Annexin A1 Alleviates CVB3-induced Viral Myocarditis Via Inhibiting Viral Replication By Restoring Tight Junction Protein ZO-1

Viral myocarditis(VMC)is inflammation and injury of the heart caused by viral infections,causing dysfunctional function and reduced contractility of cardiomyocytes.due to the onset hidden,no early diagnosis index,the lack of clinical treatment,easy to progress for chronic myocarditis,myocardial fibrosis and deadly dilated cardiomyopathy,virus myocarditis can result in about 20%of adolescents cardiovascular disease of sudden cardiac death.Coxsackievirus B3(CVB3)is the main cause of viral myocarditis and the most frequently detected virus in patients with virus myocarditis.The pathogenesis of viral myocarditis is still not clear,and effective therapeutic drugs and means are lacking.Therefore,it is urgent to develop small molecule drugs with preventive and therapeutic effects to antiviral or inhibit viral myocarditis.Virus replication and immune cell infiltration are the key reasons for the development of CVB3 viral myocarditis.Our previous study confirmed that the local innate immune response in the heart plays an important role in antiviral and initial inflammation at the early stage of CVB3 infection(0-3 days).Among them,neutrophils infiltrate into the heart in large numbers earlier than other immune cells in the first to third day of infection,and play an important pro-inflammatory role.Annexin Al,the second downstream messenger of glucocorticoids,is known to inhibit the rapid migration of neutrophils.Annexin A1 and its N-terminus mimic peptide Ac2-26 can effectively inhibit neutrophils tissue infiltration and reduce tissue inflammation in animal models such as atherosclerosis and myocardial infarction.We intended to investigate the therapeutic effect of Annexin A1 N-terminus peptide Ac2-26 in the treatment of acute viral myocarditis and its therapeutic cellular and molecular immunological mechanisms.We injected male BALB/c mice with 1000 TCID 50 dose of CVB3 intraperitoneally,and successfully induced acute viral myocarditis mouse model characterized by high level of cardiac CVB3 replication(peak on day 3),a large number of inflammatory cell infiltration in the heart,and myocardial necrosis(day7-10),significantly upregulated pro-inflammatory factor(day7-10,TNF-α,IL-1β,IL-6,IFNy)expression,impaired cardiac diastolic function as the main features of acute viral myocarditis in mice model.To demonstrate the therapeutic effects of Ac2-26 peptide on CVB3-myocarditis and the relative mechanism,in this study,using an CVB3-infected male BALB/c mice model,we treat mice with 2 doses(2.5 mg/kg)of exogenous Annexin A1-derived peptide Ac2-26 on day 0.5 and 2.5 post CVB3 infection.It has been found that:1)Annexin A1 was almost not expressed in the hearts of uninfected mice and was significantly upregulated on 0-7 days after CVB3 infection.2)Ac2-26 peptide administration increased the survival rate of mice,reduced cardiac inflammation infiltration and cardiomyocyte damage.3)Ac2-26 peptide administration potently inhibited cardiac CVB3 replication in mice and in cardiomyocytes;4)The therapeutic effect of Ac2-26 peptide was not dependent on its receptorformyl peptide receptor.5)In vitro infection assay revealed that:Ac2-26 peptide had direct antiviral effect only in cardiomyocytes,but not in other susceptible cells.And the major targeting step is the cell-entry step of virus infection cycle.6)By mass spectrometry,we identified tight junction protein ZO-1 as the potential target protein of Ac2-26.7)Ac2-26 peptide effectively blocked degradation of ZO-1 by CVB3,thus restoring ZO-1 up-regulation and its CVB3-inhibitory role in cardiomyocytes.Taken together,to explore the therapeutic effect and molecular mechanism of Annexin A1 and Ac2-26 peptide on CVB3-induced acute viral myocarditis,in this study,we treat mice with a 2 dose-treating regimen and demonstrate the potent anti-viral activity and myocarditis-alleviating effects of Ac2-26 peptide.And we find its ZO-1regulating mechanism.This may provide new insights in the future design of Annexin A1-based anti-CVB3 compound drugs and for the treatment of viral myocarditis.