Talk:Vilas-Boas 2022 Abstract Bioblast

Comments by the Reviewer

Moreno-Sanchez Rafael (2022-05-28)

I think the abstract is clear and concise, outlining some new data on the effect of Ca²⁺ on respiratory rates in isolated liver mitochondria. However, the abstract seems to require some clarifications and amendments in some minor aspects, to improve the results description, interpretation and relevance.

1. Not studied to date

"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 was, surprisingly, not studied to date."

• This statement is somewhat inaccurate. Some papers have been published on the relationship between Ca²⁺ and respiratory rates or mitochondrial matrix enzyme activities of the energy metabolism in liver mitochondria. For instance see Int J Biochem (1984) 16:477-81; FEBS Lett (1985) 180:259-64; J Biol Chem (1985) 260:4028-34; Biochem J (1985) 23:597-608.

2. Not been determined

"The effects of changes in the affinity of these enzymes on overall mitochondrial metabolic pathway flux have not been determined."

• This statement is inaccurate. The effect of Ca²⁺ on the activity of several mitochondrial matrix NAD-linked dehydrogenases and respiratory rates has been determined by Denton and McCormack, and Hansford, although mainly in heart and brain mitochondria. Why should it be expected to be different in liver mitochondria? The enzymes in liver mitochondria are identical to those expressed in heart or brain. The only expected difference should be in the enzyme V_max and respiratory maximal flux. Subtle differences but essentially very similar effects by Ca²⁺. The authors could disclose what novel effects by Ca²⁺ were considered to undertake their study in liver mitochondria.

3. Ca²⁺ concentrations

• It is recommended to specify the exact Ca²⁺ concentrations used and determined in the different experiments described. Thus, the reader may elucidate whether physiological or supraphysiological Ca²⁺ concentrations were used or were developed, leading to correct interpretations and meaning of results either under physiological or pathological settings.

Version 1

2022-05-28

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 was, surprisingly, not studied to date. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. 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. 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. But moderate Ca2+ within the organelle can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. 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 effects of changes in the affinity of these enzymes on overall mitochondrial metabolic pathway flux 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 and medium 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.