Suppression of Calpain Attenuates Coxsackievirus B3-Induced Myocarditis by Regulating RIPK1/RIPK3/MLKL Pathway

Figures

• 

• 

  Fig. 1. CVB3 replication in neonatal rat cardiomyocytes. A CVB3 RNA copy number at different viral titers (MOI). B VP1 protein expression at different viral titers (M.O.I). C CVB3 RNA copy number at different time points post-infection (MOI = 20). D VP1 protein expression (WB) at different time points post-infection (MOI = 20). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Technical replicates (RT-qPCR) n = 6; Biological replicates (WB in vitro) n = 3 independent cultures per condition

• 

  Fig. 2. Calpain attenuates cardiomyocytes necroptosis infected by CVB3 via RIPK1/RIPK3/MLKL pathway in vitro. A WB analysis of RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL expression at different time points post-infection. B-D RT-qPCR analysis of RIPK1, RIPK3, and MLKL mRNA levels at different time points post-infection. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Technical replicates (WB) n = 3 and Technical replicates (RT-qPCR) n = 6

• 

  Fig. 3. Calpain attenuates myocardial necroptosis infected by CVB3 via RIPK1/RIPK3/MLKL pathway in vivo. A WB analysis of Cast and calpain expression in neonatal rat cardiomyocytes. B, C RT-qPCR analysis of Cast and calpain mRNA levels in neonatal rat cardiomyocytes. D WB analysis of RIPK1, RIPK3, and MLKL expression in neonatal rat cardiomyocytes. Quantitative analysis was shown in (E) RIPK1, (F) RIPK3, and (G) MLKL. IF staining of RIPK1 (H), RIPK3 (I), and MLKL (J) in neonatal rat cardiomyocytes. Scale bar = 50 μm. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Technical replicates (WB) n = 3 and Technical replicates (RT-qPCR) n = 6. Immunofluorescence was evaluated by two independent blinded readers

• 

  Fig. 4. CVB3 induces myocardial necroptosis via the RIPK1/RIPK3/MLKL pathway in vivo. A IF showed p-MLKL expression site, Scale bar = 100 μm. B WB of RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL expression in mouse myocardial tissue at different time points post-infection. Technical replicates (WB) n = 3. Immunofluorescence was evaluated by two independent blinded readers. W: weeks, C: Control group, V: VMC group

• 

  Fig. 5. Calpain attenuates myocardial necroptosis infected by CVB3 via RIPK1/RIPK3/MLKL pathway in vivo. A WB analysis of Cast and calpain expression in mouse myocardial tissue. B H&E staining showing pathological changes in mouse myocardial tissue. Scale bar = 200 μm. C Masson staining assessing collagen deposition in the myocardial interstitium. Scale bar = 200 μm. D, E Serum levels of cTnI (D) and CK-MB (E). F WB analysis of RIPK1, RIPK3, and MLKL expression in mouse myocardial tissue. Quantitative analysis of protein expression: (G) RIPK1, (H) p-RIPK3, (I) RIPK3, (J) p-MLKL, (K) MLKL. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Technical replicates (WB) n = 3 and Technical replicates (RT-qPCR and ELISA) n = 6. H&E and Massonʼs trichrome were evaluated by two independent blinded readers. W weeks, C control group, V VMC group