Cecatto 2022 EBEC

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Cristiane Cecatto
Cecatto C, Cardoso LHD, Gnaiger E (2022) Mitochondrial calcium uptake capacity is lower than calcium retention capacity in the presence and absence of cyclosporin A. EBEC 2022.

Link: EBEC 2022 Conference website

Cecatto Cristiane, Cardoso Luiza HD, Gnaiger Erich (2022)

Event: EBEC2022 Marseille FR

Mitochondrial calcium homeostasis is involved in the regulation of calcium-dependent cellular processes. Defective calcium homeostasis is a cause of bioenergetic stress, cell death, and diseases, hence arousing growing interest. Mitochondria contribute to the buffering of cytosolic free calcium levels by uptake and release, or storage of osmotically inactive calcium phosphates in the mitochondrial matrix. Frequently, calcium retention capacity CaRC is measured as the amount of calcium titrated up to a threshold, when there is no more calcium uptake and permeability transition triggers the release of calcium, disruption of ion balance, dyscoupling and inhibition of respiration, and leakage into the cytosolic space of several other substances such as cytochrome c. We measured net calcium uptake and respiration simultaneously in mitochondria isolated from mouse liver using the Oroboros O2k High-Resolution Respirometer with Smart Fluo-Sensors and Calcium Green™. The incubation medium was specifically designed to measure calcium uptake and respiration. Calcium Green did not inhibit respiration in a substrate-uncoupler-inhibitor titration protocol with evaluation of LEAK, OXPHOS and ET capacity supported by NADH-linked substrates (N), succinate (S), and the combined NS-pathway [1]. In the LEAK state (absence of ADP), stepwise 5 µM calcium titrations induced a sharp increase of calcium concentration [Ca2+], followed by a gradual decline of [Ca2+] in the medium during intervals of 6 min as a result of mitochondrial net calcium uptake. Contrary to the general assumption, Ca2+ uptake was incomplete, however, as shown by the merely partial decline of [Ca2+] in the medium upon initial and final titrations before the onset of permeability transition. Consequently, the actual calcium uptake capacity CaUC was corrected for incomplete Ca2+ uptake calculated from the fluorometric signals and corresponding increases of [Ca2+] in the medium. Ca2+ uptake capacity CaUC was 45 % of the conventionally defined Ca2+ retention capacity CaRC in controls and 55 % in the presence of cyclosporin A. These results suggest that high Ca2+ retention capacities reported in the literature require critical evaluation and should be replaced by Ca2+ uptake capacities properly corrected for extramitochondrial calcium concentrations measured in the medium.


Bioblast editor: Cecatto C O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit


Affiliations

Cristiane Cecatto1, Luiza H.D. Cardoso1, Erich Gnaiger1
[email protected]
  1. Oroboros Instruments, Innsbruck, Austria


References

  1. Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. https://doi.org/10.26124/bec:2020-0002

List of abbreviations, terms and definitions - MitoPedia

» MitoPedia: Terms and abbreviations


Labels: MiParea: Respiration, Instruments;methods 

Stress:Permeability transition  Organism: Mouse  Tissue;cell: Liver  Preparation: Isolated mitochondria 

Regulation: Calcium  Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k, TIP2k, O2k-Fluorometer, Ca