Up-regulation of ectopic trypsins in the myocardium by influenza A virus infection triggers acute myocarditis

Abstract

Aims: Influenza A virus (IAV) infection markedly up-regulates ectopic trypsins in various organs, viral envelope glycoprotein processing proteases, which are pre-requisites for virus entry and multiplication. We investigated the pathological roles of trypsin up-regulation in the progression of IAV-induced myocarditis, cytokine induction, and viral replication in the hearts, and also investigated the protective effects of trypsin inhibitor on cardiac dysfunction in vivo and selective knockdown of trypsin on IAV-induced cellular damage in cardiomyoblasts.

Methods and results: The relationship of the expression among IAV RNA, trypsins, matrix metalloproteinase (MMP)-9, MMP-2, pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumour necrosis factor-α was analysed in mice hearts and cardiomyoblasts after IAV infection. The severity of myocarditis was most noticeable during Day 6-9 post-infection, along with peak expression of viral RNA, trypsins, particularly trypsin₂, MMPs, and cytokines. Cardiac ATP levels were the lowest at Day 9. Up-regulated trypsins, viral protein, and tissue-injured loci in the myocardium were closely localized. Trypsin inhibitor aprotinin treatment in vivo and selective trypsin₁- and trypsin₂-knockdown, particularly the latter, in H9c2 cardiomyoblasts significantly suppressed viral replication, up-regulation of MMPs, and production of active MMP-9 and cytokines, resulting in marked protection against cellular damage, ATP depletion, and apoptosis. IAV infection-induced cardiac dysfunction monitored by echocardiography was improved significantly by aprotinin treatment.

Conclusions: IAV-induced trypsins, particularly trypsin₂, in the myocardium trigger acute viral myocarditis through stimulation of IAV replication, proMMP-9 activation, and cytokine induction. These results suggest that up-regulation of trypsins is one of the key host pathological findings in IAV-induced myocarditis.

Figure 1

Increased vascular hyperpermeability, tissue oedema, and inflammatory cell infiltration in acute myocarditis, and kinetics of up-regulation of trypsins in myocardium after IAV infection. Vascular hyperpermeability monitored by Evan's blue extravasation during the course of infection from Day 0 (d 0) to 12 (d 12) (A). Haematoxylin and eosin staining (B). Bar = 50 μm. Heart oedema determined by wet/dry weight ratio (C). Data are mean ± SD of 10 mice in each group. **P < 0.01 vs. D 0; ^††P < 0.01 vs. D 9. Detection by RT-PCR of trypsin_1–3, trypsin isoforms T₁, T₂, and T₃ and IAV NS1 gene in the hearts from Day 0 to 12 post-infection (D1, E, and F1, respectively). Detection by zymography of trypsin activity (D2), by western immunoblotting of trypsinogen and trypsin (D3) and viral nucleoprotein (NP) (F2). Each result is a representative of three experiments.

Figure 2

Close distribution of up-regulated trypsins with viral antigen and inflammatory injury, and kinetics of up-regulation of MMPs and cytokines as well as ATP depletion in the hearts after IAV infection. (A) Immunohistochemical detection of up-regulated trypsins in the myocardium from Day 0 (d 0) to 12 (d 12) post-infection. Bar = 50 μm. (B) Immunofluorescence detection of viral antigen (green), trypsins (blue), and injured loci monitored by Evan's blue extravasation (red) in the hearts at Day 0 and 6. Bar = 50 μm. Right panel shows merge. (C) Kinetics of up-regulation of proMMP-9, proMMP-2, and actMMP-9 in the hearts determined by zymography (C1) and western immunoblotting (C2) from Day 0 to 12 post-infection. (D) Kinetics of up-regulation of IL-6, IL-1β, and TNF-α in the hearts determined by western immunoblotting from Day 0 to 12 post-infection. (E) Changes in cardiac ATP levels during IAV infection. Data are mean ± SD of three repeated experiments from 10 mice per each group. *P<0.05, **P < 0.01 vs. Day 0; ^†P < 0.05 vs. Day 9.

Figure 3

Effects of trypsin inhibitor administration on viral replication and up-regulation of trypsins, MMPs, and cytokines as well as ATP depletion in the hearts. Intraperitoneal administration of aprotinin (Apr) at 2 mg/kg twice a day for 9 days significantly suppressed viral replication and trypsin up-regulation analysed by RT-PCR (A1 and B), by western immunoblotting (A2), and zymography (A3), up-regulation of proMMP-9, actMMP-9, proMMP-2, IL-6, IL-1β, and TNF-α analysed by western immunoblotting (C1) and ATP depletion (D) at Day 9 post-infection. β-Actin as an internal control. Relative levels of MMPs and cytokines after IAV infection vs. mock infection (C2 and C3). *P < 0.05, **P < 0.01 vs. mock infection. ^†P < 0.05, ^††P < 0.01 vs. IAV infection without Apr treatment.

Figure 4

Suppression of cardiac function by IAV infection and its restoration by trypsin inhibitor. Representative M-mode echocardiogram images of mice infected with mock and IAV with or without intraperitoneal administration of aptrotinin (Apr) at 2 mg/kg twice a day for 9 days (A1). Measurements of LVEDD and LVESD at Day 9 post-infection (A2) and %FS (A3). Comparison of blood pressure (B) and heart rate (C) at Day 9 post-infection among mock and IAV-infected mice treated with or without Apr. SBP, systolic blood pressure; DBP, diastolic blood pressure. *P < 0.05, **P < 0.01 vs. mock infection; ^††P < 0.01 vs. IAV infection without Apr treatment.

Figure 5

Effects of trypsin knockdown on IAV replication, MMPs up-regulation, and actMMP-9 production in H9c2 cardiomyoblasts. Silence efficiency of T₁ and T₂ genes was determined by RT-PCR in T₁-sh, T₂-sh, and C-sh cell lines (A). Effects of T₁- and T₂-knockdown on viral replication monitored by NS1 gene in cell lysate by RT-PCR, NP in culture media by western Immunoblotting (B1), and relative values of NS1 and NP vs. infection control (C-sh) (B2). Immunofluorescent detection of viral antigen (red) in H9c2 cells (C1) at 24 h post-infection. Nuclei were stained by DAPI. Bar = 25 μm. Percentage of infected cells (C2). Data are mean ± SD of three independent experiments. Effects of T₁- and T₂-knockdown on the expression of proMMP-9, proMMP-2, and actMMP-9 in cell lysate and culture medium of T₁-sh, T₂-sh, and C-sh cells infected with IAV or mock monitored by western immunoblotting (D). *P < 0.05, **P < 0.01 vs. mock infection; ^††P < 0.01 vs. infection control; ^##P < 0.01 vs. T₁-knockdown.

Figure 6

IAV infection-induced cytokine up-regulation, mitochondrial membrane depolarization, ATP depletion and apoptosis, and abrogation by T₁- and T₂-knockdown in H9c2 cardiomyoblasts. Effects of T₁- and T₂-knockdown on IL-6, IL-1β, and TNF-α levels in culture medium were analysed by ELISA at 24 h post-infection (A). ▵Ψ_m of C-sh, T₁-sh, and T₂-sh cells infected with mock or IAV were analysed after IAV infection for 24 h (B). Top: ▵Ψ_m was monitored by treatment for 15 min with JC-1 (red). Bottom: apoptotic cells detected by TUNEL staining (light blue). Nuclei were stained by DAPI. Bar = 25 μm. Right: percentage of apoptotic cells in total cells. ATP levels under the same experimental conditions in B (C). Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. mock infection; ^†P < 0.05, ^††P < 0.01 vs. infection control; ^#P < 0.05, ^##P < 0.01 vs. T₁-knockdown.