The Role And Mechanism Of Cathepsin B In Coxsackievirus B3-Induced Viral Myocarditis

Background:Viral myocarditis is a diffuse or focal inflammatory disease in the myocardium caused by viral infection.It is not only one of the most important reasons for sudden cardiac death in the youths but also the most common cause for secondary dilated cardiomyopathy.However,it still lacks effective and specific clinical treatment at present.Apoptosis and necrosis of the cardiomyocytes as well as the inflammatory responses are now believed as the major pathological characteristics of viral myocarditis.Cathepsin B(CatB)is a widely expressed cysteine protease mainly located in the lysosome and late endosome.It takes part in many pathophysiological processes,such as apoptosis,extracellular matrix degradation,antigen presentation,inflammatory response and autophagy,etc.,so that it is involved in the pathogenesis of many kinds of diseases.However,the role of CatB in viral myocarditis is to be elucidated.This study aims to explore whether CatB plays a role in Coxsackievirus B3(CVB3)-induced viral myocarditis,and if so,how it influences the occurrence and development of this disease.Methods:We built the viral myocarditis model by intraperitoneal injection of 1000 median tissue culture infective dose(TCID50)of CVB3,using 4-week-old male CatB knockout(Ctsb-/-)mice,cystatin C knockout(Cstc-/-)mice which overexpress CatB,as well as the wildtype C57BL/6 mice.At the corresponding time points after viral infection,we did the echocardiography to evaluate the cardiac function of all the mice,and then harvested their serums and hearts for further examination.Hematoxylin-eosin staining,tissue pathological scoring,echocardiography and enzyme linked immunosorbent assay(ELISA)were employed to observe the effects of CatB or cystatin C deletion on the survival and severity of murine viral myocarditis.By using western blot,enzymatic activity assay,ELISA,terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL)and cardiac virus titration,the possible mechanism of how CatB influenced the severity of viral myocarditis was further investigated.Results:At day 7 and day 28 after virus infection,we found CatB was significantly activated in the cardiac tissue from mice with viral myocarditis,which was supported by higher activated CatB protein level and CatB enzymatic activity than the control mice.The deletion of CatB or its endogenous inhibitor cystatin C in mice had no impact on their short-time survival and baseline cardiac structure and function.However,compared with the wildtype mice receiving equal amount of CVB3,genetic ablation of CatB in mice significantly improved their survival,reduced cardiac inflammatory cell infiltration,decreased serum level of cardiac troponin I and ameliorated cardiac dysfunction.Conversely,genetic deletion of cystatin C,which markedly enhanced CatB levels in hearts,distinctly increased the mortality and severity of murine viral myocarditis.Furthermore,compared with the control,we found the NOD-like receptor family,pyrin domain containing 3(NLRP3)inflammasome was activated in the hearts of wildtype mice with viral myocarditis,which was attenuated in the infected hearts of Ctsb-/-mice but was further enhanced in infected Cstc-/-mice.Consistently,the caspase-1-initiated pyroptosis was also reduced in the infected hearts of Ctsb-/-mice and further increased in infected Cstc-/-mice.However,neither CatB nor cystatin C deletion altered cardiac virus titers at both day 7 and day 28 after virus infection.Conclusion:These results suggest that CatB is activated in the cardiac tissue from mice with CVB3-induced viral myocarditis.Without altering cardiac virus titers,CatB aggravates murine viral myocarditis partially through activating the NLRP3 inflammasome and promoting pyroptosis.This finding might provide a novel strategy for treating viral myocarditis.