Mitochondrial injury and complement dysregulation are drivers of pathological inflammation in viral myocarditis

Figures

• 

  Fig 1. CVB3-induced cardiomyocyte injury is associated with pro-inflammatory macrophage activation. (A) Schematic representation of monoculture or co-culture experiments with murine HL-1 cardiomyocytes and murine RAW 264.7 macrophages. Cells were infected with CVB3 (multiplicity of infection [MOI] = 100, 48 h) and subsequently subjected to inflammatory gene expression analysis and presented as relative quantitation (RQ) (mean ± SD, n = 3). Statistical analysis was performed using analysis of variance with Tukey post hoc test. (B) HL-1 cardiomyocyte (C), RAW264.7 macrophages (M), and co-culture (C + M) were infected as above and subjected to RT- qPCR analysis for the target gene Il6 and normalized to Actb. (C) Schematic illustration depicting sham- or CVB3-infected (MOI = 100, 8 h) HL1 cardiomyocytes subjected to hypotonic lysis. (D and E) Hypotonic lysate (B) was diluted in culture medium and incubated with RAW264.7 cells for 4 h, and cells were then subjected to Western blot analysis for macrophage activation marker, COX2 (C), and qPCR for anti-viral type I IFN gene (D). (F–H) Low mitochondrial gene expression is associated with increased inflammation in human left ventricle hearts, as…

• 

  Fig 2. Mitochondrial dsDNA is released upon CVB3 infection, but mtDNA depletion alone is not sufficient to block CVB3-mediated macrophage activation. (A) HL-1 murine cardiomyocytes were sham or CVB3-infected (MOI = 100, 8 h), followed by confocal microscopy analysis for mtDNA release (cytosolic dsDNA) and the mitochondrial marker TOM20. Scale bar = 20 µm. (B) Extracellular mtDNA is increased following CVB3 infection. HeLa and HEK293 cells were infected with CVB3 (MOI = 10 and 100, respectively, 5 h) and then subjected to fractionation of cytosolic and mitochondrial compartments. Cytosolic mtDNA was assessed via qPCR analysis of the mitochondrial gene marker mtCO2 and normalized to total cell protein. Extracellular mtDNA was assessed in supernatants of control or CVB3-infected HeLa cells (MOI = 10, 5 h) using qPCR analysis of the mitochondrial gene marker mtCO2. Statistical analysis between sham and CVB3 groups was performed using an unpaired Student t-test. (C) qPCR assessment of inflammatory gene expression in RAW264.7 macrophages transfected with either poly I:C (1 µg/mL) or herring testes (HT)-DNA (3 µg/mL) (mean ± SD, n = 3). (D) Extracted mtDNA from control or ddC-treated HeLa…

• 

  Fig 3. Incubation with mitochondrial proteins and lipids induces macrophage pro-inflammatory activation. (A) Bright field image of mitochondria isolated using mito-IP protocol (left). THP1-derived macrophages were treated with mitochondria purified from mito-IP diluted in culture medium (right). Total RNA was purified from cells and subjected to qPCR analysis for inflammatory gene markers. (B) Proteins were extracted from mito-IP preparations and subjected to Western blot analysis for mitochondrial markers VDAC and TOM20. Statistical analysis between control and mito-IP groups was performed using an unpaired Student t-test (mean ± SD, n = 3). (C) Purified mitochondrial protein fractions (B) were diluted in culture medium and used to treat THP1-derived macrophages for 4 h. RNA was purified from cells following treatment and subjected to qPCR measurement for inflammatory gene expression. Statistical analysis between control and mito protein groups was performed using an unpaired Student t-test (mean ± SD, n = 3). (D) Schematic illustration of lipid isolation protocol from purified crude mitochondria extracts. (E) RAW264.7 macrophage cells were treated with mitochondrial lipid extracts…

• 

  Fig 4. CVB3 infection is associated with pathological complement activation. (A) Volcano plot demonstrating differential gene expression between sham- and CVB3-infected male BALB/c mice 6–8 weeks old at 10 days post-infection, with red points representing significantly upregulated genes and blue points indicating significantly downregulated genes (n = 3 mice per group). (B) Analysis of differentially upregulated genes in panel A was plotted using R studio and presented in rank order of highest enrichment using Panther’s annotated Reactome data set. (C) CVB3-positive human myocarditis tissues exhibit elevated levels of membrane attack complex C5b9. Tissues were stained with double-stranded RNA (dsRNA) as a marker of viral RNA replication and confirmed with in situ hybridization CVB3-specific probes (see reference [16]). Scale bar = 50 µm. Results are representative of three biologically independent samples for each group. C5b9 optical density was quantified in the right panel and presented as mean ± SD, n = 3. (D) TOM20-expressing HEK293 cells were sham or CVB3-infected for 8 h at an MOI of 100 and then analyzed by immunofluorescence staining with anti-TOM20 (red) and anti-C5b9 (green)…

• 

  Fig 5. Mitochondria-mediated activation of complement exacerbates inflammation. (A) Serum complement factors enhance CVB3-induced pro-inflammatory macrophage activation. RAW264.7 macrophages were either sham or CVB3-infected in the presence (+) or absence (−) of 10% normal human sera (NHS). RNA was extracted and subjected to qPCR analysis for IL-6 and presented as mean fold change relative to −NHS/sham (group 1). Statistical analysis between groups was performed using analysis of variance with Tukey post hoc test. (B) Purified mitochondria from stable TOM20-HEK293 cells were subjected to Western blot analysis for mitochondrial proteins VDAC and TOM20. Ponceau red staining was used as a total protein loading control. (C) NHS was incubated with or without purified mitochondria for 1 h at 37°C, and the mixture was subjected to Western blot analysis for complement protein C5. (D and E) Purified mitochondria were subjected to in vitro complement time-course assay in the presence of anti-TOM20 antibody. Lysates were subjected to Western blot analysis for complement factors component 1R (C1R) (D) and component 4 (C4) (E). Complement activation was assessed and quantified in the right panels.…

• 

  Fig 6. Complement protective factors are downregulated following CVB3 infection. (A) Schematic illustration of cardiac protein expression analysis in control and 7 day CVB3-infected A/J male mice (n = 3 per group). (B) Protein expression of complement protective factors CD59 and CD55 was assessed by Western blot analysis from cardiac tissue of sham or CVB3-infected A/J mice (A). Ponceau red stain of membrane was used to assess total protein. (C) HL1 cardiomyocytes were sham or CVB3-infected (MOI = 100) for 12 or 24 h. Proteins were harvested from cells and subjected to Western blot analysis for complement protective factors CD55 and CD59. VP1 and ACTB were used as viral replication marker and loading control, respectively. (D) Control and CVB3-infected human heart tissues were subjected to immunohistochemistry to detect the complement protective factors CD59 and CD55. Scale bar = 60 µm. CD59 and CD55 optical density was quantified in the right panel and presented as mean ± SD, n = 3. (E) In vitro cleavage assay was performed on HeLa cell lysates using purified CVB3 viral proteinase 2A (300 ng), 3C (100 ng), or 2A + 3C (left). Previously reported substrates EIF4G and SNAP29 were used as…

• 

  Fig 7. Schematic illustration of the novel mechanism that can act as a potential contributor to CVB3 pathogenesis through mitochondrial injury-mediated autoimmunity.