All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1,2,3,4, a phenomenon that occurs in hypoxia4,5,6,7,8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.
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We thank M. Kowalewski (Institute of Veterinary Anatomy, UZH) for allowing us the use of the microscope for live-cell imaging; A. Alfuzzi, J. Prieto, A. Mellado (IIS-IP) and B. Barreira (CIBERES) for collaboration in experiments; E. Fuertes-Yebra (IIS-IP) for technical assistance; M. E. Soriano and F. Caicci (University of Padova) for performing electron microscopy; R. Rizzuto and D. De Stefani (University of Padova) for MCU KO and control cell lines; M. T. Alonso (IBGM, University of Valladolid and CSIC) for the pcDNA3-erGAP3 plasmid; J. Langer from CIC biomaGUNE for fruitful discussion and support with the IR spectroscopy measurements; I. Sekler (Ben-Gurion University), C. Rueda and J. Satrústegui (CMBSO, UAM-CSIC) for providing plasmids and other material and for helpful discussions; M. Cano and A. G. García (IIS-IP and UAM), M. Murphy (MRC and University of Cambridge), I. Wittig (Goethe Universität), J. Miguel Mancheño (IQFR, CSIC), A. Pascual and J. López-Barneo (IBIS, US-CSIC) for helpful discussions; and L. del Peso (UAM) and F. Sánchez-Madrid (IIS-IP and UAM) for their support. This research has been financed by Spanish Government grants (ISCIII and AEI agencies, partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R. and J.A.E.); CP07/00143, PS09/00101, PI12/00875, PI15/00107 and RTI2018-094203-B-I00 (to A.M.-R.); CP12/03304 and PI15/01100 (to L.M.); CP14/00008, CPII19/00005 and PI16/00735 (to J.E.); SAF2016-77222-R (to A. Cogolludo); PI17/01286 (to P.N.); SAF2015-65633-R, RTI2018-099357-B-I00 and CB16/10/00282 (to J.A.E.); RTI2018-095793-B-I00 (to M.G.L.); and SAF2017-84494-2-R (to J.R.-C.), by the European Union (ITN GA317433 to J.A.E. and MC-CIG GA304217 to R.A.-P.), by grants from the Comunidad de Madrid B2017/BMD-3727 (to A. Cogolludo) and B2017/BMD-3827 (to M.G.L.), by a grant from the Fundación Domingo Martínez (to M.G.L. and A.M.-R.), by the Human Frontier Science Program grant HFSP-RGP0016/2018 (to J.A.E.), by grants from the Fundación BBVA (to R.A.-P. and J.R.-C.), by the UCM-Banco Santander grant PR75/18-21561 (to A.M.-d.-P.), by the Programa Red Guipuzcoana de Ciencia, Tecnología e Información 2018-CIEN-000058-01 (to J.R.-C.) and from the Basque Government under the ELKARTEK Program (grant no. KK-2019/bmG19 to J.R.-C.), by the Swiss National Science Foundation (SNF) grant 310030_124970/1 (to A.B.), by a travel grant from the IIS-IP (to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). The CNIC is supported by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (Spanish Government award SEV-2015-0505). CIC biomaGUNE is supported by the María de Maeztu Units of Excellence Program from the Spanish Government (MDM-2017-0720). P.H.-A. was a recipient of a predoctoral FPU fellowship from the Spanish Government. E.N. is a recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM). A.M.-R., L.M. and J.E. are supported by the I3SNS or ‘Miguel Servet’ programmes (ISCIII, Spanish Government; partially funded by the FEDER/ERDF).
The authors declare no competing interests.
Peer review information Nature thanks Israel Sekler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Interference of Slc8b1 affects NCLX function, but inhibition of the NCLX has no effect on general mitochondrial function.
a–c, Assessment of the effect of different siRNAs against Slc8b1 mRNA (siNCLX) on mitochondrial Ca2+ influx and efflux rates in BAECs transfected with the mitochondria-directed Ca2+ reporter protein Cepia 2mt (siSCR n = 4, siNCLX1 n = 5, siNCLX2 n = 4, siNCLX3 n = 3, siNCLX4 n = 5), after addition of histamine; mean traces plotted as fluorescence change relative to initial fluorescence (F/F0) (a); mitochondrial Ca2+ peak amplitude (calculated from the highest value of fluorescence minus the first basal value of fluorescence, from panel a) (b); percentage of mitochondrial Ca2+ efflux (calculated from the highest value of fluorescence minus the lowest value of fluorescence after histamine application, relative to percental siSCR values, from panel a) (c). d, Assessment of NCLX protein amount after interference in whole-cell extracts from BAECs, by western blot (Representative image of three independent experiments). e–g, Mitochondrial Ca2+ influx and efflux rates in WT, NCLX KO, KO+pNCLX or WT+dnNCLX MEFs transfected with Cepia 2mt (n = 4, except WT n = 5 and KO+pNCLX n = 3) after addition of histamine, as in a–c. h–j, Assessment of NCLX protein amount in MEFs mitochondrial extracts by western blot (representative images of n = 4 in h and n = 2 in i and j). k, Mitochondrial membrane potential in BAECs measured with 20 nM TMRM in non-quenching mode (n = 3). l, Oxygen consumption rate (OCR) in BAECs with NCLX inhibition by 10 μM CGP-37157 (upper panel, n = 4), or NCLX interference with siNCLX1 (lower panel, n = 3). All data are represented as mean ± s.e.m. One-way ANOVA with Tukey’s test (f, g and k for the siNCLX inset) and two-tailed Student’s t-test (b, c, k for the CGP inset). For gel source data, see Supplementary Fig. 1.
Cytosolic Ca2+ (Ca2+i) or Na+ (Na+i) were measured by live imaging fluorescence microscopy with Fluo-4 AM or CoroNa Green AM, respectively, in normoxia or acute hypoxia (2% O2). a–d, BAECs not treated (No treat) or transfected with siSCR or siNCLX. e–h, BAECs treated or not with the NCLX inhibitor CGP-37157 (10 μM). a, c, e, g, time-course traces; b, d, f, h, slopes. All data are represented as mean ± s.e.m. of n = 3 independent experiments. Two-tailed Student’s t-test (b, d, f, h): * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant.
Extended Data Fig. 3 Inhibition of the NCLX prevents the increase in ROS production triggered by hypoxia.
a–c, Superoxide detection by fluorescence microscopy after incubation with DHE in 10-min time windows in normoxia (Nx) or hypoxia (1% O2); AA = antimycin A. BAECs treated or not with 10 μM CGP-37157, images of one experiment (a) and mean intensity of three independent experiments (b). HUVECs treated or not with 10 μM CGP-37157, mean intensity of three independent experiments (c). d, Detection of H2O2 by live confocal microscopy in CytoHyPer-transfected BAECs either not treated or treated with 10 μM CGP-37157 in normoxia (Nx) or acute hypoxia (1% O2, Hp); representative images and time-course traces as mean of four independent experiments. e, Detection of ROS by live fluorescence microscopy with DCFDA in normoxia or acute hypoxia (2% O2); time-course traces and slopes, mean of three independent experiments. f, g, Superoxide detection by fluorescence microscopy with DHE in normoxia (Nx) or 10 min hypoxia (1% O2) or normoxia with AA in primary (f) or immortalized (g) HUVECs; mean of three independent experiments. All data are represented as mean ± s.e.m. Two-tailed Student’s t-test for pairwise comparisons (d, e) and one-way ANOVA with Tukey’s test for multiple comparisons (b, c, f, g): n.s. not significant, * P < 0.05, **P < 0.01, *** P < 0.001. In b, c, statistical comparisons shown only for Nx vs 0-10 groups.
a, b, Assessment of interference of subunits of CI (NDUFS4) or CIII (RISP) by western blot in whole-cell extracts from BAECs. Representative images of two independent experiments. c, d, Cytosolic Ca2+ (Ca2+i; c) or Na+ (Na+i; d) measured by live imaging confocal microscopy with Fluo-4 AM or CoroNa Green AM, respectively, in normoxia or acute hypoxia (1% O2); time-course traces and slopes, n = 4 (c), n = 3 (d). e, f, Effect of OXPHOS inhibitors on cytosolic Ca2+ (e; n = 3) or Na+ (f; n = 6) measured as in c and d. All data are represented as mean ± s.e.m. Two-tailed Student’s t-test or Mann–Whitney U test (c; siSCR). n.s. not significant, * P < 0.05, **P < 0.01. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Acute hypoxia promotes A/D transition in CI and matrix acidification independently of NCLX activity.
a–c, ND3·Cys39 exposure, which reflects the D conformation of CI, measured as the ratio between TMR signal (Cys39 labelling) and Sypro Ruby staining (total protein for the ND3 band, identified by mass spectrometry11). Thermal deactivation is used as a positive control of CI D state. Scheme of the technique11 (a); BAECs (b; n = 5 and n = 4 for de-activated samples) or HepG2 (c; n = 2) exposed to normoxia (Nx), 5 min of hypoxia (1% O2, H5), normoxia with CGP-37157 (NxCGP), 5 min of hypoxia with CGP-37157 (H5CGP). d, Complex I reactivation rate measured in the presence of Mg2+ in isolated mitochondrial membranes from BAECs subjected to normoxia, 10 min of hypoxia (1% O2) and NCLX inhibition with CGP-37157 (n = 4). e–g, Mitochondrial matrix pH measured using calibrated mitosypHer in WT or CI KO cybrid cell lines (e; n = 8), MEFs preincubated or not with rotenone (f; n = 4) or NCLX WT or KO MEFs (g; n = 8). h, Mitochondrial matrix acidification using mitosypHer in BAECs in normoxia or acute hypoxia (1% O2) treated or not with NCLX inhibitor CGP-37157; time-course traces and slopes (n = 4). i, j, Effect of the CI inhibitor rotenone on cytosolic Ca2+ (i) or cytosolic Na+ (j) measured by live confocal microscopy with Fluo-4 AM or CoroNa Green AM, respectively, in BAECs in normoxia or acute hypoxia (1% O2), n = 3. All data are represented as mean ± s.e.m. Two-tailed Student’s t-test (b, d, h, i, j): n.s. not significant, * P < 0.05, **P < 0.01, *** P < 0.001.
Extended Data Fig. 6 Mitochondrial matrix acidification promotes mitochondrial Na+/Ca2+ exchange via the NCLX.
a, TEM-EDX determination of calcium element (Ca) versus carbon (C), oxygen (O) and lead (Pb) content in regions of mitochondria with (empty circles) or without (filled circles) electron dense spots (n = 7). b, Frequency of CaP precipitates per mitochondrion in BAECs, seen by TEM during normoxia (741 mitochondria), 10 min of hypoxia (1% O2; 619 mitochondria) or 30 min with 1 μM FCCP (393 mitochondria), three independent experiments. c, Total Ca2+ content of mitochondria extracted from mouse adult fibroblasts (MAFs) which had been treated for 10 min with 1 μM FCCP or 1 μM rotenone, measured in a hypotonic buffer at pH 6.8 (n = 4). d, Western blot showing MCU and Fp70 in CRISPR Control, MCU KO2 and MCU KO3 cells (representative image of two independent experiments). e, Mitochondrial Ca2+ measured by live cell confocal microscopy in MCU WT or KO human breast cancer cells transfected with Cepia2mt (fluorescence signal relative to starting signal –F/F0–, representative traces of n = 5 for WT, n = 9 for MCU KO2 and n = 5 for KO3, independent experiments). f, Total Ca2+ content of mitochondria extracted from MAFs which had been subjected to normoxia or 10, 30 or 60 min of hypoxia (1% O2), measured in a hypotonic buffer at pH 6.8 (n = 5). g, Ca2+ content of mitochondria extracted from MAFs which had been subjected to 10 min normoxia or 10 min hypoxia (1% O2) in a medium without Ca2+, measured in a hypotonic buffer at pH 6.8 (total mitochondrial Ca2+) or pH 7.5 (soluble mitochondrial Ca2+; n = 4). h, Cytosolic Ca2+ measured by live cell confocal microscopy in MEFs transfected with cyto-GEM-GECO in the absence of Ca2+ in the incubation medium (n = 4). i, Endoplasmic reticulum Ca2+ measured by live cell confocal microscopy in MEFs transfected with erGAP3 in the presence or absence of Ca2+ in the incubation medium (n = 8). j, Superoxide detection with DHE in MCU WT or MCU KO immortalized breast cancer cells in normoxia (Nx) or after 10 min of hypoxia (1% O2; Hp; n = 4). All data except e are represented as mean ± s.e.m. One-way ANOVA with Tukey’s test (c and f) and two-tailed Student’s t-test (a, b, g and j): n.s. not significant, * P < 0.05, ** P < 0.01. Two- tailed Student’s t-test (CRISPR Control Hp vs MCU KOs Hp in j): & P < 0.05, && P < 0.01. For gel source data, see Supplementary Fig. 1.
a, Effect of NaCl and/or CaCl2 additions on H2O2 production detected with Amplex Red in isolated rat heart mitochondria (500 μg) respiring after addition of glutamate/malate (GM) in KCl-EGTA buffer. Representative traces of five independent experiments. b–e, Effect of NCLX activation by 10 mM NaCl/0.1 mM CaCl2 on glutamate/malate- (b, d) or TMPD-based (c, e) oxygen consumption rate (OCR) in isolated coupled mitochondria from BAECs (b, c; n = 5) or MEFs (d, e; n = 6). f, g, Effect of 0.1 mM CaCl2 (f; n = 5) or NaCl (g; n = 4) additions on CII+III activity in isolated mitochondrial membranes from BAECs. h, i, Cytosolic Ca2+ (h) or cytosolic Na+ (i) measured by live cell confocal microscopy of BAECs transfected with cyto-GEM-GECO (h) or treated with ANG2-AM (i), either not treated (No treat) or treated with 10 μM DMM during normoxia and hypoxia (1% O2; n = 4). j, Superoxide detection by fluorescence microscopy after incubation with DHE in 10-min time windows in non-treated BAECs (No treat) or treated with 10 μM DMM during normoxia (Nx) or hypoxia (1% O2; n = 3, except AA n = 2). k, l, Effect of 10 mM NaCl addition on succinate dehydrogenase activity (k) or ubiquinone 2-cytochrome c activity in the presence of n-Dodecyl β-D-maltoside (DDM; l) from BAECs mitochondrial membranes (n = 4). m, n, Effect of hypoxia (1% O2) in mitochondrial Na+ content measured with SBFI in BAECs transfected with siSCR or siNCLX (m) or treated with CGP-37157 (n) (n = 4). o, Effect of hypoxia (1% O2) in mitochondrial Na+ content in WT and KO MEFs, measured with CoroNa Green adapted for mitochondrial loading and live fluorescence (n = 10 MEFs WT and n = 12 MEFs KO). p, Representative images of three independent experiments showing colocalization of CoroNa Green-AM and TMRM signals after application of a long incubation protocol for CoroNa Green AM. All data are represented as mean ± s.e.m. Two-tailed Student’s t-test (f, k, l) and one-way ANOVA with Tukey’s test for multiple comparisons (b–e, h–j, m–o): n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t-test (No treat vs DMM; h, i): n.s. not significant, & P < 0.05. j, statistical comparisons shown only for Nx vs 0-10 groups. Pearson correlation coefficient in g: R = -0.9753.
a, FRAP of BAECs expressing mitoRFP in normoxia or hypoxia for 20 min (1% O2), with or without CGP-37157 (n = 15 for Nx, n = 16 for Hp, n = 13 for NxCGP and n = 10 for HpCGP, obtained from four independent experiments). b, NAO quench FRAP signal of BAECs exposed to normoxia or hypoxia for 15 min (1% O2; n = 5), with or without CGP-37157. c, CoQ10 redox state of HUVECs subjected to normoxia (Nx), 10 min of hypoxia (Hp 10 min; 1% O2), or normoxia with antimycin A (AA), n = 4. d, e, Anisotropy of phosphatydilcholine (PC) or PC:cardiolipin (PC:CL) liposomes treated with increasing concentrations of NaCl measured by TMA-DPH (d; n = 3) or DPH (e; n = 4) fluorescence. f–i, ESR spectra of 5-, 12- and 16-Doxyl PC in DOPC liposomes. 5-Doxyl PC exhibited an increased restricted motion (broadening of the hyperfine splitting, 2Amax) as a function of the NaCl concentration whereas the correlation time for 12- and 16-Doxyl PC remained unchanged. Hyperfine splitting (2Amax) of 5-Doxyl PC measured by ESR in PC liposomes treated with increasing concentrations of NaCl (n = 2) (f). Chemical structures of 5-, 12- and 16-Doxyl PC (g). ESR of 5-, 12- and 16-Doxyl PC in PC liposomes (h) and rotational times (i), τc, of 12- and 16-Doxyl PC in PC liposomes treated with increasing concentrations of NaCl as measured by ESR (n = 2). j, IR spectroscopy absorption spectra of PC liposomes treated or not with 16 mM NaCl (n = 2). Data are represented as mean ± s.e.m., except mean ± s.d. in d–f and i. Two-tailed Student’s t-test (c) and one-way ANOVA with Tukey’s test (a, b, d, e): n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Extended Data Fig. 9 Mitochondrial Na+ influx inhibition abolishes hypoxic pulmonary vasoconstriction.
a, Effectiveness of NCLX silencing in mice pulmonary artery smooth muscle cells (PASMCs) measured by quantitative RT–PCR analysis, n = 3. b, Superoxide detection by fluorescence microscopy after incubation with DHE in PASMCs treated with siSCR or siNCLX in normoxia (Nx) or hypoxia (Hp; 1% O2), n = 4 (except AA, n = 2). c, d, Representative traces (c) and average values (d) of hypoxic pulmonary vasoconstriction (HPV) measured in rat pulmonary arteries in the absence of pretone (precontraction). Each artery was exposed twice to hypoxia, and the second hypoxic challenge was performed in the absence or the presence of 30 μM CGP-37157, n = 7 (except peak CGP, n = 8; steady state CGP, n = 9). All data are represented as mean ± s.e.m. One-group t-test vs. 1 (a), two-tailed Student’s t-test (a, d) and one-way ANOVA with Tukey’s test (b): * P < 0.05, ** P < 0.01, *** P < 0.001. In b, statistical comparisons shown only for Nx vs 0-10 groups.
a, Complex I undergoes the A/D transition, this leads to mitochondrial matrix acidification and free Ca2+ release from the CaP precipitates. b, The increase in mitochondrial matrix free Ca2+ activates NCLX which introduces Na+ into the mitochondrial matrix. c, Na+ interacts with phospholipids in the inner leaflet of the inner mitochondrial membrane, decreasing diffusion of CoQH2, uncoupling CoQ cycle only in free complex III and promoting superoxide production at the Qo site of complex III. d, Mitochondrial ROS in hypoxia activate downstream targets. In addition, the entry of extracellular Ca2+ promotes a positive feed-back loop that further increases mitochondrial Na+ import and ROS production in hypoxia.
Source data for images for gels and blots.
FRAP of normoxic WT MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of wild type MEFs expressing mitoRFP subjected to normoxia (n=14).
FRAP of hypoxic WT MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of wild type MEFs expressing mitoRFP subjected to 10 min of hypoxia (1% O2; n=14).
FRAP of normoxic KO MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of NCLX KO MEFs expressing mitoRFP subjected to normoxia (n=14).
FRAP of hypoxic KO MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of NCLX KO MEFs expressing mitoRFP subjected to hypoxia (1% O2; n=14).
FRAP of normoxic KO+pNCLX MEFs expressing mito- RFP. Time-lapse showing a representative FRAP of NCLX KO MEFs expressing pNCLX and mitoRFP, subjected to normoxia (n=14).
FRAP of normoxic KO+pNCLX MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of NCLX KO MEFs expressing pNCLX and mitoRFP, subjected to hypoxia (1% O2; n=14).
FRAP of normoxic WT+dnNCLX MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of wild type MEFs expressing dnNCLX and mitoRFP, subjected to normoxia (n=14).
FRAP of hypoxic WT+dnNCLX MEFs expressing mitoRFP. Time-lapse showing a representative FRAP of wild type MEFs expressing dnNCLX and mitoRFP, subjected to 10 min of hypoxia (1% O2; n=14).
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Hernansanz-Agustín, P., Choya-Foces, C., Carregal-Romero, S. et al. Na+ controls hypoxic signalling by the mitochondrial respiratory chain. Nature 586, 287–291 (2020). https://doi.org/10.1038/s41586-020-2551-y
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