Role Of Calpain In Coxsackievirus B3-induced Myocarditis

Viral myocarditis (VMC) is the inflammation of cardiac muscles due to viral infection. Coxsackievirus B3(CVB3) infection presents to be a significant cause of myocarditis. Most patients could completely recover from VMC, whereas there are still portion (10-15%) of patients have residual myocardial injury and develop to dilated cardiomyopathy (DCM). Among idiopathic DCM, evidence demonstrated that virus RNA could be detected in60%of DCM patients, indicating that virus infection is crucial in viral cardiomyopathy pathogenesis.Various viruses could lead to the onset of myocarditis, in which CVB3is the most common indicated by an increased virus titer in patients of acute viral myocarditis. Thus, it is important for the treatment of viral cardiomyopathy to looking for potential drug target and possible intervention pathways.Virus infection triggered myocardium injury, caused low level of persistent inflammation, lead to consequential fibrotic remodeling and the final left ventricle dilation and heart failure (HF), forming the continuous pathological process of "viral myocarditis-DCM-HF". Thus, persistent virus replication, inflammatory cell infiltration and myocardium fibrosis are three important mechanisms during the onset and progression of viral cardiomyopathy.Calpain is a family of neutral cysterine proteases and is involved in many forms of physiological and pathological processes. Although studies demonstrated that calpain participated in viruses’replication, host immune inflammation response and tissue fibrosis, role of calpain in CVB3-induced myocarditis is still not clear. As such, this study was designed to discuss this issue aiming at clarifying the potential of calpain to be a novel drug target for viral myocarditis.Part I:Establishment of Acute and Chronic Coxsackievirus B3Myocarditis Model and Dilated Cardiomyopathy Model in C57BL/6Mouse[Objective] To discuss the method of establishing acute Coxsackievirus B3myocarditis model in C57BL/6mouse strain as well as the methodology of chronic viral myocarditis (CVM) and dialted cardiomyopathy (DCM).[Methods] According to initial body weight/week ages, mouse were grouped to group A (3-4weeks age,11-15grams)and group B (5-6weeks age,15-20grams) with3different virus titers infection subgroups respectively:1,000TCID50/ml,10,O00TCID50/ml,100,000TCID50/ml and control groups. Virus capsid protein VP1 and myocardium inflammatory infiltration conditions were observed and evaluated7days post infection. Mouse was infected with CVB3. And2and3months later, heart function, myocardium inflammation and fibrosis were observed respectively to verify the establishment of CVM and DCM.[Results] Immunohistochemistry staining showed that virus capsid protein VP1appeared in the myocardium of virus-infected mouse.Three subgroups in lower body weight group have a higher mortality (40-100%), while the higher body weight subgroups showed a tendency of body weight decline. Histopathologically, in the lower body weight group myocardium of the survival cases were inflammatorily infiltrated, comparing with a negative observation in death cases. All the virus-infected cases were found virus capsid protein VP1in the myocardium via immunohistochemistry method. Myocardium of higher body weight group showed a tendency of deterioration with the increase of virus titer and bared a sound survival rate. CVM and DCM model were not established in C57BL/6mouse strain.[Conclusion] In C57BL/6mouse of5-6week age/15-20grams body weight, infection of coxsackievirus B3in the dose of100,000TCID50/ml×0.3ml is able to establish the acute viral myocarditis model while it is not practical to establish CVM and DCM model in C57BL/6mouse strain. Part Ⅱ:Cardioprotective Effect of Calpastatin-overexpression in Coxsackievirus B3-induced Myocarditis[Objective] Calpain activity is negatively regulated by the endogenous inhibitor of calpastatin.Role of calpain in the pathogenesis of Coxsackievirus B3(CVB3)-induced myocarditis is still unknown. Using a novel transgenic mouse overexpressing calpastatin in myocardium, this project was devoted to deal with this issue and to further find the possible underlying mechanisms.[Methods] An in vivo model of CVB3-induced myocarditis was established via direct CVB3infection of Tg-CAST mouse with parallel controls set simultaneously. Parameter of heart weight/body weight ratio (HW/BW) was calculated. Heart sections were stained using HE and myocardium inflammation was observed and scored. Circulating myocardium injury markers of CK-MB and cTnl were detected. Besides, virus capsid protein VP1, fibrogenic factors of Smad3and MMP2and inflammatory factors of IL17, IFNγ and perforin (PFN) were also detected.[Results] Calpastatin is upregulated in the myocardium of Tg-CAST mouse with a significant decrease of calpain activity. Comparing with CVB3-infected wild type mouse, Tg-CAST mouse showed amelioration of myocardium injury indicated by lower HW/BW, decreased inflammation infiltration and moderate peripheral CK-MB and cTnI. In the myocardium of Tg-CAST mouse, CVB3capsid protein VP1was downregulated (P<0.05), as well as fibrogenic factors of Smad3and MMP2and inflammatory factors of IL17, IFNγ and PFN (P<0.05).[Conclusions] These data showed a significant cardioprotective effect of calpastatin overexpresssion in CVB3-induced myocarditis indicating role of calpain in the pathogenesis of viral myocarditis. This protective effect might be attributing to roles of calpain in several mechanisms like CVB3replication, inflammatory cytokine production as well as a certain fibrogenic factors expression. As such, calpain might be a novel drug target to viral myocarditis treatment that still needs further study. Part Ⅲ:Coxsackievirus B3-induced Calpain Activation Hinders Apoptosis via Enhancing Autophagy in the Early Phase of Infection in H9c2Cells[Objective] Calpain is a family of neutral cysterine proteases involved in many physiological and pathological processes. It’s reported that calpain participates in virus replication, autophagosome formation and apoptosis. However, role of calpain in Coxsackievirus B3(CVB3) replication through autophagy-apoptosis cross-talk has not yet been elucidated that was discussed here.[Methods] Primary rat cardiomyocytes and H9c2cell line were infected with CVB3in vitro, dynamic changes of calpain activity, caspase3activity, apotosis and autophagy parameters were observed followed by effect of calpain activity manipulation on apoptosis and autophagy and autophagy regulation on apoptosis. At last, effects of calpain activity and autophagy manipulation on CVB3replication in cardiomyocytes were observed.[Results] The data showed that calpain was activated in a time-course of24hours post-infection. Inhibition and activation of calpain decreased and enhanced autophagy respectively, indicating that calpain enhanced autophagy in the context of CVB3 infection. Besides, calpain activity also hindered apoptosis in the early10hours post CVB3infection. And induction or inhibition of autophagy prevented or enhanced apoptotic cell death respectively indicating that autophagy following calpain activation hinders apoptosis in the early phase of CVB3infection.[Conclusion] In summary, the present study demonstrated that CVB3infection of H9c2cells hinders host cell apoptosis via calpain-mediated autophagy enhancement in the early phase. Part IV:Role of Calpain in the Myocardium Fibrosis in Coxsackievirus B3Myocarditis[Objective] To observe the effect of calpain inhibiton on the myocardium fibrosis in murine coxsackievirus B3(CVB3) myocarditis and the proliferation and. migration ability of myocardium fibroblast in vitro.[Methods] An in vivo model of CVB3-induced myocarditis was established via direct CVB3infection of Tg-CAST mouse with parallel controls set simultaneously. Mice were harvested on the seventh day. Then hearts were taken out and slices were prepared for Mallory staining to observe the degree of myocardium fibrosis with total fibrosis area calculated through imaging system and for picrosirus red staining to observe the collegen type alterations.Myocardium fibroblasts were cultured and synchronized via fasting for24hours before inoculating with culture medium containing serially-diluted ALLN (40μg/ml,20μg/ml,10μg/ml,5μg/ml,2.5μg/ml,1.25μg/ml,0.625μg/ml) and20%fetal calf serum for another24-hour’s inoculation. The same control groups without ALLN intervention were set at the same time. The effects of calpain inhibition on the proliferation and migration of cardiac fibroblasts were detected respectively by Cell Counting Kit-8(CCK-8) and cell scratch experiment.[Results] Total degree of myocardium fibrosis of virus myocarditis group is significantly (P<0.01) more severe than normal controls and was ameliorated in Tg-CAST (P<0.01). Experiments in vitro indicated that calpain activity inhibition decreased the absorbance of fibroblast (P<0.001) and demonstrated an inhibition effect on fibroblast migration (P<0.001) in a concentration dependent manner. [Conclusion] Calpain is involved in the pathogenesis of fibrosis in Coxsackievirus B3myocarditis probably through its promotion effect on both cardiac fibroblast proliferation and migration.