Cytocrome P450 Epoxygenase 2J2 And Its Metabolites EETs Attenuate Coxsackievirus B3-induced Acute Viral Myocarditis

Background and objective Viral myocarditis occurs mainly in the population of young adults.According to American epidemiological investigation,incidence of viral myocarditis was approximately22 in 100,000 persons,death rate was up to 4.8 in 100,000 people.Clinical symptoms vary from subtle chest pain to sudden death due to many factors such as age,gender,region,race,virus strain and genetic susceptibility.Vast majority of myocarditis has self-limitation and good prognosis.However,about 9~12 % myocarditis evolved to acute fulminant myocarditis with high mortality.Through three years' follow-up,21 % myocarditis patients were found to progress to chronic myocarditis and finally dilated cardiomyopathy.The common etiology of myocarditis is viral infection,including enterovirus,adenovirus,parvovirus B19(PVB 19),herpes virus type 6(HPV6)and cytomegalovirus,of which coxsackievirus B 3(Coxsackievirus B3 CVB3)was the most dominant causative agent.The pathogenesis of viral myocarditis has three sequential stages: the first stage is acute phase,characterized by the procession that CVB3 virus binds to respective viral receptor embed in cellular surface,enters into myocardial cells via endocytosis and leads to direct myocardial cell toxicity and necrosis;the second stage is subacute phase with activation of immune system(innate immunity,humoral immunity and cell immunity)and infiltration of great deal of inflammatory cells like macrophages and T cells in heart.Inflammatory cells release various cytokines,aggravate the apoptosis and necrosis of cardiomyocytes and secrete different extracellular matrix hydrolases contributing to degradation of interstitial extracellular matrix,which finally leading to cardiac dysfunction and ventricular dilation;the third stage is chronic phase with persistent low titer viral replication and high susceptibility to DCM.Some studies verified that the patients of severe myocarditis with significant hemodynamic abnormalities and inflammatory cell infiltration recovered within a short period and have good prognosis owing to clearing of virus by overwhelming inflammation.But patients with persistent viral replication and few inflammatory cells infiltration had higher risk in progressing to chronic viral myocarditis and dilated cardiomyopathy.Above all,persistent viral replication,inflammatory cell infiltration and apoptosis and necrosis of cardiomyocyte are prominent mechanism of CVB3-induced acute viral myocarditis,responsible for the development of CVB3-induced myocarditis: viral invasion—activation of immunity—chronic myocarditis—dilated cardiomyopathy—heart failure.At present,the pathogenesis of viral myocarditis has not yet been elucidated.Cytochrome P450 oxidase 2J2(CYP2J2)is highly expressed in the human heart and vascular endothelial cells and exert the function of metabolizing arachidonic acid(arachidonic,acid,AA)to four kinds of epoxy-twenty carbon three acid(Epoxyeicosatrienoic Acids,EETs),respectively 5,6-,8,9-,11,12-and 14,15-EET.EETs could be hydrolyzed by soluble epoxide hydrolase(s EH)to dihydmxyeicosatrienoicacacik(DHET),which harbors weak biological activity.Studies have shown that overexpression of CYP2J2 or inhibition of s EH activity increase EETs' level and exert protective effect on heart: 1,EETs can activate potassium channels to promote hyperpolarization of vascular smooth muscle cells,dilating blood vessels and lower blood pressure;2,EETs inhibit the translocation of NF-kappa B into the nucleus and reduce the migration and adhesion of inflammatory cells;3,EETs can inhibit smooth muscle cell proliferation and migration;4,EETs promotes phosphorylation of endothelial nitric oxide synthase(e NOS)at site Ser1177 and Thr495,thus improving endothelial function in vascular endothelial cells;5,EETs enhanced heart function by reducing macrophage infiltration and phenotype change in lipopolysaccharide-induced septic cardiomyopathy;6,The expression of 2J2 relieved the vascular tension of abdominal aortic aneurysm induced by Ang II infusion;7,EETs can activate AMPK alpha,promote the nuclear translocation of AKT1 and exert anti-hypertrophic effect;8,EETs can inhibit cardiac fibroblasts TGF-beta /smad pathway,alleviate the formation of ROS and ameliorate the deterioration of cardiac fibrosis caused by Ang II infusion.s EH is a negative regulator of tissue and serum concentration of EETs,if inhibited,the degradation of EETs is impaired and concentration of EETs is upregulated.Study confirmed that spontaneously hypertensive rats were administrated with AUDA(25mg/L/d)for 6 weeks,AUDA can reduce the systolic blood pressure and reduce the infarction area.When s EH gene were knocked out,PI3K/AKT/NOS3 and AMPK were activated and apoptosis of renal tubular epithelial cells was decreased,which in turn alleviated STZ-induced diabetic nephropathy.Gavage of TPPU(3mg/kg/d)for 7 days can inhibit bombesin or arginine-induced acute pancreatitis by decreasing the infiltration of inflammatory cells.In our study,we accomplished the overexpression of CYP2J2 or gavage of TPPU in order to determine whether it could reduce CVB3-induced viral myocarditis by decreasing inflammatory cells infiltration and activation of inflammatory factor expression,inhibiting the replication of Coxsackie virus,alleviating the injury of myocardial cells,cardiac troponin release,and impaired heart function.Experimental methods and results1,To establish the model of viral myocarditis: 4~6 week old Balb/c male mice were divided into seven groups: con,GFP,2J2,CVB3,CVB3+GFP,CVB3+2J2,CVB3+TPPU,including which n=10 in each group in con,GFP,2J2 three groups,n=15 in each group within CVB3,CVB3+GFP,CVB3+2J2,CVB3+TPPU four groups.Intravenous injection of r AAV-GFP and r AAV-2J2 recombinant adeno-associated at the concentration of 1x1011 PFU was done 2 weeks before the intraperitoneal injection of 100 ul DMEM in containing105TCID50 CVB3.In group CVB3+TPPU,TPPU(3mg/kg/d)was given orally for 10 days in advance.Then CVB3 was injected into the abdominal cavity with 100 ul 105TCID50CVB3.In the duration of 10 days after CVB3 injection,we observed and recorded the body weight and survival of mice,performed echocardiography and hemodynamic measurements and finally harvested heart,liver and spleen tissue for HE staining,Sirius red staining,immunohistochemistry and TUNEL staining.The results showed that CVB3 successfully induced the formation of acute viral myocarditis.Heart / body weight ratio in myocarditis was decreased.Survival rate of CVB3 myocarditis group was about 25~30%.Echocardiographic left ventricular ejection fraction(EF)and Short axis shortening fraction(FS)were significantly reduced,while the left ventricular systolic diameter(LVID-s)and end diastolic diameter(LVID-d)were significantly increased,and cardiac function was impaired.Hemodynamic tests showed that the maximal rate of left ventricular pressure(dp/dt max)and the maximum rate of left ventricular pressure(dp/dt min)decreased significantly.In HE,diffuse infiltration of inflammatory cells,local tissue necrosis and calcium deposition were found in myocarditis groups,of which pathological score was 2.3.Immunohistochemistry showed macrophages,neutrophils,T cell infiltration increased in myocarditis cardiac tissue.In picrosirius red,we found the myocardial fibrosis area of about 20% in myocarditis group.TUNEL staining showed a higher ratio of apoptosis in myocarditis.The overexpression of CYP2J2 or gavage of TPPU can improve the cardiac function,alleviate the inflammatory cell infiltration of myocarditis and reduce pathological score and lower myocardial apoptosis.2,Detection of inflammatory cytokines in cardiac tissues.4~6 mice in each group were selected and the relative expression levels of inflammatory cytokines were detected by realtime PCR.Plasma and tissue homogenates of each group were used to detect the levels of inflammatory cytokines in blood and heart tissues by ELISA.Real-time PCR showed that pro-inflammatory factor TNF-alpha,IL-1 beta,IL-12 A,IL-23,IL-21 and IL-17 A m RNA levels were significantly increased in heart,while the level of anti-inflammatory cytokines such as IL-4,IL-10,IL-13,IFN-alpha and IFN-beta were increased.Overexpression of 2J2 or gavage of TPPU ameliorated the increase of pro-inflammatory cytokines(TNF-alpha,IL-1 beta,IL-12 A,IL-23,IL-21 and IL-17A)and increased the level of anti-inflammatory cytokines(IL-4,IL-10 and IL-13)and IFN-alpha /IFN-beta inhibiting replication of CVB3.By ELISA,we verified inflammatory cytokines(TNF-alpha,IL-1beta,IL-17 A and IL-10)of heart homogenates and plasma elevated.Overexpression of 2J2 or administration of TPPU,compared with the CVB3 group,reduced the level of inflammatory cytokines and upregulated anti-inflammatory cytokines.3,Western Blot was used to detect the activation of inflammatory pathway,translocation of transcription factor NF-kappa B,oxidative stress,fibrosis and apoptosis in viral myocarditis.Inflammatory pathway is activated with increased nuclear translocation of P65,phosphorylation of I?B?,STAT1 and STAT3.Besides,fibrosis related molecules such as collagen I,TGF-beta and phosphorylated Smad expression also elevated.Pro-apoptotic proteins-activated cleaved Caspase3 and Bax increased,while anti-apoptotic protein Bcl-2expression was decreased.Representative oxidative stress protein gp91,gp67,gp40 and gp22 were significantly increased.Meanwhile,both AMPK and AKT1 were activated.After overexpression of 2J2 or administration of TPPU,the phosphorylation level of remarkable molecules in the inflammatory pathway was decreased and activation of oxidative stress,fibrosis and apoptosis pathway was inhibited to some degree.4,Detection of troponin c Tn I and CK-MB in plasma.The levels of plasma c Tn I and CK-MB were significantly increased in myocarditis group,which suggested severe damage on cardiomyocyte.Overexpression of 2J2 or TPPU alleviated the elevation of myocardial enzymes in viral myocarditis.5,Test of viral titer in heart and liver tissue homogenate with Reed and Muench gradient dilution method.Overexpression of 2J2 or gavage of TPPU reduced burden of CVB3 in heart and liver.6,Flow cytometry was used to detect percentage of Th1/Th2/Th17/Treg and CD4—CD8+ T cells in spleen and macrophage in heart.Macrophage accumulated in myocarditis group,particularly M1 macrophages.Pro-inflammatory subsets of T cell(Th1/Th17/CD8+ T cell)were promoted in the progression of myocarditis.The percentage of regulatory T cells also increased in response to CVB3 infection.Overexpression of 2J2 or TPPU gavage reduced the proportions of Th1/Th17/CD8+ T cells in spleen and macrophages in heart and increased the proportions of Th2 cells and regulatory T cells in spleen.7,Measurements of CVB3 virus replication in primary rat cardiomyocytes and fibroblasts.Primary rat myocardial cells and fibroblasts were cultured on plate,11,12-EET and14,15-EEZE intervention were done one hour before CVB3(MOI=1)infection.After 12 hours,realtime PCR was used to test viral titer in two primary cells.It was found that11,12-EET could alleviate the replication of CVB3 virus and the protective effect of11,12-EET on the replication of CVB3 could be blocked by 14,15-EEZE.Conclusion Overexpression of CYP2J2 or administration of oral s EH inhibitor effectively alleviates cardiac injury in viral myocarditis,mainly through ameliorating inflammatory cell infiltration,apoptosis of myocardial cell,viral replication and differentiation of splenic T cells,which provides a new therapeutic target for acute viral myocarditis.