Time-dependent and somatically acquired mitochondrial DNA mutagenesis and respiratory chain dysfunction in a scleroderma model of lung fibrosis

Abstract

Reactive oxygen species (ROS) have been implemented in the etiology of pulmonary fibrosis (PF) in systemic sclerosis. In the bleomycin model, we evaluated the role of acquired mutations in mitochondrial DNA (mtDNA) and respiratory chain defects as a trigger of ROS formation and fibrogenesis. Adult male Wistar rats received a single intratracheal instillation of bleomycin and their lungs were examined at different time points. Ashcroft scores, collagen and TGFβ1 levels documented a delayed onset of PF by day 14. In contrast, increased malon dialdehyde as a marker of ROS formation was detectable as early as 24 hours after bleomycin instillation and continued to increase. At day 7, lung tissue acquired significant amounts of mtDNA deletions, translating into a significant dysfunction of mtDNA-encoded, but not nucleus-encoded respiratory chain subunits. mtDNA deletions and markers of mtDNA-encoded respiratory chain dysfunction significantly correlated with pulmonary TGFβ1 concentrations and predicted PF in a multivariate model.

Figure 1. Simplified hypothesis of mitochondrial involvement in the pathogenesis of bleomycin induced PF.

Bleomycin induces ROS that are perpetuated by positive feedback loops involving respiratory impairment and mitochondrial mutagenesis. ROS, respiratory chain dysfunction, or a combination of both, then induce TGFβ1 and interstitial lung disease.

Figure 2. Effects of bleomycin on lung histology.

Representative electron micrographs (A, B) demonstrate mitochondrial enlargement, disrupted crystal architecture and intracytoplasmic vacuoles in an alveolar epithelial cell at day 14. Panels C and D demonstrate depressed COX activity in pulmonary tissue at day 14 (brown stain in COX/SDH histochemistry) and an upregulation of SDH activity (blue stain) not visible at day 0 (insert in panel D). Panels E and F show the expression of αSMA. In normal lung, brown staining was only observed in the smooth muscle around the airways (E); a representative slide of lung tissue 14 days after bleomycin however documents excess αSMA expression in the thickened interstitial space (F). Magnification bars: 4 μm (A,B), 40 μm (C,D, insert D); 100 μm (E,F).

Figure 3. Correlations between PF, pulmonary TGFβ1 content, mitochondrial mutagenesis and mitochondrial function.