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Reply to Rutter et al.: The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylation

Open AccessPublished:July 24, 2020DOI:https://doi.org/10.1074/jbc.RL120.014342
      Each model used in the work referred to by Rutter et al. (
      • Rutter G.A.
      • McCormack J.G.
      • Halestrap A.P.
      • Denton R.M.
      The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylation.
      ) addressed certain aspects of mitochondrial biology, and together, they fully support the conclusions made. Please note that we describe Ca2+-mediated regulation of oxidative phosphorylation (OXPHOS) fluxes (
      • Gellerich F.N.
      • Gizatullina Z.
      • Trumbekaite S.
      • Korzeniewski B.
      • Gaynutdinov T.
      • Seppet E.
      • Vielhaber S.
      • Heinze H.-J.
      • Striggow F.
      Cytosolic Ca2+ regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle.
      ,
      • Szibor M.
      • Gizatullina Z.
      • Gainutdinov T.
      • Endres T.
      • Debska-Vielhaber G.
      • Kunz M.
      • Karavasili N.
      • Hallmann K.
      • Schreiber F.
      • Bamberger A.
      • Schwarzer M.
      • Doenst T.
      • Heinze H.-J.
      • Lessmann V.
      • Vielhaber S.
      • et al.
      Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.
      ) and do not question Ca2+-responsiveness of pyruvate dehydrogenase en-zyme activity (
      • McCormack J.G.
      • Halestrap A.P.
      • Denton R.M.
      Role of calcium ions in regulation of mammalian intramitochondrial metabolism.
      ). To address concerns such as those raised by Rutter et al. (
      • Rutter G.A.
      • McCormack J.G.
      • Halestrap A.P.
      • Denton R.M.
      The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylation.
      ), we studied glutamate/malate-dependent OXPHOS in the absence of exogenous pyruvate in mitochondria, omitted pyruvate from cell experiments, and implemented the working rat heart model perfused by Krebs–Henseleit (glucose) buffer. This unequivocally demonstrates in a broad range of models that MAS (malate-aspartate shuttle) inhibition induces a state of mitochondrial pyruvate starvation (
      • Szibor M.
      • Gizatullina Z.
      • Gainutdinov T.
      • Endres T.
      • Debska-Vielhaber G.
      • Kunz M.
      • Karavasili N.
      • Hallmann K.
      • Schreiber F.
      • Bamberger A.
      • Schwarzer M.
      • Doenst T.
      • Heinze H.-J.
      • Lessmann V.
      • Vielhaber S.
      • et al.
      Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.
      ).
      An unresolved observation is that mitochondria of MCU knockout mice show negligible activity of Ca2+-uptake (5), which we confirm (
      • Szibor M.
      • Gizatullina Z.
      • Gainutdinov T.
      • Endres T.
      • Debska-Vielhaber G.
      • Kunz M.
      • Karavasili N.
      • Hallmann K.
      • Schreiber F.
      • Bamberger A.
      • Schwarzer M.
      • Doenst T.
      • Heinze H.-J.
      • Lessmann V.
      • Vielhaber S.
      • et al.
      Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.
      ). We attributed this activity to residual expression of wild-type Mcu transcripts (
      • Szibor M.
      • Gizatullina Z.
      • Gainutdinov T.
      • Endres T.
      • Debska-Vielhaber G.
      • Kunz M.
      • Karavasili N.
      • Hallmann K.
      • Schreiber F.
      • Bamberger A.
      • Schwarzer M.
      • Doenst T.
      • Heinze H.-J.
      • Lessmann V.
      • Vielhaber S.
      • et al.
      Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.
      ) as the result of a rare event of gene-trap excision during mRNA splicing, since this activity was sensitive to ruthenium red, an inhibitor of the MCU. Besides, please also note the low MCU Ca2+ affinity (
      • Carafoli E.
      The interplay of mitochondria with calcium: an historical appraisal.
      ). In vivo, the endoplasmic reticulum is thought to facilitate the generation of microcompartments of high Ca2+ concentration to allow Ca2+ uptake via MCU (
      • Carafoli E.
      The interplay of mitochondria with calcium: an historical appraisal.
      ). This mechanism is compromised in MCU knockout mice and can be ruled out in isolated mitochondria. Thus, our data support the notion that, depending on tissue, model system and pathophysiological status, a combination of mechanisms (e.g., mitochondrial gas pedal and MCU) control OXPHOS substrate supply.
      Notably, the viability of MCU knockout mice (
      • Szibor M.
      • Gizatullina Z.
      • Gainutdinov T.
      • Endres T.
      • Debska-Vielhaber G.
      • Kunz M.
      • Karavasili N.
      • Hallmann K.
      • Schreiber F.
      • Bamberger A.
      • Schwarzer M.
      • Doenst T.
      • Heinze H.-J.
      • Lessmann V.
      • Vielhaber S.
      • et al.
      Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.
      ,
      • Pan X.
      • Liu J.
      • Nguyen T.
      • Liu C.
      • Sun J.
      • Teng Y.
      • Fergusson M.M.
      • Rovira I.I.
      • Allen M.
      • Springer D.A.
      • Aponte A.M.
      • Gucek M.
      • Balaban R.S.
      • Murphy E.
      • Finkel T.
      The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.
      ), albeit living in a laboratory cage, indicates that MCU-dependent pathways are dispensable for a sedentary life. It remains interestingto elucidate, however, why MCU-dependent activation of matrix dehydrogenases is indispensable for high activity states (
      • Wu Y.
      • Rasmussen T.P.
      • Koval O.M.
      • Joiner M.-L.A.
      • Hall D.D.
      • Chen B.
      • Luczak E.D.
      • Wang Q.
      • Rokita A.G.
      • Wehrens X.H.T.
      • Song L.-S.
      • Anderson M.E.
      The mitochondrial uniporter controls fight or flight heart rate increases.
      ) and how this may allow stressful life in the wild.

      References

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        • Chen B.
        • Luczak E.D.
        • Wang Q.
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      Linked Article

      • The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylation
        Journal of Biological ChemistryVol. 295Issue 30
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          Szibor et al. (1) concluded that mitochondrial pyruvate oxidation is regulated primarily by cytosolic Ca2+ ([Ca2+]cyt) activation of the malate-aspartate shuttle, rather than by mitochondrial Ca2+ ([Ca2+]mit) activation of intramitochondrial dehydrogenases. Pyruvate dehydrogenase (PDH) activity largely reflects the ratio of active nonphosphorylated PDH to inactive phosphorylated PDH (PDHP) (2), but Szibor et al. (1) did not measure PDH/PDHP ratios. Moreover, their studies used unphysiological conditions with isolated mitochondria (saturating ADP); with synaptosomes, thymocytes, and fibroblasts (uncoupler and high pyruvate); and with perfused hearts (high pyruvate).
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