Cathepsin B aggravates coxsackievirus B3-induced myocarditis through activating the inflammasome and promoting pyroptosis

Abstract

Cathepsin B (CatB) is a cysteine proteolytic enzyme widely expressed in various cells and mainly located in the lysosomes. It contributes to the pathogenesis and development of many diseases. However, the role of CatB in viral myocarditis (VMC) has never been elucidated. Here we generated the VMC model by intraperitoneal injection of coxsackievirus B3 (CVB3) into mice. At day 7 and day 28, we found CatB was significantly activated in hearts from VMC mice. Compared with the wild-type mice receiving equal amount of CVB3, genetic ablation of CatB (Ctsb-/-) significantly improved survival, reduced inflammatory cell infiltration, decreased serum level of cardiac troponin I, and ameliorated cardiac dysfunction, without altering virus titers in hearts. Conversely, genetic deletion of cystatin C (Cstc-/-), which markedly enhanced CatB levels in hearts, distinctly increased the severity of VMC. Furthermore, compared with the control, we found the inflammasome was activated in the hearts of wild-type mice with VMC, which was attenuated in the hearts of Ctsb-/- mice but was further enhanced in Cstc-/- mice. Consistently, the inflammasome-initiated pyroptosis was reduced in Ctsb-/- mice hearts and further increased in Cstc-/- mice. These results suggest that CatB aggravates CVB3-induced VMC probably through activating the inflammasome and promoting pyroptosis. This finding might provide a novel strategy for VMC treatment.

Fig 1. The establishment of CVB3-induced VMC model.

4-week-old male C57BL/6 mice were injected intraperitoneally with 1000 TCID₅₀ of CVB3 virus or an equal amount of DMEM on day 0. (A) Representative HE staining of paraffin sections of heart tissues from VMC mice and control mice on both day 7 and day 28 after virus injection. Scale bar: 50μm. (B) The pathologic score of myocarditis was assessed according to the mononuclear infiltration foci and myocardial necrosis. (C) Representative M-mode echocardiographic images of VMC mice and control mice on day 7 and day 28 post-infection. (D, E) Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) from the echocardiographic data for both day 7 and day 28 post-infection. (Con: control group; CVB3: coxsackievirus B3 group; Con: n = 10; CVB3: n = 10; ***P<0.001 vs. Con-day 7; ##P<0.01 vs. Con-day 28; ###P<0.001 vs. Con-day 28).

Fig 2. CatB was activated in hearts of CVB3-induced VMC mice.

Hearts were harvested on day 7 and day 28 after virus inoculation. Western blot analysis showed that cardiac expression of activated CatB was significantly enhanced in the CVB3 group on day 7 (A) and day 28 (B) post-infection compared with the control group. (Con: control group; CVB3: coxsackievirus B3 group; Con: n = 10; CVB3: n = 10; **P<0.01 vs. Con; ***P<0.001 vs. Con).

Fig 3. CatB deficiency increased while cystatin C deficiency decreased the survival of CVB3-induced VMC.

Mice were randomly assigned into 4 groups: WT group (n = 10); WT+CVB3 group (n = 14); Ctsb^-/-+CVB3 group (n = 12) and Cstc^-/-+CVB3 group (n = 11). Each mouse of the latter three groups received 1000 TCID₅₀ of CVB3 intraperitoneally, while the WT group received an equal amount of DMEM. (A) Western blot analysis of actived CatB expression to verify the deletion and overexpression of CatB in Ctsb^-/- mice and Cstc^-/- mice. (B) Survival was monitored daily until day 28 post-infection. The Kaplan-Meier curve showed a significant increase in survival in Ctsb^-/-+CVB3 group and a remarkable decrease in Cstc^-/-+CVB3 group, compared with WT+CVB3 group. (WT: wild-type; *P<0.05 vs. WT group; #P<0.05 vs. WT+CVB3 group; &&P<0.01 vs. Ctsb^-/-+CVB3 group).

Fig 4. CatB deficiency alleviated while cystatin C deficiency aggravated the severity of CVB3-induced VMC.

(A, B) Representative HE staining in hearts from mice of indicated intervention. Scale bar: 50μm. The HE staining showed the infiltration of inflammatory cells and pathologic score was significantly decreased in Ctsb^-/-+CVB3 group while increased in Cstc^-/-+CVB3 group on day 7 pi, compared with WT+CVB3 group. (C) The serum cTnI level was significantly lower in Ctsb^-/-+CVB3 group and higher in Cstc^-/-+CVB3 group than the WT+CVB3 group on day 7 pi. (D-F) The echocardiographic data showed that cardiac dysfunction caused by CVB3 infection was significantly alleviated in Ctsb^-/-+CVB3 group, though there was no statistical difference between the Cstc^-/-+CVB3 group and the WT+CVB3 group. (WT group: n = 10; WT+CVB3 group: n = 14; Ctsb^-/-+CVB3 group: n = 12; Cstc^-/-+CVB3 group: n = 11; ***P<0.001 vs. WT group; #P<0.05 vs. WT+CVB3 group; &P<0.05 vs. Ctsb^-/-+CVB3 group; &&P<0.01 vs. Ctsb^-/-+CVB3 group).

Fig 5. CatB deficiency reduced while cystatin C deficiency increased the activation of the NLRP3 inflammasome induced by CVB3 infection.

(A) Western blot analysis of the expression of the components of the inflammasome including NLRP3, ASC, caspase-1 p-20 in hearts from mice receiving different treatments. (B) Cardiac caspase-1 activity was determined by a commercial assay kit and presented as fold change compared with the WT mice receiving DMEM. (C) Compared with WT+CVB3 group, CatB deficiency significantly decreased the serum IL-1β level while cystatin C deletion increased the level of IL-1β. (WT group: n = 10; WT+CVB3 group: n = 14; Ctsb^-/-+CVB3 group: n = 12; Cstc^-/-+CVB3 group: n = 11; **P<0.01 vs. WT group; ***P<0.001 vs. WT group; #P<0.05 vs. WT+CVB3 group; ##P<0.01 vs. WT+CVB3 group; ###P<0.001 vs. WT+CVB3 group; &&P<0.01 vs. Ctsb^-/-+CVB3 group; &&&P<0.001 vs. Ctsb^-/-+CVB3 group).

Fig 6. CatB deficiency reduced while cystatin C deficiency increased myocardial pyroptosis caused by CVB3 inoculation.

(A) Representative cardiac TUNEL staining of mice with different treatments. Scale bar: 50μm. (B) The statistical result showed the increased TUNEL positive staining cells in the WT+CVB3 group were significantly decreased in the Ctsb^-/-+CVB3 group, but further increased in the Cstc^-/-+CVB3 group. (WT group: n = 10; WT+CVB3 group: n = 14; Ctsb^-/-+CVB3 group: n = 12; Cstc^-/-+CVB3 group: n = 11; ***P<0.001 vs. WT group; ##P<0.01 vs. WT+CVB3 group; &&&P<0.001 vs. Ctsb^-/-+CVB3 group).