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. 2013 Dec;15(12):1464-72.
doi: 10.1038/ncb2868. Epub 2013 Nov 10.

The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter

Xin Pan  1 , Jie Liu, Tiffany Nguyen, Chengyu Liu, Junhui Sun, Yanjie Teng, Maria M Fergusson, Ilsa I Rovira, Michele Allen, Danielle A Springer, Angel M Aponte, Marjan Gucek, Robert S Balaban, Elizabeth Murphy, Toren Finkel
Affiliations

The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter

Xin Pan et al. Nat Cell Biol. 2013 Dec.

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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Figures

Figure 1
Figure 1
MCU-/- mice lack MCU expression and evidence for rapid mitochondrial calcium uptake. a) Besides their size, MCU-/- mice (on right) lack a discernible phenotype. b) MCU-/- mice are smaller than WT mice (mean +/- S.E.M., p<0.01 by ANOVA; n=14 female WT and n=13 female MCU-/- mice). c) MCU mRNA expression by RT-PCR analysis in various tissues of WT, heterozygous and MCU-/- mice (n=3 animals per genotype, mean +/- S.E.M., *p<0.05, **p<0.01 by ANOVA compared to WT expression). d) MCU protein expression in various tissues using a rabbit polyclonal antibody generated against the C-terminus of MCU. Tubulin is used as a loading control. e) Assessment of mitochondrial calcium levels using the fluorescent mitochondrial calcium sensor Fluo-4FF. Calcium addition over the physiological (micromolar) range results in increasing calcium levels in mitochondria isolated from WT skeletal muscle. This uptake in WT mitochondria is inhibited by Ru360 addition. MCU-/- mitochondria lack any demonstrable uptake. Shown is one experiment that is representative of three similar experiments. Inset-Western blot analysis of MCU expression in purified WT and MCU-/- mitochondria with cytochrome C oxidase subunit IV (isoform 1) used as a loading control. f) Similar experiment performed using cardiac mitochondria. At higher Ca2+ concentrations, there is a small, non Ru360-inhibitable, increase in Fluo-4FF fluorescence in MCU-/- mitochondria observed. g) Parallel assessment of extramitochondrial calcium measurements demonstrating that only WT cardiac mitochondria appear capable of calcium uptake.
Figure 2
Figure 2
MCU regulates mitochondrial calcium uptake in permeabilized MEFs. a) Comparison of cytosolic calcium levels in permeabilized WT and MCU-/- MEFs. Arrows indicate calcium addition. Increasing cytosolic calcium results in a rapid increase in the fluorescent signal in both cell types but the subsequent decline in the fluorescent signal, representing mitochondrial calcium uptake, is only observed in WT cells. Shown is one tracing that is representative of three similar experiments. b) Ruthenium red (Ru360; 3 μM) inhibits mitochondrial calcium uptake in permeabilized WT MEFs. c) Western blot analysis of MCU expression. MCU-/- MEFs were infected with retroviruses encoding an epitope-tagged form of either WT MCU or MCUmut containing two amino acid substitutions (at amino acid 261 and 264) known to abrogate uniporter activity. The tagged constructs migrate slightly slower than endogenous MCU but were expressed at roughly endogenous levels. d) Cytosolic calcium measurements in permeabilized WT MEFs, MCU-/- MEFs, MCU-/- MEFs reconstituted with wild type MCU and MCU-/- MEFs reconstituted with MCUmut.
Figure 3
Figure 3
MCU regulates ligand-stimulated mitochondrial calcium uptake. a) Isoproterenol stimulates an increase in mitochondrial calcium in WT cardiac myocytes. Adult WT and MCU-/- adult cardiac myocytes were freshly isolated, loaded with mitochondrial calcium sensitive probe Rhod-2 and stimulated with 1 μM isoproterenol at time zero (n=10 random fields per genotype). b) WT or MCU-/- MEFs were infected with an adenovirus encoding a mitochondrial targeted aequorin construct. Levels of mitochondrial calcium were assessed by aequorin luminescence following histamine stimulation (100 μM) for WT (n=10) and MCU-/- MEFs (n=12). Values were normalized to maximal aequorin luminescence observed in permeabilzed cells exposed to exogenous calcium. C) Cytosolic calcium levels in WT (n=14) or MCU-/- MEFs (n=17) as measured by Fluro-4 fluorescence following histamine stimulation. All pooled data represents mean +/- S.E.M.
Figure 4
Figure 4
The role of MCU in basal metabolism. a) Seahorse X-24 analysis of oxygen consumption rate (OCR) in WT and MCU-/- MEFs under basal conditions or following the addition of oligomycin, the uncoupler FCCP or the electron transport inhibitor antimycin A (n=5). b) Representative tracings of the oxygen consumption observed in isolated hepatic mitochondria given glutamate/malate as a substrate (G/M) followed by ADP (State 3). c) Quantification of the respiratory control ratio (RCR; State 3/State 4 respiration) in WT and MCU-/- mitochondria (p=NS; n=3 independent experiments). d) Average total body oxygen consumption in WT and MCU-/- mice during day and night conditions (n=5 WT and n=6 MCU-/- mice; p=NS between genotypes). All pooled data represents mean +/- S.E.M.
Figure 5
Figure 5
Altered in vivo skeletal muscle metabolism and PDH activity in MCU-/- mice. a) Mitochondrial oxygen consumption following depolarization induced by the addition of 500 μM calcium with and without the respiratory chain inhibitor Antimycin A (15 μM). The average +/- SEM of three independent experiments is shown. **p<0.01 by t-test compared to without calcium. b) Levels of matrix calcium measured in WT and MCU-/- mitochondria derived from skeletal muscle following an overnight 16 hour fast (**p<0.01 by t-test; n=3 WT and N=4 MCU-/- mice). c) Western blot determination of the levels of phospho-PDH (serine 293 of the E1-α subunit) and total PDH levels in the skeletal muscle of three pairs of WT and MCU-/- mice starved for 16 hours. d) Under starved conditions, altered PDH phosphorylation is seen various muscle types including the extensor digitorum longus (EDL) representing glycolytic/fast twitch fibers, the soleus (SOL) that is predominantly oxidative/slow twitch and the gastrocnemius (GN) that is a mix of fast and slow twitch. e) Skeletal muscle PDH activity in WT and MCU-/- mice after a 16 hour fast (*p< 0.05; n=3 mice per genotype). f) Serum lactate levels in WT and MCU-/- male mice under fed conditions or after 16 hours of starvation (n=7 WT and n=6 MCU-/-, *p<0.05 by t-test). g) Metabolomic analysis of skeletal muscle demonstrating levels of various TCA cycle intermediates in mice that were starved overnight (n=3 per genotype). There was a trend for increased lactate levels in the MCU-/- muscle. All pooled data represents mean +/- S.E.M.
Figure 6
Figure 6
MCU regulates skeletal muscle peak performance. a) Assessment of skeletal muscle function using maximal work performed on an inclined treadmill test (n=6 WT and n=11 MCU-/- mice). b) Grip strength assessment for WT and MCU-/- mice (n=5 WT and n=5 MCU-/- mice). c) Forelimb strength during a modified vertical pull up test (n=11 WT and n=12 MCU-/- mice). All mice assessed for physiological responses were female. *p<0.05 and **p<0.01 by t-test. d) Analysis of fiber-specific mRNA abundance in the gastrocnemius of WT and MCU-/- mice (n=3 mice per genotype, p=NS between genotypes). All pooled data represents mean +/- S.E.M.
Figure 7
Figure 7
MCU expression is necessary for calcium-induced PTP opening but not required for cell death. a) Only WT mitochondria undergo PTP opening after calcium addition (500 μM) as evidence by a rapid drop in absorbance. Shown is one experiment using heart mitochondria that is representative of three similar experiments. b) Average change in absorbance from three independent experiments using isolated cardiac mitochondria in the presence or absence of CsA (0.2 μM) and Ru360 (3.0 μM). c) Cell viability as measured by Annexin V/PI staining in WT and MCU-/- MEFs following a wide array of challenges including hydrogen peroxide exposure (1 mM), tunicamycin (2 μg/ml), doxorubicin (2 μM), C2-ceramide (100 μM) and thapsigargin (1 μM). The time course and magnitude of cell death was not altered by the absence of MCU expression (p=NS, n=3 per genotype). d) Cytosolic cytochrome C levels in WT or MCU-/- MEFs following the addition of hydrogen peroxide. Tubulin is shown as a loading control. e) Caspase-3 activity was measured under basal conditions or 24 hours after treatment with tunicamycin or doxorubicin (p=NS; n=3). All pooled data represents mean +/- S.E.M.
Figure 8
Figure 8
Role of MCU in ischaemia-reperfusion injury. a) Assessment of the rate pressure product (RPP, heart rate times systolic blood pressure) after ischaemia-reperfusion injury in the hearts of WT or MCU-/- mice with and without Cyclosporin A (CsA, 0.2 μM) for five minutes prior to ischemia. g) Infarct size in WT and MCU-/- mice following 20 minutes of global ischemia and 90 minutes of reperfusion. *p<0.05, **p<0.01 by ANOVA; all pooled data represents mean +/- S.E.M.
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