Knockdown of NFAT5 Transcription Factor Enhances the Coxsackievirus B3‐induced Myocarditis

Abstract

Viral myocarditis is an inflammatory heart disease caused by viral infection and Coxsackievirus B3 (CVB3) is the predominant pathogen for myocarditis. Upon infection, CVB3 modulates various cellular signaling pathways, leading to cell cycle arrest and programmed cell death. Intercalated Discs (ICDs) are substantial connections maintaining cardiac structure and mediating signal communication among cardiomyocytes. NFAT5 (Nuclear factor of activated T-cells 5) is a transcription factor that was shown to regulate some ICD proteins, and was cleaved by CVB3 viral proteases during infection. In order to delineate the interplay between NFAT5 and ICD structure in CVB3 infected conditions, we generated a conditional cardiac-specific NFAT5 knockout (KO) mouse model and verified that NFAT5 KO mice are more susceptible to CVB3 infection. Thus, we hypothesize that CVB3-induced cleavage of NFAT5 leads to the destruction of ICD proteins, which results in damages of cardiomyocytes and contributes to viral pathogenesis. In the study, we generated this specific mouse model by using a tamoxifen-inducible Cre-loxp system to induce NFAT5 KO in the heart. The littermate Cre negative NFAT5flx/flx mice were used as controls. Two weeks after tamoxifen induction and then CVB3 infection, mouse heart as well as other organs were harvested to confirm the KO of NFAT5 by semi-qPCR and western blot. The replication of CVB3 in the heart was detected by qPCR and western blot to detect viral RNA and VP1 protein, respectively. Meanwhile, H&E staining was used to determine the inflammatory response in different mice organs. The results showed that the protein level of NFAT5 in the heart dropped significantly compared to that in other organs. Moreover, the transcription levels of desmoplakin, connexion 43 and N-cadherin genes in the ICD structure were differentially regulated by NFAT5. Also, more cardiac damages and inflammatory infiltrates were observed in CVB3-infected NFAT5 KO mice than in control mice, and this difference can only be observed in the heart tissue, but not in other organs like pancreas. Taken together, our study suggests that the cardiac-specific NFAT5 KO mice are more susceptible to CVB3 infection and have more severe myocarditis compared to the non-KO group.