Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice

Abstract

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Figures

Fig. 1.

Western immunoblot probed for the hemaglutin (HA) tag of the fusion protein in nuclear and mitochondrial subcellular fractions of lungs from control mice (Contr) and mice injected with the 8-oxoguanine-DNA glycosylase 1 (OGG1) fusion protein construct. Immunoblots labeled for the nuclear protein, lamin B1, and the mitochondria protein, porin-1, are also shown.

Fig. 2.

Mouse lung extravascular plasma equivalent albumin spaces showing bronchoalveolar lavage albumin space, extravascular tissue albumin space, and the total extravascular albumin space after ventilation for different times and with different peak inspiratory pressures (PIP). A: mice received either no ventilation (No Vent.), or 1 h of ventilation with 10 cmH2O PIP (PIP10), or 40 cmH2O PIP only (PIP40), or 40 cmH2O PIP with OGG1 (PIP40 OGG1). B: mice received either no ventilation, or 2 h of ventilation with 40 cmH2O PIP only (PIP40), or 40 cmH2O PIP with OGG1 (PIP40 OGG1). C: mice received either no ventilation, or 2 h of ventilation with 50 cmH2O PIP only (PIP50), or 50 cmH2O PIP with OGG1 (PIP50 OGG1). *P < 0.05 vs. No Vent. group. **P < 0.05 vs. No Vent. group and PIP10 groups. #P < 0.05 vs. all other groups. †All animals in the untreated PIP50 group died before 2 h.

Fig. 3.

Lung wet/dry weight ratios for all experimental groups. *P < 0.05 vs. No Vent., PIP10 × 1 h Vent. only, PIP40 × 1 h Vent. only PIP40 × 1 h + OGG1, and PIP40 × 2 h + OGG1 groups. All animals in the untreated PIP50 group died before 2 h.

Fig. 4.

Kaplan-Meier survival plot for the group ventilated at 50 cmH2O PIP for 2 h with (dashed line) and without (solid line) pretreatment with the OGG1 fusion protein.

Fig. 5.

Macrophage inflammatory protein-2 (MIP-2) concentrations in bronchoalveolar lavage fluid (BALF) from lungs of mice after different ventilatory protocols. A: groups received either no ventilation, ventilation for 1 h without treatment at 10 cmH2O PIP (PIP10), or ventilation for 1 h with 40 cmH2O PIP (PIP40) with and without pretreatment with OGG1. B: groups received either no ventilation or ventilation for 2 h with 40 cmH2O PIP (PIP40) with and without pretreatment with OGG1. C: groups received either no ventilation or ventilation for 2 h with 50 cmH2O PIP (PIP50) with and without pretreatment with OGG1. *P < 0.05 vs. No Vent. group. #P < 0.05 vs. all other groups. †All animals in the untreated PIP50 group died before 2 h.

Fig. 6.

Interleukin-6 (IL-6) concentrations in BALF from lungs of mice after different ventilatory protocols. A: groups received either no ventilation, ventilation for 1 h without treatment at 10 cmH2O PIP (PIP10), or ventilation for 1 h with 40 cmH2O PIP (PIP40) with and without pretreatment with OGG1. B: groups received either no ventilation or ventilation for 2 h with 40 cmH2O PIP (PIP40) with and without pretreatment with OGG1. C: groups received either no ventilation or ventilation for 2 h with 50 cmH2O PIP (PIP50) with and without pretreatment with OGG1. *P < 0.05 vs. No Vent. group. #P < 0.05 vs. all other groups. †All animals in the untreated PIP50 group died before 2 h.

Fig. 7.

Lung tissue glutathione (GSH) concentrations in nonventilated controls and groups ventilated for 2 h with 40 cmH2O PIP and 2 h with 50 cmH2O PIP with and without OGG1 pretreatment. Shown are the total glutathione pool (A) and GSH/GSSG ratios (B). *P < 0.05 vs. nonventilated controls; †all untreated PIP50 group members died before 2 h.

Fig. 8.

Southern blot analysis of mitochondrially targeted DNA (mtDNA) in lung tissue in groups ventilated for 2 h with 40 cmH2O PIP. Samples were treated with alkali to reveal mtDNA strand breaks and alkali + formamidopyrimidine glycosylase (Fpg) to indicate base damage. Line on blot denotes that the OGG1 portion of the blot was moved over in the same blot picture. Bar graph summarizes ratios of blot intensities of alkali+EFG/alkali bands in lungs of nonventilated controls (No Vent.), and mice either untreated (PIP40 × 2 h Vent.) or OGG1 treated (PIP40 × 2 h + OGG1) ventilated for 2 h at 40 cmH2O PIP. #P < 0.05 vs. all other groups.