Mitochondrial dysfunction contributes to alveolar developmental arrest in hyperoxia-exposed mice - PubMed (original) (raw)

Mitochondrial dysfunction contributes to alveolar developmental arrest in hyperoxia-exposed mice

Veniamin Ratner et al. Am J Respir Cell Mol Biol. 2009 May.

Abstract

This study investigated whether mitochondrial dysfunction contributes to alveolar developmental arrest in a mouse model of bronchopulmonary dysplasia (BPD). To induce BPD, 3-day-old mice were exposed to 75% O2. Mice were studied at two time points of hyperoxia (72 h or 2 wk) and after 3 weeks of recovery in room air (RA). A separate cohort of mice was exposed to pyridaben, a complex-I (C-I) inhibitor, for 72 hours or 2 weeks. Alveolarization was quantified by radial alveolar count and mean linear intercept methods. Pulmonary mitochondrial function was defined by respiration rates, ATP-production rate, and C-I activity. At 72 hours, hyperoxic mice demonstrated significant inhibition of C-I activity, reduced respiration and ATP production rates, and significantly decreased radial alveolar count compared with controls. Exposure to pyridaben for 72 hours, as expected, caused significant inhibition of C-I and ADP-phosphorylating respiration. Similar to hyperoxic littermates, these pyridaben-exposed mice exhibited significantly delayed alveolarization compared with controls. At 2 weeks of exposure to hyperoxia or pyridaben, mitochondrial respiration was inhibited and associated with alveolar developmental arrest. However, after 3 weeks of recovery from hyperoxia or 2 weeks after 72 hours of exposure to pyridaben alveolarization significantly improved. In addition, there was marked normalization of C-I and mitochondrial respiration. The degree of hyperoxia-induced pulmonary simplification and recovery strongly (r(2) = 0.76) correlated with C-I activity in lung mitochondria. Thus, the arrest of alveolar development induced by either hyperoxia or direct inhibition of mitochondrial oxidative phosphorylation indicates that bioenergetic failure to maintain normal alveolar development is one of the fundamental mechanisms responsible for BPD.

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Figures

Figure 1.

Figure 1.

(A) Experimental timeline and design. (B) Electron microscopy of pulmonary mitochondria isolated from naïve and hyperoxic Postnatal Day (P)18 mice. Scale bar = 500 nm. (C) Immunoblots of representative samples from naïve (room air [RA]-exposed) and O2-exposed mice and optical density graphs of MnSOD and COX IV expression in mitochondrial isolates from naïve P18 mice (open bars, n = 4) and P18 mice subjected to hyperoxia for 2 weeks (solid bars, n = 4). (D and E) Representative images of hematoxylin and eosin–stained lung sections with corresponding radial alveolar count (RAC) analysis; in naïve mice (open bars, P3 n = 4, P6 n = 7, P18 n = 10, P39 n = 4), mice subjected to hyperoxia (solid bars, P6 n = 8, P18 n = 8), and recovery (hatched bar, P39 n = 4). Scale bar = 500 μm. Data are mean ± SEM. *P ≤ 0.002 compared with naïve littermates, **P = 0.0001 compared with naïve P6 mice, #P < 0.0001 compared with P6 and P18 hyperoxia-exposed mice, §P = 0.0003 compared with P3 naïve mice.

Figure 2.

Figure 2.

(A) Pulmonary mitochondrial respiration rates during phosphorylation of ADP (State 3), after acceleration by DNP (DNP-rate), and during resting (State 4) respiration in naïve (open bars) and hyperoxia-exposed mice (solid bars) measured: at 72 hours (naïve n = 4, hyperoxic mice n = 5), at 2 weeks (naïve n = 6, hyperoxic mice n = 7), and after 3 weeks of recovery (hatched bars, naïve = 4, hyperoxic mice n = 4). (B) Representative recording of mitochondrial respiration in a mouse subjected to hyperoxia for 2 weeks (dashed line) and naïve mouse (solid line). Time-points at which mitochondria, ADP, and DNP were added are indicated. (C and D) RCR and ADP:O ratio in mice subjected to hyperoxia for 72 hours, 2 weeks (solid bars), and after 3 weeks of recovery (hatched bar) compared with naïve littermates (open bars). (E) Complex I activity in pulmonary mitochondria isolated at 72 hours of hyperoxia (solid bar, n = 5) compared with naïve mice (open bar, n = 4), at 2 weeks of hyperoxia (solid bar, n = 5) compared with naïve mice (open bar, n = 5), and after 3 weeks of recovery in RA (hatched bar, n = 4) compared with naïve littermates (open bar, n = 4). (F) Correlation between RAC and C-I activity in mice subjected to hyperoxia for 2 weeks (solid circles) and in mice recovered for 3 weeks after 2 weeks of hyperoxia (hatched circles). All data are mean ± SEM. *P < 0.01 compared with naïve littermates, **P < 0.01 compared with mice subjected to hyperoxia for 2 weeks, ¶P = 0.04 compared with P6 naïve mice, #P = 0.004 compared with mice subjected to hyperoxia for 72 hours, *P = 0.0002 compared with mice exposed to hyperoxia for 72 hours.

Figure 3.

Figure 3.

(A) Respiration rates in pulmonary mitochondria isolated from mice subjected to hyperoxia (solid bar, n = 8), pyridaben (hatched bar, n = 6) for 72 hours or 2 weeks (n = 5 in each group) compared with mice recovered from 72 hours of pyridaben exposure for 2 weeks (bricked bar, n = 6). Data expressed as percentage of mean values (100%) in vehicle-treated littermates (n = 5 for each age group). *P < 0.05 compared with vehicle controls, **P < 0.01 compared with 72 hours of pyridaben exposure. (B) ATP-production rate in mitochondria after 72 hours of exposure to pyridaben (hatched bar, n = 4) or hyperoxia (solid bar, n = 5). Data expressed as percentage of mean value (100%) in vehicle-treated littermates. (C) Correlation between phosphrylating respiration and ATP production rates: open circle, the mean value of State 3 respiration rate in vehicle-treated mice (n = 4); hatched circles, pyridaben; solid circles, hyperoxia-exposed mice. All data are mean ± SEM. *P ≤ 0.03 compared with vehicle-treated controls.

Figure 4.

Figure 4.

(A) Representative images of lung sections in mice exposed to hyperoxia (O2), pyridaben for 72 hours or 2 weeks, and mice allowed to recover after 72 hours of pyridaben exposure compared with age-matched vehicle-treated controls. Scale bar = 500 μm. (B and C) RAC and Lm in mice subjected to hyperoxia (solid bar, n = 8), pyridaben (hatched bar, n = 6) for 72 hours or 2 weeks (n = 5), and in mice after recovery from 72 hours of pyridaben exposure (bricked bar, n = 6) compared with vehicle-treated controls (open bar, n = 4 and n = 6). (D) Recording of mitochondrial respiration in mouse subjected to pyridaben for 2 weeks (dashed line) and vehicle-treated littermate (solid line). Time points at which mitochondria and ADP were added, and end-points of ATP production, are indicated. (E) ADP:O ratio (% of mean value in vehicle-treated controls, n = 6) in mitochondria from mice exposed to hyperoxia for 72 hours (solid bar, n = 5), pyridaben (hatched bar, at 72 h n = 6 and at 2 wk n = 5), and mice recovering from 72 hours of pyridaben exposure (bricked bar, n = 6). All data are mean ± SEM. *P ≤ 0.002 compared with age-matched vehicle-treated controls, **P ≤ 0.03 compared with P6 vehicle-treated controls, §P < 0.04 compared with mice exposed to pyridabed for 2 weeks.

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