Abstract Body (Do not enter title and authors here): Objectives: Viral myocarditis (VMC) caused by Coxsackievirus B3 (CVB3) is a common cardiovascular disease that can progress to heart failure. Necroptosis is crucial for viral infection with limited evidence regarding its role in VMC progression. We demonstrated that protein levels of Calpain and phosphorylated MLKL (p-MLKL) in cardiomyocytes after CVB3 infection was significantly increased. Therefore, we aimed to investigate the effect of Calpain on necroptosis in VMC.

Methods: Cardiac tissue derived from the left ventricles of mice and neonate rat cardiomyocytes infected with CVB3 was assessed by pathological and molecular biological analysis. CVB3-infection-related gene expression after intervention was evaluated in cardiomyocytes by quantitative Real-time PCR (RT-qPCR) and Western blot (WB). Myocardial inflammation and fibrosis were evaluated in mice cardiac tissue. Mice and neonate rat cardiomyocytes were treated with calpain activator PMMA and the inhibitor ALLN before CVB3 infection. Expression of Calpain/Calpastatin (Cast, an endogenous inhibitor of Calpain) and RIPK1/RIPK3/MLKL signaling were determined by WB, RT-qPCR and immunofluorescence (IF).

Results: CVB3 mRNA, VP1, Calpain and Cast expression levels gradually increased after CVB3 infection with severe cardiac injury, inflammatory infiltration, fibrosis in rat cardiomyocytes and mouse hearts compared with control. CVB3 induced cardiomyocyte necroptosis dynamically through the RIPK1/RIPK3/MLKL pathway which increased at the early acute stage and potentially alleviated at the later stage of VMC, positively correlated with the change of Calpain system. IF results revealed translocation of pMLKL from nucleus at the early acute stage to membrane at the late acute stage in cardiomyocytes. Furthermore, we found Calpain induced myocardial necroptosis via RIPK1/RIPK3/MLKL pathway after infection of CVB3 in vivo and in vitro, which was exacerbated by PMMA treatment. ALLN ameliorated the excessive activation of the RIPK1/RIPK3/MLKL pathway, suppressed cardiomyocyte necroptosis, and ultimately alleviated cardiac dysfunction after CVB3 infection. We demonstrated that the inhibition of Calpain alleviates CVB3-induced VMC by modulating the RIPK1/RIPK3/MLKL pathway.

Conclusions: A novel regulatory role for Calpain in CVB3-induced VMC was revealed by dynamically modulating the RIPK1/RIPK3/MLKL signaling pathway. Selective Calpain inhibitor may serve as a promising therapeutic target in viral cardiomyopathy.