shown that activation of CB2R plays an antioxidant role in alleviating tissue damage in animal models of cisplatin induced renal injury,degenerative and ischemic nerve injury,and isoproterenol-induced myocardial infarction.Meanwhile,it has been found that CaMKII could be activated by CB2R agonists to regulate Ca2+channels in the nervous system.However,whether CB2R plays a role in AF by regulating CaMKII needs to be further investigated.Objective:1.To assess the expression levels of CB1R and CB2R in the atrial tissues of AF patients and Ang Ⅱ-induced AF model mice.2.To evaluate the effect of CB2R activation on Ang Ⅱ-induced atrial remodeling and susceptibility to AF in mice,3.To study the effect and mechanism of CB2R activation on Ang Ⅱ-induced mitochondrial damage and oxidative stress in HL-1 cells.Materials and methods:1.6 patients with degenerative valvular disease who were expected to undergo mitral valve replacement admitted to the Department of Cardiovascular Surgery of the Northern Theater Command General Hospital from May 2021 to September 2021 were enrolled.General information about patients and data from echocardiography were collected.Patients were divided into the sinus rhythm group and AF group according to their medical history and preoperative electrocardiogram examination.Atrial appendage were obtained during the operations.The expressions of CB1R and CB2R in the two groups were detected by immunohistochemistry,immunofluorescence and Western blot.Meanwhile,twenty healthy male C57BL/6 mice were randomly divided into the Control group and the Ang Ⅱ group.Ang Ⅱ[2000 ng/(kg·min)]was infused via osmotic pumps into mice in the Ang Ⅱ group to establish AF model.On the 21st day after medication,programmed electrical stimulation was performed to detect the difference in susceptibility to AF between the two groups,and immunohistochemistry,immunofluorescence and Western blot were performed to detect the expression changes of CB 1R and CB2R in atrial tissue.2.Seventy-five healthy male C57BL/6 mice were randomly divided into the Control group,Ang Ⅱ group,Ang Ⅱ+Vehicle(Veh)group,Ang Ⅱ+AM1241(CB2R agonist)group and Ang Ⅱ+AM630(CB2R antagonist)group.Ang Ⅱ[2000 ng/(kg·min)]was infused via osmotic pumps into mice except for the control group.The mice in the Control group and the Ang Ⅱ group were injected intraperitoneally with 0.1 ml of PBS daily.The mice in the Ang Ⅱ+Veh group were injected intraperitoneally with 0.1 ml of solvent containing DMSO,Tween-20,PBS(1:1:8))daily.The mice in the Ang Ⅱ+AM1241 group was injected intraperitoneally with 0.1 ml of AM1241 solution(20mg/kg).The mice in the Ang II+AM630 group was injected intraperitoneally with 0.1 ml of AM630 solution(3mg/kg).Twenty-one days after medication,noninvasive blood pressure measurement was performed to observe the fluctuations of blood pressure;programmed electrical stimulation was performed to evaluate the susceptibility to AF;electrophysiological mapping was performed to assess the atrial electrical changes;left atrial diameter detected by transthoracic echocardiography,myocardia fibrosis detected by Masson staining,and the expression levels of TGF-β、MMP9、Collagen Ⅰ、Collagen Ⅲ detected by Western blot were used to assess the atrial structural remodeling;MDA content,SOD activity and GSH content detected by serologically,and ROS level in atrial tissue detected by DHE staining were used to assess the atrial oxidative stress;mitochondrial structural changes detected by transmission electron microscopy,and the expression level of p-Drp1 detected by Western blot were used to assess the mitochondrial damage.Besides,the expression levels of NOX2,NOX4,and ox-CaMKII were detected by Western blot to explore the mechanism of AM1241 activating CB2R affecting Ang Ⅱ-induced atrial remodeling in mice.3.CaMKII overexpression(CaMKII-OE)plasmid was prepared.The CCK-8 kit was performed to assess cell viability in response to drug intervention to determine the optimal concentration of Ang Ⅱ and AM1241.HL-1 cells were divided into four groups according to the intervention conditions:Control group,Ang Ⅱ group,Ang Ⅱ+AM 1241 group,Ang II+AM1241+CaMKII-OE group.Cells in Ang Ⅱ+AM1241+CaMKII-OE group were transfected with the CaMKII-OE plasmid.Then all cells were treated with corresponding drugs.Fluo-4 AM was performed to detect the intracellular calcium homeostasis.Western blot was performed to assess the expression of TGF-β.A fluorescent probe DCFH-DA was used to measure reactive oxygen species.Mito-Tracker Red CMXRos was performed to estimate mitochondrial network.JC-1 was performed to evaluate the mitochondrial membrane potential.Meanwhile,the expression levels of CB2R,NOX2,NOX4,and oxCaMKII were detected by Western blot to explore the molecular mechanism of CB2R activation by AM 1241 on mitochondrial damage and oxidative stress in HL-1 cells induced by Ang Ⅱ.Results:1.There was no significant difference in general information and systolic and diastolic function indicated by echocardiography in patients between the sinus rhythm group and the AF group.The results of immunohistochemistry,immunofluorescence and Western blot indicated that the expression level of CB1R in atrial tissue of AF patients was higher than that of patients with sinus rhythm,the expression level of CB2R was lower than that of patients with sinus rhythm,and the difference was statistically significant.In addition,the AF induction rate of mice in the Ang Ⅱ group was significantly higher than that in the Control group.The results of immunohistochemistry,immunofluorescence and Western blot showed that the expression level of CB1R in atrial tissue of the Ang Ⅱ group was significantly higher than that in the Control group,while the expression level of CB2R was slightly lower than that in the Control group.2.After Ang Ⅱ stimulation,blood pressure and serum Ang Ⅱ and aldosterone levels were elevated in mice,and the administration of solvent,AM1241 and AM630 did not significantly impact on the levels of above.AM 1241 reduced the susceptibility to AF induced by Ang Ⅱ in mice.It alleviated the changes in atrial electrical and structural remodeling,decreased the oxidative stress level of atrial tissue,and improved the mitochondrial damage of atrial tissue.However,the application of solvent and AM630 had no significant effect on AF susceptibility and atrial remodeling induced by Ang Ⅱ.We also found that the Ang Ⅱ infusion elevated the expression levels of NOX2,NOX4 and oxCaMKII in mouse atrial tissue,and that the expression levels were significantly decreased after AM 1241 activation of CB2R.3.The cell viability test indicated that the survival rate of HL-1 cells after 24-hour intervention with 10-5 M Ang Ⅱ was about 50%.When AM1241 was co-treated with Ang II,the protective effect reached a plateau at the concentration of 7 μM.Ang Ⅱ intervention in HL-1 cells can induce calcium homeostasis imbalance,increased expression of TGF-β,elevated reactive oxygen species and mitochondrial damage.The activation of CB2R in cells of the Ang Ⅱ+AM 1241 group improved the above changes induced by Ang Ⅱ.The above changes did not significantly improve in cells overexpressing CaMKII even treated with AM1241 and Ang Ⅱ.At the same time,we found that overexpression of CaMKII enhanced the expression level of ox-CaMKII,and did not significantly affect the expression level of CB2R,NOX2 and NOX4.Conclusion:1.The atrial tissue of AF patients and Ang Ⅱ-induced AF model mice showed high expression of CB1R,while low expression of CB2R.2.Activation of CB2R by AM 1241 alleviated Ang Ⅱ-induced atrial remodeling through antioxidative stress and reduced AF susceptibility in Ang Ⅱ infused mice.3.The activation of CB2R by AM 1241 reduced CaMKII oxidation via NOX inhibition,alleviated mitochondrial damage,reduced ROS levels and improved HL-1 cell damage induced by Ang Ⅱ.4.Activation of CB2R plays a protective role in AF by inhibiting NOX/CaMKII signaling pathway,and CB2R is expected to become a potential target for AF therapy. |
PDF Full Text Request