Mitochondrial DNA damage mediates hyperoxic dysmorphogenesis in rat fetal lung explants

Abstract

Background: Numerous studies in cultured cells indicate that damage to mitochondrial DNA (mtDNA) dictates cellular responses to oxidant stress, yet the consequences of mtDNA damage have not been studied directly in the preterm lung.

Objective: We sought to determine whether hyperoxia-induced fetal lung dysmorphogenesis is linked to mtDNA damage and establish mtDNA repair as a potential therapeutic approach for treating lung dysplasia in the preterm neonate.

Methods: Hyperoxia-induced mtDNA damage was assessed by quantitative alkaline gel electrophoresis in normoxic (3% O2) and hyperoxic (21% O2) fetal rat lung explants. A fusion protein construct targeting the DNA repair enzyme endonuclease III (Endo III) to the mitochondria was used to augment mtDNA repair. Fetal lung branching and surfactant protein C (SFPTC) were assessed in these tissues.

Results: Hyperoxia induced mtDNA damage in lung explants and was accompanied by impaired branching morphogenesis and decreased SFPTC mRNA expression. Treatment of lung explants with Endo III fusion protein prevented hyperoxia-induced mtDNA damage and restored normal branching morphogenesis and SFPTC mRNA expression.

Conclusion: These findings support the concept that mtDNA governs cellular responses to oxidant stress in the fetal lung and suggest that modulation of mtDNA repair is a potential pharmacologic strategy in the prevention of hyperoxic lung injury.

Copyright © 2012 S. Karger AG, Basel.

Figures

FIGURE 1

Fusion protein is targeted to the mitochondria. The HA tag in the fusion protein was used to track protein partitioning to total cellular lysate (T), mitochondrial (M), and nuclear (N) fractions isolated by centrifugation from fetal lung explants at 24 hours. HA immunoreactivity was detected by western analysis in the mitochondrial fraction (cytochrome c positive) indicating that the DNA repair enzyme concentrates within explant mitochondria. Representative of 3 experiments.

FIGURE 2

Nuclear DNA integrity is maintained in hyperoxic fetal lung explants. Quantitative alkaline gel electrophoresis was used to determine the mean fragment length of BamHI-restricted DNA isolated from fetal lung explants. A) Representative ethidium bromide stain of total DNA isolates from fetal lung explants cultured in normoxic (3% O2lane 1) or hyperoxic (21% O2) conditions in the absence (lane 2) or presence of vehicle-buffer (composed of 20 mM Tris-HCl, pH 8.0, 500 nM NaCl, and 10% glycerol, lane 3), or mitochondrially-targeted Endonuclease III fusion protein construct (Endo III, lane 4). B) Quantitative analysis of mean fragment length of total DNA isolated from fetal lung explants. No shift in mean fragment length was detected in nuclear DNA from fetal lung explants as a function of treatment. N=6

FIGURE 3

Hyperoxia causes mtDNA damage in fetal rat lung explants that is prevented by the Endo III fusion protein construct. A) Representative slot blot analysis of mtDNA content in fetal rat lung explants cultured for 24 hours in normoxic or hyperoxic conditions in the absence (control and buffer) or presence of Endo III fusion protein construct (EndoIII). No change in mitochondrial DNA content was detected in fetal lung explants as a function of treatment. Representative of 3 experiments. B) Representative Southern blot of alkali-labile mtDNA damage in fetal rat lung explants. Note decreased hybridization intensity in hyperoxia compared to normoxia indicating increased mtDNA damage in hyperoxic fetal lung explants. Note, too, restoration of band intensity when hyperoxic explants were pretreated with the Endo III-fusion protein. C) Histogram depicting the changes in equilibrium lesion density calculated using the Poisson equation Break frequency= -ln (Po) where Po is the quotient of the band intensity of the hyperoxic cultures (control, buffer, or EndoIII) relative to the band intensity of the normoxic explant culture. Calculated changes in equilibrium lesion density normalized to normoxic explants also indicate that hyperoxia increases mtDNA damage which is prevented when mtDNA repair is enhanced *P <0.05 N=4.

FIGURE 4

The Endo III fusion protein restores lung branching and SFPTC mRNA in hyperoxic fetal lung explants. A) Photomicrograph of fetal lung explants cultured in normoxia or hyperoxia in the absence (control) or presence of mitochondrially-targeted Endo III fusion protein (Endo III) or fusion protein vehicle (buffer). Bar=150µm. Fetal oxygen tension promotes branching in fetal lung explant cultures whereas branching is inhibited in explants cultured in hyperoxia under either control or buffer treated conditions. In contrast, lung branching is restored in explants treated with the mitochondrially-targeted Endo III. B) Quantitation of branching morphogenesis in fetal lung explants assessed by counting peripheral branch points in the right upper lobe (6–10 replicates per experiment; N=6). C) SFPTC mRNA expression is decreased in hyperoxia exposed fetal lung explants and treatment with the fusion protein returns mRNA levels to that of normoxia.