Abstract
In warm-blooded species, heat released as a by-product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. The energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, with a variable proportion released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain was fully functional, both in HEK cells and primary skin fibroblasts. This differential was abolished by respiratory inhibitors or in cells lacking mitochondrial DNA, but enhanced by expressing thermogenic enzymes such as the alternative oxidase or the uncoupling protein UCP1. The activity of various RC enzymes was maximal at, or slightly above, 50 °C. Our study prompts a re-examination of the literature on mitochondria, taking account of the inferred high temperature.
Main Text
Mitochondrial targeting of the recently developed, temperature-sensitive fluorescent probe (Fig. S1A), Mito-Thermo-Yellow (MTY)(1) was confirmed in both HEK293 cells and primary skin fibroblasts, based on colocalization with the well-characterized MitoTracker green (MTG; Fig. 1A). Initial mitochondrial capture was dependent on the maintenance of a minimal membrane potential(1). Fluorescence remained stable over 2 hours in HEK293 cells, but mitochondrial MTY retention varied between cell lines (Fig. S2A). The exact sub-mitochondrial location of the probe is yet to be established, although it has been postulated to reside within the inner membrane. No toxicity of MTY (100 nM in culture medium) could be detected after 3 days.
The fluorescence of MTY (measured in solution at 562 nm) was progressively and reversibly decreased by as the medium temperature increased (about 50% from 25 to 45°C; Fig. 1B, a, b). Using a thermostated, magnetically stirred, closable 750 μl quartz-cuvette fitted with an oxygen sensitive optode device(2) we simultaneously studied oxygen consumption or tension and changes in MTY fluorescence (Fig. 1C). Adherent cells were harvested and pre-loaded for 20 min with 100 nM MTY, harvested and washed, then kept as a concentrated pellet at 38°C for 10 min, reaching anaerobiosis in < 1 min. When cells were added to the oxygen-rich medium they immediately started to consume oxygen (red trace), accompanied by a progressive decrease of MTY fluorescence (blue trace; phase I; Fig. 1C). In the absence of any inhibitor, the fluorescence gradually reached a stable minimum (phase II). Once all the oxygen in the cuvette was exhausted (red trace) the shift of MTY fluorescence reversed (phase III), returning gradually almost to the starting value (phase IV). To calibrate the fluorescence signal, the temperature of the extra-cellular medium was increased stepwise (green trace). MTY fluorescence returned to the prior value when the medium was cooled to 38 °C (Fig. 1C, phase V). Based on this calibration, we estimate the rise in mitochondrial temperature due to the activation of respiration as ∼10 °C (n=10, range 7-12 °C). At the lowest (phase II) and highest fluorescence value (38°C, imposed by the water bath; phase IV), the signal was proportional to the amount of added cells (Fig. 1D, a). Cell number did not affect the maximal rate of fluorescence decrease (phase I), but, once anaerobic conditions had been reached (phase III, initial), it was inversely related to the initial rate of fluorescence increase (Fig. 1D, b). To confirm that the observed fluorescence changes were due to mitochondrial respiration and not some other cellular process, we depleted HEK cells of their mtDNA with ethidium bromide (EtBr) to a point where cytochrome c oxidase activity was less than 2% of that in control cells (Fig. 1E, a; Fig. S1C). In EtBr-treated cells, no MTY fluorescence changes were observed under aerobiosis and cyanide treatment had no effect (Fig. 1E, b). Because MTY is derived from the membrane potential-sensitive dye rhodamine, whose fluorescence is essentially unaffected by temperature (Figure 1B, b), we investigated whether the observed changes MTY fluorescence could be influenced by altered membrane potential, or by an associated parameter, e.g. pH. We therefore compared the response of MTY fluorescence to cyanide or oligomycin (Fig. 2A, B; Fig. S1D) exerting opposite effects on membrane potential and pH gradient (Fig. 2C). To avoid the possibly confounding effect of anaerobiosis, the quartz cuvette was kept uncapped in this experiment, with the oxygen tension rather than the rates of oxygen uptake being recorded (red traces). Once MTY fluorescence was stabilized (maximal mitochondrial heating), and the medium re-oxygenated, cyanide was added, causing a progressive increase in MTY fluorescence to the starting value (Fig. 2A). Note that, when cyanide was present from the start of the reaction, fluorescence changes and oxygen uptake were both abolished (Fig. 2A, dotted lines). Adding oligomycin in lieu of cyanide decreased oxygen consumption and, as observed with cyanide, brought about an increase in MTY fluorescence (Fig. 2B). Added first, oligomycin strongly decreased oxygen uptake, abolishing MTY fluorescence decrease (Fig. 2B, dotted lines). Taken together, these experiments imply that electron flow through the respiratory chain (RC) rather than membrane potential controls mitochondrial temperature. This conclusion is supported by the similar effects observed with two other respiratory inhibitors (Fig. S1D), affecting either RC complex I (CI; rotenone, Fig. 2D) or III (CIII; antimycin, Fig. 2E). Despite their different effects on the redox state of the various RC electron carriers, these inhibitors blocked oxygen uptake and again triggered an increase in MTY fluorescence. Taking advantage of cyanide removal from cytochrome oxidase to form cyanohydrin in the presence of α-ketoacids under aerobiosis(3), we confirmed that the blockade of the respiratory chain did not result in MTY leakage from the mitochondria since pyruvate addition resulted in oxygen uptake resuming and MTY fluorescence decrease, both being inhibited by a further addition of antimycin (Fig. 2F). Noticeably the fluorescence of an endoplasmic reticulum (ER)-targeted version of the probe in HEK cells and skin fibroblasts was essentially unaffected by the activity of the mitochondria when modulated by cyanide, pyruvate or antimycin (Fig. S3).
We next studied MTY probe behavior in HEK cells made partially deficient for CI by varying amounts of added rotenone. The rate of MTY fluorescence decrease was proportional to the residual respiratory electron flux (Fig. 3A) while the maximal temperature as judged from MTY fluorescence (phase II) was essentially unchanged (Fig. 3A; inset). We next tested the effect of expressing the cyanide-insensitive non-proton motive alternative oxidase from Ciona intestinalis (AOX; Fig. 3B, S1E), whose activity is unmasked in the presence of cyanide(4). Before cyanide addition, the decrease in MTY fluorescence in AOX-expressing cells was similar to control cells, consistent with previous inferences that the enzyme does not significantly participate in uninhibited cell respiration. However upon cyanide addition, AOX-endowed cells maintained low MTY fluorescence (Fig. 3B, blue trace), despite oxygen consumption decreasing by more than 50% (red trace). The increased ratio of heat generated to respiration is consistent with the predicted thermogenic properties of AOX. Subsequent addition of 0.1 mM propylgallate, which inhibits AOX, almost completely abolished the residual respiration and brought MTY fluorescence back to its starting value (corresponding to 38°C).
So as to circumvent the fact that we were not able to use chemical uncouplers with this probe(1), we used HEK cells engineered to express the uncoupling protein 1 (UCP1; Fig. 3C, S1F). As expected, UCP1 conferred an increased rate of respiration, which was only partially inhibited by oligomycin (red trace), and accompanied by an even greater drop in MTY fluorescence, equivalent to a temperature at least 15 °C above the cellular environment. The surprisingly high inferred mitochondrial temperatures prompted us to check the dependence on assay medium temperature of RC enzyme activities measured under Vmax conditions (Fig. 4A, B) in crude extracts, where mitochondrial membrane integrity is maintained. Antimycin-sensitive CIII, malonate-sensitive succinate:cytochrome c reductase (CII+CIII) and cyanide-sensitive cytochrome c oxidase (CIV) activities all showed temperature optima at or slightly above 50°C, whilst these activities tended gradually to decrease again as the temperatures were raised further (Fig. 4A). This was not so for those enzymes whose activities can be measured in vitro only after osmotic disruption of both outer and inner mitochondrial membranes (Fig. 4B). Oligomycin-sensitive ATPase (CV) activity was optimal around 46 °C, whereas rotenone-sensitive NADH quinone-reductase (CI) activity declined sharply at temperatures above 38 °C. Under the even more disruptive conditions of clear-native electrophoresis, which requires detergent-dispersal of the membrane-bound complexes, in-gel activity even of CII or CIV was impaired at temperatures above 42 °C and 46 °C, respectively (Fig. 4C).This strongly suggests a vital role for the lipid components of the inner mitochondrial membrane in the stabilization of the RC complexes at high temperature. We next analyzed the temperature profile of RC activity of primary skin fibroblasts. For CII+CIII, CIII and CIV (Fig. 4D), as well as CV (Fig. 4E) similar temperature optima were observed as in HEK cells, whilst MTY fluorescence (Fig. 4F) also indicated mitochondria being maintained at least 6-10 °C above environmental temperature. Our findings raise numerous questions concerning the biochemistry, physiology and pathology of mitochondria. The physical, chemical and electrical properties of the inner mitochondrial membrane and of mitochondria in general, will need to be re-evaluated, given that almost all previous literature reflects experiments conducted far from physiological temperature. Traditional views of the lipid component of the respiratory membrane as a lake in which the RC complexes are floating resulting in a random-diffusion model of electron transfer or, more recently, as a sealant occupying the space between tightly packed proteins(5), need to be revised in favour of one that considers it more as a glue that maintains the integrity of the respiratory complexes. The effects of respiratory dysfunction need to be reconsidered, to include those attributable to temperature changes, such as effects on membrane fluidity, electrical conductance and transport. RC organization into supercomplexes(6, 7) should be re-examined at more realistic temperatures, using methods other than CNE, which does not maintain functional integrity even of the RC complexes individually. Finally, whilst the subcellular distribution of mitochondria (e.g. perinuclear, or synaptic) has previously been considered to reflect ATP demand, mitochondria should also be considered as a source of heat, potentially relevant in specific cellular or physiological contexts, not just in specifically thermogenic tissues like brown fat.
Material and Methods
Cell culture
Human cells derived from Embryonic Kidney (HEK293), Hepatoma Tissue Culture (HTC-116) and from large cell lung cancer (NCI-H460) cells (American Type Culture Collection, Manassas, VA 20108 USA) were cultured in DMEM medium containing 5 g/l glucose supplemented by 2 mM glutamine (as Glutamax™), 10% foetal calf serum, 1 mM pyruvate, 100 μg/ml penicillin/streptomycin each. AOX-(9) or UCP-endowed(10, 11) HEK293 cells were obtained as previously described.
Primary skin fibroblasts were derived from healthy individuals and grown under standard condition in DMEM glucose (4.5 g/l), 6 mM glutamine, 10% FCS, 200 μM uridine, penicillin/streptomycin (100 U/ml) plus 10 mM pyruvate.
Immunoblot analyses and in gel enzyme activity assays
For the Western blot analysis, mitochondrial proteins (50 μg) were separated by SDS–PAGE on a 12% polyacrylamide gel, transferred to a nitrocellulose membrane and probed overnight at 4°C with antibodies against the protein of interest, AOX 1:10,00010, UCP1 1:10,00011. Membranes were then washed in TBST and incubated with mouse or rabbit peroxidase-conjugated secondary antibodies for 2 h at room temperature. The antibody complexes were visualized with the Western Lightning Ultra Chemiluminescent substrate kit (Perkin Elmer). For the analysis of respiratory chain complexes, mitochondrial proteins (100 μg) were extracted with 6% digitonin and separated by hrCN-PAGE, on a 3.5–12% polyacrylamide gel. Gels were stained by in gel activity assay (IGA) detecting CI, CII and CIV activity as described12.
Staining procedures and life cell imaging
Cells (HEK293, MTC, NIC, primary skin fibroblasts) were seeded on glass coverslips and grown inside wells of a12 well-plate dish for 48 h in standard growth media at 37 °C, 5% CO2. The culture medium was replaced with pre-warmed medium containing fluorescent dyes, 100 nmol MitoTracker Green (Invitrogen M7514) and 100 nmol MitoThermo Yellow(1) or 100 nmol ER Thermo Yellow(12). After 10 min the staining medium was replaced with fresh pre-warmed medium or PBS buffer and cells were observed immediately by Leica TCS SP8 confocal laser microscopy.
Assay of mitochondrial respiratory chain activity
The measurement of RC activities was carried out using a Cary 50 spectrophotometer (Varian Australia, Victoria, Australia), as described(13). Protein was estimated using the Bradford assay.
Simultaneous spectrofluorometric, temperature, oxygen uptake assay
Detached sub-confluent HEK (NCI, HTS) cells (25 cm2 flask) or trypsinized sub-confluent skin fibroblasts (75 cm2 flask) were treated for 20 min with 100 μM MTY in 10 ml DMEM, and spun down (1,500 g x 5 min). The pellet is resuspended in 1 ml PBS, cells being next spun down (1,500 g x 5 min) and kept as a concentrated pellet. After anaerobiosis (checked by inserting an optic fiber equipped with an oxygen-sensitive fluorescent terminal sensor (Optode device; FireSting O2, Bionef, Paris, France) was established (10 min incubation of the pellet at 38°C;), cells (1 mg prot) were added to 750 μl of 38°C-thermostated medium. The fluorescence (excitation 542 nm, emission 562 nm for MTY; excitation 559 nm, emission 581 nm for ERTY), the temperature of the medium in the cuvette and the respiration of the intact cell suspension were simultaneously measured in a magnetically-stirred, 38°C-thermostated 1 ml-quartz cell in 750 μl of PBS using the Xenius XC spectrofluorometer (SAFAS, Monaco). Oxygen uptake was measured with an optode device fitted to a handmade cap, ensuring either closing of the quartz-cell yet allowing micro-injections (hole with 0.6 mm diameter) or leaving the quartz-cell open to allow for constant oxygen replenishment. Alternatively, untreated HEK293 cells (250 μg protein) were added to 750 μl of medium consisting of 0.25 M sucrose, 15 mM KCl, 30 mM KH2PO4 (pH 7.4), 5 mM MgCl2, EGTA 1 mM, followed by addition of rhodamine to 100 nM and digitonin to 0.01 % w/v. The permeabilized cells were successively given a mitochondrial substrate (10 mM succinate) and ADP (0.1 mM) to ensure state 3 (phosphorylating) conditions, under which either 6.5 μM oligomycin or 1 mM cyanide was added.
Statistics
Data are presented as mean ± SD statistical significance was calculated by standard unpaired one-way ANOVA with Bonferroni post-test correction; a p< 0.05 was considered statistically significant (GraphPad Prism).
Data availability statement
The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files.
Authors contribution
M.R., HT.J. and P.R. conceived the project. HT.J., M.J., YT. C. and P.R. wrote the manuscript. D.C., P.B., R. EK., HH.H., S.K., M.R. and P.R. conducted research. All authors contributed to data analysis and manuscript preparation.
Author information
Reprints and permissions information is available at www.nature.com/reprints.
We confirm that there are no known conflicts or competing financial interests of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Correspondence and requests for materials should be addressed to Pierre Rustin, pierre.rustin{at}inserm.fr, or Malgorzata Rak, malgorzata.rak{at}inserm.fr.
Acknowledgements
This work was supported by French (ANR MITOXDRUGS to DC, PB, MR and PR) and European (E-rare Genomit to DC, PB, MR and PR) institutions and patient’s associations to PB and PR Association d’Aide aux Jeunes Infirmes (AAJI), Association contre les Maladies Mitochondriales (AMMi), Association Française contre l’Ataxie de Friedreich (AFAF), and Ouvrir Les Yeux (OLY).
Abbreviations
- AOX
- Alternative oxidase
- CNE
- Clearnative Electrophoresis
- ERTY
- Endoplasmic Reticulum Thermo Yellow
- EtBr
- Ethidium bromide
- MTG
- MitoTracker Green
- MTY
- MitoThermo Yellow
- RC
- Respiratory chain
- UCP1
- Uncouplin protein 1