Abstract
Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU−/− mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU−/− mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU−/− mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU−/− mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not seem to protect MCU−/− cells and tissues from cell death, although MCU−/− hearts fail to respond to the PTP inhibitor cyclosporin A. Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.
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Acknowledgements
We are grateful to C. Brantner, P. S. Connelly and M. P. Daniels of the NHLBI Electron Microscopy Core Facility for assistance with electron microscopy, C. Petucci of the Metabolomics Core Facility Sanford-Burnham Medical Research Institute for aiding in the metabolomic profiling, C. Combs and the NHLBI Microscopy Core for help with the Rhod-2 fluorescent measurements and A. Wiederkehr for the original mito-aequorin adenovirus. This work was supported by NIH Intramural funds.
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X.P., J.L. and T.N. designed, performed and analysed the experiments and aided in writing the manuscript, C.L. helped construct the mouse model, J.S., Y.T., M.M.F., I.I.R., M.A., D.A.S., A.M.A. and M.G. contributed to the completion of various experiments, and R.S.B., E.M. and T.F. conceived the study, supervised the research and contributed to writing the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Disruption of the MCU gene by gene-trapping.
(a) Graphical representation of the mouse MCU genomic locus and the trapping vector insertion site. The Omnibank Gene Trap Vector 76, which contains a b-Geo (fusion of b-Gal gene and neomycin-resistance gene) selection marker flanked by a splice acceptor (SA), a transcription termination sequence (pA), as well as the retroviral LTR sequences was inserted within Intron 1 of the mouse MCU gene. (b) The genomic DNA sequence surrounding the gene trap insertion site identified in the IST11669F8 mouse ES clone. The insertion site is denoted with a red asterisk. (c) Individual organ weight at necropsy normalized to total body weight of WT (MCU+/+) and MCU−/− mice (n = 4 per genotype). d) Body composition as determined by quantitative magnetic resonance in young mice (3-4 months of age, n = 7 per genotype) and older mice (10-12 months, n = 5 per genotype). All pooled data represents mean +/− S.E.M.
Supplementary Figure 2 Mitochondrial morphology and MCU expression in MCU−/− mice.
(a) Electron micrographic images of both fetal (E12.5) and adult liver and heart. WT and MCU−/− tissues show similar mitochondrial morphology and abundance. Scale bars = 500 nm. (b) Analysis of MCU expression in tissues from WT and MCU−/− mice. Protein lysates were identical to those shown in Figure 1d, however a different antibody against MCU (Sigma) recognizing the N-terminal region of MCU was used. Tubulin, identical to Figure 1d, is again used as a loading control. (c) Protein expression in MEFs obtained from wild type or MCU−/− embryos. GAPDH is used as a loading control.
Supplementary Figure 3 Mitochondrial calcium uptake using isolated mitochondria and intact cells.
(a) Cardiac mitochondria were loaded with the calcium indicator Fluo-4FF. Calcium addition over the physiological (micromolar) range results in increasing calcium levels in mitochondria isolated from WT hearts. This uptake in WT mitochondria is blocked by Ru360 addition. MCU−/− mitochondria lack any demonstrable uptake at low calcium concentrations. At higher Ca2+ concentrations, there is a small, non Ru360-inhibitable, increase in Fluo-4FF fluorescence in MCU−/− mitochondria observed. Addition of EGTA (20 mM) returns fluorescence back to baseline, consistent with dye leakage as the basis of this non Ru360-inhibitable increase in Fluo-4FF fluorescence. (b) A similar experiment where the MCU−/− mitochondria were briefly pelleted at the end of the experiment. All fluorescence seen with the MCU−/− mitochondria appears to reside in the supernatant, suggesting again that the fluorescence observed following the addition of higher concentrations of calcium is most consistent with dye leakage out of the mitochondria. (c) Cardiac myocytes were isolated from WT or in MCU−/− hearts and adult myocytes were loaded with Rhod-2 to measure mitochondrial calcium levels. Shown is a representative experiment following addition of KCl (50 mM) with average fluorescence calculated from WT myocytes (n = 9 cells) or MCU−/− myocytes (n = 11 cells). (d) Similar analysis using caffeine (20 mM) as a stimulus to increase cytosolic calcium with subsequent imaging of WT myocytes (n = 16 cells) and MCU−/− myocytes (n = 8 cells). All pooled data represents mean +/− S.E.M.
Supplementary Figure 4 Basal oxygen consumption and authophagic flux in MCU−/− cells and tissues.
(a) Relative oxygen consumption measured using a Seahorse X-24 analyzer analyzing WT and MCU−/− MEFs under basal conditions with glucose as the substrate (25 mM), or a similar analysis using pyruvate (2 mM) or in the presence of galactose (25 mM). Results are the average +/− S.E.M. of three independent experiments each performed in quadruplicate. b) Autophagic flux as determined by levels of p62 and the ratio of LC3I/LC3II. MEF cells were shifted to a low nutrient media (Hanks buffered salt solution; t = 0) and subsequently assessed for markers of autophagic flux. Consistent with increased autophagic flux, starvation led to decreased levels of p62 and an increased LC3II/LC3I ratio. However, this response was similar between WT and in MCU−/− MEFs. A representative example from three similar experiments is shown. (c) Similar assessment in vivo in both liver and heart tissues under randomly fed conditions. Again, no consistent differences were observed between genotypes. GAPDH is shown as a loading control.
Supplementary Figure 5 Regulation of skeletal muscle PDH phosphorylation by MCU.
(a) Measurement of matrix calcium levels under fed conditions reveals that the differences between WT and in MCU−/− mitochondria are less pronounced then under fasting conditions (n = 3 mice per genotype). (b) Mice were fasted overnight, re-fed and then harvested four hours later. Phosphorylation of serine 293 of skeletal muscle PDH E1- α subunit was determined. (c) Densitometric quantification of the intensity of phosphorylated to total PDH was determined. n = 3; p = NS. (d) Isolated skeletal muscle mitochondria were analyzed in the absence or presence of extramitochondrial calcium (500 μM for 1 minute). Calcium addition led to a brisk reduction in PDH phosphorylation in WT skeletal muscle mitochondria but this effect was not observed in MCU−/− mitochondria. PDH phosphorylation was determined by a phospho-specific antibody. (e) Analysis of citrate synthase activity in WT and in MCU−/− skeletal muscle mitochondria. n = 3 mice per genotype, p = NS by t-test, all pooled data represents mean +/− S.E.M.
Supplementary Figure 6 Fiber type profile of the gastrocnemius WT and MCU−/− mice.
Immunohistochemical analysis of soleus (SOL) and extensor digitorum longus (EDL) muscle using antibodies for slow and fast twitch muscle. No differences were observed between genotypes. One representative experiment from two similar experiments is shown. Scale bar equals 100 μm.
Supplementary Figure 7 MCU expression and cell death.
( a) Calcium-induced PTP opening in hepatic mitochondria isolated from WT or MCU−/− mice. One representative experiment from four similar experiments is shown. Experiments were performed in the presence or absence of cyclosporin A (CsA; 0.2 μM) and the arrow indicates the addition of calcium (500 μM). (b) Cytosolic extracts were prepared from WT and MCU−/− MEFs at the indicated time points after the addition of doxorubicin (2 μM). Cytochrome C release was similar in magnitude and kinetics between WT and MCU−/− cells. Tubulin is shown as a loading control. (c) Measurement of cytosolic calcium using Fluo-4AM fluorescence in primary MEF cells following the addition of 2 mM hydrogen peroxide. Cells were analyzed at the indicated times by flow cytometry. While calcium levels rose after hydrogen peroxide, no discernable differences were observed between genotypes.
Supplementary Figure 8 Role of MCU in ischaemia/reperfusion injury.
(a) Regions of infracted myocardium were assessed for TUNEL positive cells (n = 3 mice per genotype). Analysis was from three mice per genotype using at least three random sections per mouse. (b) Magnitude of ischemic contracture, a measure of cytosolic calcium levels, was assessed in WT and MCU−/− hearts. There does not appear to be an appreciable difference between WT (n = 7) and MCU−/− (n = 6) mice suggesting that under ischemic conditions, MCU−/− hearts do not develop significantly higher levels of cytosolic calcium. All pooled data represents mean ± S.E.M., statistical significance was evaluated by t-test.
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Pan, X., Liu, J., Nguyen, T. et al. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol 15, 1464–1472 (2013). https://doi.org/10.1038/ncb2868
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DOI: https://doi.org/10.1038/ncb2868
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