Hypoxia-adaptation involves mitochondrial metabolic depression and decreased ROS leakage

Abstract

Through long-term laboratory selection, we have generated a Drosophila melanogaster population that tolerates severe, normally lethal, level of hypoxia. This strain lives perpetually under severe hypoxic conditions (4% O(2)). In order to shed light on the mechanisms involved in this adaptation, we studied the respiratory function of isolated mitochondria from the thorax of hypoxia-adapted flies (AF) using polarographic oxygen consumption while monitoring superoxide generation by electron paramagnetic resonance (EPR) techniques. AF mitochondria exhibited a significant 30% decrease in respiratory rate during state 3, while enhancing the resting respiratory rate during State 4-oligo by 220%. The activity of individual electron transport complexes I, II and III were 107%, 65%, and 120% in AF mitochondria as compared to those isolated from control flies. The sharp decrease in complex II activity and modest increase in complexes I and III resulted in >60% reduction in superoxide leakage from AF mitochondria during both NAD(+)-linked state 3 and State 4-oligo respirations. These results provide evidence that flies with mitochondria exhibiting decreased succinate dehydrogenase activity and reduced superoxide leakage give flies an advantage for survival in long-term hypoxia.

Figure 1. Mitochondrial respiration is depressed in hypoxia-adapted flies (AF) in comparison with normoxia-adapted flies (NF).

Mitochondria were isolated from the thoraxes of ∼200 NF or AF as described (Methods), and respiratory O₂ consumption measured using a Clark-type electrode. (A) Representative oxygen consumption traces from NF and AF mitochondria, using malate+pyruvate (5 mM each) and ADP (250 µM) to stimulate State 3 respiration, oligomycin (2.5 µg/ml) to initiate State 4-oligo respiration, and CCCP (0.2 µM) to produce maximal uncoupled respiration (State 3_U). Analysis of oxygen consumption (µM/min/mg protein) by NF and AF mitochondria during State 3 (B) and State 4-oligo (C). State 3 respiration in AF was 31.1±14% lower than NF, p<0.05 but NF exhibited significantly faster resting (State 4-oligo) respiration (218.8±24.8% increase, p<0.05). N = 3 or 6 independent runs for AF or NF groups, respectively, with approximately 200 thoraxes utilized per run.

Figure 2. Decreased reactive oxygen species production by isolated mitochondria from AF as detected by electron paramagnetic resonance (EPR) spectroscopy.

Isolated thorax mitochondria prepared as for Figure 1 were analyzed by EPR 15 minutes after addition of the spin-trap, DIPPMPO (70 mM) and mitochondrial substrates malate+pyruvate (10 mM final each) in the absence (State 4-oligo respiration ) or the presence of 500 mM ADP (state 3 respiration ). The mixture was introduced into the EPR cavity, and spectra acquired. (A) EPR spectra from NF (lower two spectra) or AF (upper two spectra) under State 3 or State 4-oligo respiration. Addition of Mn-SOD (100 U/ml) to samples in State 4-oligo respiration almost completely eliminated the signal, indicating that it is derived from superoxide (third spectrum). Quantification of the EPR signal from both NF and AF groups during State 3 (B) or State 4-oligo (C). Superoxide yield in mitochondria from hypoxia-adapted flies was 60.8% lower than that from naïve flies (F_(1,5) = 8.0107, p<0.05, n = 4 for control, n = 3 for hypoxia-adapted flies) during State 4-oligo, and that during state 3 was lower by 70.2% (F_(1,7) = 8.0107, p<0.05, n = 6 for control, n = 3 for hypoxia-adapted flies). Effects of electron transport chain complex inhibitors on mitochondrial ROS production in NF versus AF groups (D). Two markers of oxidative stress, carbonylated proteins and peroxidized lipids, are not increased in the AF group relative to the NF group (E, F). (*) indicate statistically significant difference in superoxide yield from AF relative to NF mitochondria at the given respiratory states.

Figure 3. Effect of adaptation to hypoxia on mitochondrial respiratory chain complex activities.

The enzymatic activity of each respiratory chain complex was measured in isolated mitochondria as described in the Methods section. (A) Complex I: no significant changes in complex activity was observed (n = 3, p>0.05) between controls (NF) and hypoxia-adapted flies (AF). (B) Complex II: significant down-regulation of complex activity was determined in complex II in the hypoxia-selected flies (AF) (n = 3, p<0.05). (C) Complex III: significant up-regulation of complex activity in the hypoxia-selected flies (AF) (n = 3, p<0.05). (D) Complex IV: significant up-regulation of complex activity in the hypoxia-selected flies (AF) (n = 3, p<0.05). All activities are presented as percent of the naive control values (mean ±SEM).