Inhibition of coxsackievirus infection in cardiomyocytes by small dsRNA targeting its cognate coxsackievirus adenovirus receptor

BACKGROUND & OBJECTIVES: Coxsackievirus B (CVB), a member of human Enterovirus group, is the most common cause of viral myocarditis. Coxsackievirus adenovirus receptor (CAR) is identified as a key determinant for the entry of CVB in the target cells. Thus, blockade of receptor by RNA interference (RNAi) may inhibit the entry and pathogenesis of CVB in cardiac cells. The present study was aimed to determine the effect of CAR small dsRNA (siRNA) on coxsackieviral load and CAR expression in coxsackievirus-infected cardiomyocytes. METHODS: Transfection efficiency in rat cardiomyocytes (H9c2) was determined by the fluorescent microscopy and flow cytometry. CAR siRNA dose was optimized based on cell viability and relative CAR messenger RNA (mRNA) expression. Cardiomyocytes were transfected with CAR siRNA followed by infection with 100 multiplicity of infection of CVB, which were harvested after 24, 48 and 72 h post-infection (p.i.). RNA was extracted for relative CAR mRNA expression. Cells were freeze-thawed thrice for estimating coxsackieviral load. RESULTS: The efficiency of transfection was optimized to be >80 per cent and CAR siRNA dose of 60 pmol was standardized. The knockdown of CAR by siRNA decreased its expression twice the expression in normal cardiomyocytes after 24 h p.i. of CVB. The treatment with CAR siRNA resulted in significant two log reduction of CVB load in cardiomyocytes infected with CVB at 24 h p.i. and retained till 72 h p.i. INTERPRETATION & CONCLUSIONS: The inhibition of CAR by siRNA was found to be effective against CVB in cardiomyocytes. However, this treatment strategy has to be evaluated in vivo to develop a new treatment strategy for patients suffering with viral myocarditis.