Vilas-Boas 2022 Abstract Bioblast

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P05.
Vilas-Boas Eloísa Aparecida
Vilas-Boas Eloísa Aparecida, Cabral-Costa JV, Ramos VM, Caldeira da Silva CC, Kowaltowski AJ (2022) Energy metabolism regulation by mitochondrial Ca2+ transport in the liver. Bioblast 2022: BEC Inaugural Conference. In: https://doi.org/10.26124/bec:2022-0001

Link: Bioblast 2022: BEC Inaugural Conference

Vilas-Boas Eloisa Aparecida, Cabral-Costa JV, Ramos VM, Caldeira da Silva Camille C, Kowaltowski Alicia J (2022)

Event: Bioblast 2022

Mitochondria are a major hub for Ca2+ handling and energy metabolism, however, the precise relationship between these two processes under physiologically relevant conditions in the liver is still not completely clear. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients [1]. Ca2+ uptake across the mitochondrial inner membrane into the matrix occurs through the mitochondrial Ca2+ uniporter complex (MCU), and Ca2+ efflux occurs through the Na+/Ca2+ exchanger (NCLX). Mitochondria participate in crosstalk signals with the endoplasmic reticulum (ER), through microdomains formed between both organelles in which ions exchange mediates inter-organelle communication [2]. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mtPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture and cell death [3]. But moderate Ca2+ within the organelle can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production [4]. However, direct studies so far are limited to uncovering the modulation of isolated enzyme activity, and only show increases in substrate affinity, not maximal velocity. The specific effects of changes in the affinity of these enzymes on overall oxidative phosphorylation in vivo have not been determined.

We explore this gap here, to determine if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation in the liver. We used isolated mouse liver mitochondria and living AML12 hepatocytes, and measured oxygen consumption under different conditions using Oroboros O2k and Seahorse Extracellular Flux systems, respectively.

We found that isolated mitochondria present increased respiratory control ratios (1-L/P, a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 ± 0.6 µM) and medium (22.0 ± 2.4 µM) Ca2+ concentrations in the presence of NADH-linked substrates pyruvate & malate & α-ketoglutarate, respectively, but not Complex II-linked succinate. We investigated next if the increase in oxidative phosphorylation efficiency observed was dependent on mitochondrial Ca2+ uptake or cycling, using pharmacological inhibitors of the MCU (Ruthenium red, RuRed), which prevents Ca2+ uptake, and of the NCLX (CGP-37157, CGP), which prevents Ca2+ extrusion from mitochondria. Both RuRed and CGP reversed the increase in 1-L/P promoted by Ca2+ in the presence of pyruvate & malate & α-ketoglutarate, indicating that Ca2+ must enter the mitochondrial matrix to exert this effect. Interestingly, in living AML12 hepatocytes, both the decrease of cytosolic Ca2+ by chelation with BAPTA-AM and the increase of cytosolic Ca2+ due to thapsigargin (TG)-induced ER Ca2+ depletion led to decreased respiratory rates. In addition, we show that the decrease of mitochondrial respiration in the presence of high Ca2+ can be modulated by cytosolic Ca2+ chelation, inhibition of Ca2+ entry into the mitochondrial matrix with RuRed and MCU siRNA, or inhibition of mtPTP formation with cyclosporin A.

Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific “just right” concentrations that activate oxidative phosphorylation.

  1. Giorgi C, Marchi S, Pinton P (2018) The machineries, regulation and cellular functions of mitochondrial calcium. https://doi.org/10.1038/s41580-018-0052-8
  2. Csordás G, Weaver D, Hajnóczky G (2018) Endoplasmic reticulum-mitochondrial contactology: structure and signaling functions. https://doi.org/10.1016/j.tcb.2018.02.009
  3. Filadi R, Greotti E (2021) The yin and yang of mitochondrial Ca2+ signaling in cell physiology and pathology. https://doi.org/10.1016/j.ceca.2020.102321
  4. Denton RM (2009) Regulation of mitochondrial dehydrogenases by calcium ions. https://doi.org/10.1016/j.bbabio.2009.01.005

Keywords: calcium transport, mitochondria, electron transfer system, oxidative phosphorylation, metabolic flux

O2k-Network Lab: BR Sao Paulo Kowaltowski AJ


Affiliation

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil - elovilasboas@usp.br

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