Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia

Abstract

Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to 'deactive' form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na⁺/H⁺ antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia.

Keywords: Hypoxia; Mitochondrial complex I; Oxygen sensing; Redox signalling; Superoxide.

Graphical abstract

Fig. 1

Silencing of complex I subunits specifically affects the assembly of complex I-containing supercomplexes (a and b) Protein extracts from BAECs treated with siSCR, siNDUFS4 or siNDUFS2 were immunoblotted for NDUFS4 or NDUFS2 proteins with tubulin as loading control. Up: representative image; down: quantification of three independent experiments (mean±s.e.m.). (c and d) BN-PAGE of siSCR-treated or siNDUFS4-treated BAECs, analyzed by western blotting with antibodies against NDUFB6 (complex I; c) or Core I (complex III; d). Representative image of three independent experiments.

Fig. 2

Interference or inhibition of complex I prevent the increase in ROS production triggered by hypoxia. (a-c) Detection of superoxide production by fluorescence microscopy in fixed cells. Cells were incubated for 60 min in normoxia (Nx), for 30 min in normoxia with antimycin A (AA 10 µM) or incubated with pre-hypoxic medium in a hypoxia chamber at 1% O₂ (Hp) for 0, 15, 30, 45 or 60 min. DHE (5 µM) was added for additional 10 min and cells were fixed in the hypoxia chamber. (a and b) BAECs were treated with scramble siRNA (siSCR; black bars) or siRNA against NDUFS4 (a) or NDUFS2 (b). (c) BAECs were untreated (Control) or treated with 1 µM rotenone (Rot 1 µM). Data are presented as the mean±s.e.m. of three independent experiments. n.s. non-significant difference, *p<0.05, **p<0.01 and ***p<0.001 (ANOVA with Tukey post hoc test); only the significances between control normoxia and control hypoxia 0–10 min or treated hypoxia 0–10 min groups are shown. (d-f) Detection of ROS by the ratiometric fluorescent protein HyPer. (d) BAECs were transfected with CytoHyPer, treated with 2 mM of dithiothreitol (DTT) and with 30 µM antimycin A (AA). (e and f) CytoHyPer-transfected BAECs either untreated (e) or treated with 1 µM rotenone (f) were subjected to normoxia (Nx, •) or hypoxia (1% O₂; Hp, ○). Data are presented as the mean±s.e.m. of four independent experiments. n.s. non-significant difference, *p<0.05 Hp vs. Nx (Mann-Whitney U test).

Fig. 3

ROS production in acute hypoxia is not due to reverse electron transport or PINK1 function. (a) Untreated (black) or BAECs treated with 1 µM FCCP (white) were subjected to the same protocol as in Fig. 2a. Data are presented as the mean±s.e.m. of three independent experiments. n.s. non-significant difference, *p<0.05, **p<0.01 (ANOVA with Tukey post hoc test); only the significances between control normoxia and control hypoxia 0–10 min or treated hypoxia 0–10 min groups are shown. (b and c) Detection of ROS production by live fluorescence microscopy with DHE. BAECs untreated (b) or treated with 1 µM FCCP (c) were subjected to normoxia (Nx, •) or hypoxia (2% O₂; Hp, ○). (Insets) Slopes considering all time points of each replicate (n=3). The slope for each replicate was estimated by linear regression of the data for all the ROI and time points. Data are presented as the mean±s.e.m. of three independent experiments. n.s. non-significant difference, *p<0.05, ***p<0.001 (Student's t-test). (d and e) HIF-1α stabilization measured by western blotting in BAECs treated or not with 1 µM FCCP and exposed for 4 h to normoxia (Nx), normoxia with 1 mM DMOG or to hypoxia (1% O₂, Hp). Tubulin was used as loading control. (d) Representative images; (e) quantification of three independent experiments (mean±s.e.m.). (f) Protein extracts from BAECs treated with siSCR or siPINK1 were immunoblotted for PINK1 protein with tubulin as loading control. Up: representative image; down: quantification of three independent experiments (mean±s.e.m.). (g) BAECs were treated with scramble siRNA (siSCR; black bars) or siRNA against PINK1 (white bars) and subjected to the same protocol as in Fig. 2a. Data are presented as the mean±s.e.m. of three independent experiments. n.s. non-significant difference, **p<0.01 (ANOVA with Tukey post hoc test); only the significances between control normoxia and control hypoxia 0–10 min or treated hypoxia 0–10 min groups are shown.

Fig. 4

Acute hypoxia deactivates complex I in BAECs. (a) Cys-39 of ND3 remains buried in active complex I (yellow), while it is exposed in deactive complex I (red). Mal-Bodipy-TMR was used to label exposed Cys before electrophoretic protein separation. TMR fluorescence signal for the ND3 band was higher when complex I was deactive (grey picture). Protein amount for the same band is detected with Sypro Ruby staining (red picture). (b, c) Mitochondrial membranes from BAECs treated for 5 min in normoxia (Nx) or hypoxia (1% O₂, Hp or H5) were split in two equal parts; one part was incubated for 1 h at 37 °C to fully deactivate complex I (Thermal deactivation), whereas the other was kept on ice. (b) Bodipy-TMR signal reflects exposed Cys (left) and Sypro Ruby signal detects total protein (right). Arrows (➙) mark the band corresponding to ND3 identified by LC-MS/MS; the lower image on the left is a more exposed photograph of the Bodipy-TMR signal. (c) Band corresponding to TMR-labelled ND3 was quantified and normalized to total ND3. Data are presented as the mean±s.e.m. of six independent experiments. n.s. non-significant difference, **p<0.01 H5 vs. Nx (Mann-Whitney U test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 5

Acute hypoxia deactivates complex I. (a) Mitochondrial membranes from HepG2 treated as in Fig. 4. Up: representative image; down: quantification of ND3 Cys exposure (mean±s.e.m. of four independent experiments). *p<0.05, H5 vs. Nx (Mann-Whitney U test). (b) Mitochondrial membranes of BAECs treated for 5 min in normoxia (Nx) or in hypoxia (1% O₂) for 5 (H5), 15 (H15) or 30 min (H30) and treated as in Fig. 4. Up: representative image showing; down: quantification of ND3 Cys exposure (mean±s.e.m. of three independent experiments). (c) Mitochondrial membranes of BAECs subjected to normoxia or different hypoxia conditions (3% or 0.5% O₂) for 5 min; NxD: thermal deactivation of normoxic sample. Up: representative image; down: quantification of ND3 Cys exposure (mean±s.e.m. of four independent experiments). Arrows (➙) mark the band corresponding to ND3 in total protein.

Fig. 6

Acute hypoxia enhances complex I Na⁺/H⁺antiporter activity. (a) A cytosolic version of sypHer was transfected in BAECs to analyse pH change in control conditions. BAECs were treated with two subsequent additions of 30 µM NaOH and one of 8 mM HCl. Data are represented as mean±s.e.m. of eight different ROIs. (b) BAECs transfected with mitosypHer were incubated with 25 nM MitoTracker CMTMRos for 20 min and fixed. Representative fluorescence confocal microscopy images show mitochondrial localisation of mitosypHer. Estimated Pearson and Mander's correlation coefficients for colocalisation of both signals are shown. (c) 488/405 signals ratio reflecting intramitochondrial pH in BAECs transfected with mitosypHer either untreated (No treat) or treated with 1 µM FCCP (FCCP). Data are represented as mean±s.e.m. of five independent experiments. ***p<0.001 (Student's t-test). (d and e) Intramitochondrial pH measured with mitosypHer by live confocal microscopy in BAECs either untreated (d) or treated with 1 µM rotenone (e) and subjected to normoxia (Nx, •) or hypoxia (1% O₂; Hp, ○). Data are represented as mean±s.e.m. of the ratio between the fluorescence signals with excitation at 488 nm and 405 nm of four independent experiments. (Insets) Slopes considering all time points of each replicate (n=4) are plotted as mean±s.e.m. The slope for each replicate was estimated by linear regression of the data for all the ROI and time points. *p<0.05 (Student's t-test). (f) Non treated BAECs or treated with 10 µM monensin for 30 min in normoxia were subjected to the same procedure as in Fig. 2a. Data are represented as mean±s.e.m. of three independent experiments. **p<0.01 (ANOVA with Tukey post hoc test); only the significance between non-treated normoxia and monensin-treated normoxia is shown. (g) HIF-1α stabilization measured by western blotting in BAECs treated or not with 10 µM monensin (Mon) or with 1 mM DMOG and exposed for 4 h to normoxia (Nx). Tubulin was used as loading control. Representative images of three independent experiments are shown. (h) Quantification of (g); mean±s.e.m. of three independent experiments.

Fig. 7

Complex I deactivation correlates with ROS production and occursex vivoandin vivo. (a) Hippocampal slices were incubated for 30 min in normoxia (Nx), in normoxia with antimycin A (AA 10 µM) or in hypoxia at 1% O₂ (Hp 30–40 min). DHE (5 µM) was added for additional 10 min, and slices were fixed in the hypoxia chamber. (upper panel) Representative images show DHE fluorescence. (lower panel) Quantification of DHE fluorescence signal. Data are presented as mean±s.e.m. of four independent experiments. ***p<0.001 (ANOVA with Tukey post hoc test); only the significance between Nx and Hp 30–40 min is shown is shown. (b) Hippocampal slices were subjected to 30 min of normoxia (Nx) or hypoxia (1% O₂; H30) and treated as in Fig. 4. Left: representative image; right: Quantification of ND3 Cys exposure (mean±s.e.m. of three independent experiments). *p<0.05 (Mann-Whitney U test). (c) Mice were subjected to photothrombotic stroke induction and ND3 Cys exposure was estimated as in Fig. 4 from samples of different regions of the brain: infarct, ipsilateral (IPSI) and contralateral (Contra). Left: representative image; down: quantification of ND3 Cys exposure (mean±s.e.m. of three independent experiments). *p<0.05, **p<0.01 vs. Contra (ANOVA with Tukey post hoc test).

Fig. S1

Identification of ND3 band. (a and b). MS/MS identification of a peptide of bovine ND3 containing Cys-39, obtained from tryptic digestion of the ND3 band of a gel from the experiment in Fig. 5b. CID fragmentation MS/MS spectrum (a) and table (b) indicating the assigned fragments from the b and y series are shown.