A neuroglobin-based high-affinity ligand trap reverses carbon monoxide-induced mitochondrial poisoning

Abstract

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC-treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 μm) and nitric oxide (100 μm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 μm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

Keywords: CO poisoning; antidotes; carbon monoxide; hemoglobin; hypoxia; hypoxia-inducible factor (HIF); medical toxicology; mitochondria; mitochondrial disease; mitochondrial respiratory chain complex; neuroglobin; nitric oxide.

Figure 1.

A, severe CO-poisoning experiment design as described by Azarov et al. (46). Ventilated mice, sedated with 1.5% isofluorane, were exposed for 4.5 min of 30,000 parts per million CO gas. After exposure, Ngb-H64Q-CCC or PBS were infused for 2 min. Mouse arterial blood pressure and heart rate were measured for 40 min or until death. Using hearts recovered from animals in these experiments, we evaluated whether Ngb-H64Q-CCC reversed CO-dependent inhibition of respiration in an in vivo CO exposure with ex vivo biochemical measurement. Control animals were sedated with 1.5% isofluorane but not exposed to CO gas. The hearts were immediately removed upon death of the animal or sacrificed after 40 min of exposure at the cessation of the experiment (or 20 min in sedated control mice). The mouse heart was homogenized, and tissue respiration was measured after the addition of pyruvate, malate and ADP with a Clark-like electrode respiration system. B, rates were adjusted for protein level confirmed with BCA (representative raw traces). C, in CO-treated mice infused with PBS, respiration of heart tissue was significantly inhibited at 58 ± 19% of the rate of control (Mann–Whitney test; *, p = 0.032). Treatment with Ngb-H64Q-CCC restored tissue respiration to the level of control (Mann–Whitney test; p = 0.55) and to rates significantly higher than CO-poisoned mice treated with PBS (p = 0.032, *). D, to evaluate the effects of severe CO poisoning on the components of the electron transport chain, the same heart homogenate tissue underwent spectrophotometric kinetic assays. Complex I activity in animals treated with Ngb-H64Q-CCC was significantly higher than those treated with PBS (400 ± 52 versus 150 ± 64 pmol/min/mg protein; Mann–Whitney test; ***, p = 0.001) and similar to control (290 ± 130; p = 0.13). E, CO-poisoned animals treated with neuroglobin showed complex II activity levels higher than those treated with PBS (110 ± 35 versus 71 ± 11 pmol/min/mg protein; *, p = 0.029) and similar to control (120 ± 48; p = 0.72). F, animals treated with Ngb-H64Q-CCC after CO exposure showed significantly higher complex IV activity compared with those treated with PBS (330 ± 80 versus 210 ± 79 k/min/ng protein; *, p = 0.042) and similar to sedated controls (310 ± 90; p = 0.83). G, citrase synthase activity, was similar between all groups. All experiments were performed with at least n = 4 animals in each group. All statistical analyses were using the Mann–Whitney test for these in vivo CO exposure with ex vivo biochemical measurement studies. rNgb, Ngb-H64Q-CCC.

Figure 2.

In vitro effects of NO on mitochondrial respiration and reversal by Ngb-H64Q-CCC treatment. Four experimental arms were used. For control, isolated liver mitochondria was respired to hypoxia after addition of succinate and ADP. A, after hypoxia, 50 μl of aerobic PBS was added and allowed to respire to hypoxia three times (labeled raw data). B, for NO only, initially 50 μl of aerobic PBS was added and respired to hypoxia, and then aerobic PROLI-NONOate (50 μm) was added into the chamber to inhibit respiration. The small amount of oxygen dissolved in the PROLI-NONOate solution increased the oxygen but demonstrated inhibition of respiration. Further injection of 50 μl of aerobic PBS into the chamber increased oxygen concentration; however, respiration remained inhibited until the NO released from PROLI-NONOate began diffusing from the chamber (not pictured). C, for Ngb-H64Q-CCC only, 50 μl of aerobic PBS was added and respired to hypoxia twice. Oxygen-bound Ngb-H64Q-CCC was added to a concentration of 50 μm to the chamber and respired again to hypoxia. D, for NO + Ngb-H64Q-CCC, after the initial aerobic PBS (step 1), PROLI-NONOate (step 2) was added, followed by oxygen-bound Ngb-H64Q-CCC to a concentration of 50 μm (step 3) (labeled raw data). Inset, comparison of NO-inhibited respiration versus Ngb-H64Q-CCC–treated post-NO exposure respiration). The rate of the final respiration rate was compared with the initial reoxygenation respiration rate for each arm (rate respiration final step/rate of respiration initial step). E, the ratios were compared between groups. Control rates had no effect of respiration versus initial reoxygenation. Exposure to PROLI-NONOate with reoxygenation by aerobic PBS decreased respiration to 2 ± 1% of the initial rate. In mitochondria that were exposed to PROLI-NONOate and then treated with Ngb-H64Q-CCC, respiration was 61 ± 22% of the initial rate (unpaired Welch's t test NO-exposed and NO-exposed, Ngb-H64Q-CCC–treated mitochondria; ***, p = 0.0001). In an unmatched regular two-way ANOVA, we determined that there was a significant interaction between Ngb-H64Q-CCC and exposure to NO (interaction term; ****, p < 0.0001; single line) (F, representative raw traces compared). rNgb, Ngb-H64Q-CCC.

Figure 3.

In vitro effects of CO on mitochondrial respiration and reversal by Ngb-H64Q-CCC treatment. Experiments were performed in a similar manner to the NO testing; however, instead of PROLI-NONOate, 100 μl of CO-saturated PBS (to a concentration of ∼160 μm) was added to the chamber. Additionally, because CO has a higher affinity for cytochrome c oxidase in hypoxia, the chamber was left in hypoxia for 60 s before adding additional aerobic PBS. The CO-saturated PBS did not contain oxygen; therefore 50 μl of PBS was added to demonstrate CO-slowed mitochondrial respiration. B, an additional 50 μl of aerobic PBS was used to show persistent inhibition of respiration (labeled raw data). A and C, controls were similar to the NO experiments (Fig. 2): no exposure to CO or Ngb-H64Q-CCC and addition of 100 μm Ngb-H64Q-CCC without the presence of CO. D, to demonstrate the effect of Ngb-H64Q-CCC, in the final step, instead of PBS, aerobic, oxygen-bound Ngb-H64Q-CCC was added to a concentration of 100 μm to the chamber. This increased the oxygen concentration in the chamber, and the mitochondria respired at an increased rate (labeled raw data). Inset, comparison of CO-inhibited respiration versus Ngb-H64Q-CCC–treated post-CO exposure respiration). E, Control rates had little effect of respiration versus initial reoxygenation. Exposure to CO with reoxygenation by aerobic PBS decreased respiration to 45 ± 8% of the initial rate. In mitochondria that were exposed to CO but treated with Ngb-H64Q-CCC, respiration was 77 ± 14% of the initial rate (unpaired Student's t test CO-exposed and CO-exposed, Ngb-H64Q-CCC–treated mitochondria; p < 0.0001, ****). In an unmatched regular two-way ANOVA, we determined that there was a significant interaction between Ngb-H64Q-CCC and exposure to CO (interaction term; ***, p = 0.0007; single line) (F, representative raw traces compared). rNgb, Ngb-H64Q-CCC.

Figure 4.

In vitro effects of CO on complex I, II, and IV activity. Activity levels were determined at hypoxic conditions (2% oxygen) with and without the presence of CO-saturated buffer solution. A, complex I was not significantly reduced by CO in isolation (79 ± 4% of control; Welch's t test; p = 0.22). B, complex II was not significantly reduced by CO in isolation (88 ± 7% of control; Student's t test; p = 0.24). C, complex IV activity after CO exposure was significantly reduced, with the CO-exposed activity level 33 ± 5% of control (Welch's t test; **, p = 0.0011).

Figure 5.

In vitro effects of NO on complex IV activity and reversal by Ngb-H64Q-CCC treatment. To demonstrate the specific effect of NO on complex IV activity in isolated rate mitochondria, a closed chamber with a Clark-like oxygen electrode was utilized. Here, instead of succinate and ADP as substrates for oxidative phosphorylation, a combination of FCCP, TMPD, and ascorbic acid was added. These substrates facilitate the direct transfer of electrons to cytochrome c and vis-à-vis reflect cytochrome c oxidase activity. A–D, four conditions were measured: baseline level of TMPD-ascorbate–driven respiration (labeled raw data, A), respiration after the addition of 50 μm of PROLI-NONOate (B), respiration after the addition of 50 μm chamber concentration of Ngb-H64Q-CCC (C), and finally, respiration after the addition of 50 μm of PROLI-NONOate, followed by the addition of 50 μm of Ngb-H64Q-CCC (D). These rates were compared with the average rate of baseline TMPD-ascorbate–driven respiration for a given day of experiments and the same animal (measured TMPD-ascorbate–driven respiration rate/average baseline TMPD-ascorbate–driven respiration rate). E, these ratios were compared between experimental arms. Here the addition of Ngb-H64Q-CCC itself slowed complex IV activity to 77 ± 10% of baseline. The addition of NO slowed complex IV activity to 2 ± 1% of baseline. The addition of Ngb-H64Q-CCC after NO lead to a respiration rate of 70 ± 9% of baseline, markedly higher than NO untreated complex IV activity (unpaired Welch's t test; ****, p < 0.0001). In an unmatched regular two-way ANOVA, we determined that there was a significant interaction between Ngb-H64Q-CCC and exposure to NO (interaction term; ****, p < 0.0001; single line) (F, representative raw traces compared). rNgb, Ngb-H64Q-CCC.

Figure 6.

In vitro effects of CO on complex IV activity and reversal by Ngb-H64Q-CCC treatment. To demonstrate the specific effect of CO on complex IV activity in isolated rate mitochondria, a closed chamber with a Clark-like oxygen electrode was utilized. A combination of FCCP, TMPD, and ascorbate were added to demonstrate cytochrome c oxidase activity. A–D, four conditions were measured: baseline level of TMPD-ascorbate–driven respiration (labeled raw data, A), respiration after the addition of 200 μm CO in CO-saturated PBS followed by 100 μl of aerobic PBS (B), respiration after the addition of 100 μm chamber concentration of oxygenated-Ngb-H64Q-CCC (C), and finally, respiration after the addition of 200 μm CO in CO-saturated PBS followed by 100 μm of oxygenated-Ngb-H64Q-CCC (D). These rates were compared with the average rate of baseline TMPD-ascorbate–driven respiration for a given day of experiments and the same animal. E, the ratios between experimental arms were compared. In these experiments, the addition of Ngb-H64Q-CCC itself slowed complex IV activity to 62 ± 7% of baseline. The addition of CO slowed complex IV activity to 24 ± 5% of baseline. The addition of Ngb-H64Q-CCC after CO lead to a respiration rate of 39 ± 6% of baseline, markedly higher than CO-exposed, untreated complex IV activity (unpaired Student's t test; ****, p < 0.0001). In an unmatched regular two-way ANOVA, we determined that there was a significant interaction between Ngb-H64Q-CCC and exposure to CO (interaction term; ****, p < 0.0001; single line) (F, representative raw traces compared). rNgb, Ngb-H64Q-CCC. #, Figs. 5A and 6A show the same control conditions, represented for clarity.