Calcium is the most abundant mineral in the human body and has several biological functions, participating in important physiological and biochemical processes. It is a key regulator of several intracellular signaling pathways and has been implicated in metabolic control and mitochondrial function. In the form of calcium ions (Ca²⁺), it activates mitochondrial respiration by inducing the activity of enzymes involved in oxidative metabolism, thus regulating the generation of adenosine triphosphate (ATP). ATP is the main source of energy for our cells. The issue is that calcium effects on cellular respiration were known overall, but had not yet been determined quantitatively, i.e., nobody had measured them yet.

That was the challenge researcher Eloisa A. Vilas-Boas faced in her post-doctoral project, under the supervision of Professor Alicia Kowaltowski, from the Instituto de Química, Universidade de São Paulo (USP), and a member of the RIDC Redoxoma. “We always write that calcium activates mitochondrial enzymes of the Krebs cycle, impacting the production of ATP. But does it? No one had measured this effect. So what we did was study respiration in isolated mouse liver mitochondria and whole hepatocytes, modulating different calcium levels. We showed that there is an ideal range of calcium concentration in which mitochondrial respiration is activated,” says Vilas-Boas. The results were published in the prestigious Journal of Biological Chemistry (JBC).

Kowaltowski emphasizes that the work is innovative in investigating how determined calcium concentrations alter metabolic fluxes. “If you look at the quantity of something, you have a snapshot of the moment, you don't know how much is being consumed and how much is being produced. In this work, Eloisa is looking at fluxes, and how much of these molecules are being transformed. It really shows how metabolic activity is, quantitatively.”

In addition, the research evaluated cellular respiration under physiological calcium levels, to understand how the metabolism of a normal cell is regulated. “Many times we look for diseases, but there are gaps in the knowledge of what happens in an absolutely normal cell”, said Kowaltowski.

Goldilocks

Calcium affects almost all aspects of cellular life, and its intracellular concentrations are tightly controlled. In cells, most calcium is sequestered in the endoplasmic reticulum and mitochondria. Mitochondria are one of the central regulators of calcium homeostasis, as they can take up, absorb and release calcium ions.

According to the researchers, excessive calcium concentrations in mitochondria are known to lead to a process called mitochondrial permeability transition, in which the mitochondrial membrane loses selectivity, compromising ATP synthesis and leading to cell death. Moderate calcium levels, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting ATP production.

To understand the effects of calcium on mitochondrial respiration more globally, they monitored the effect of different calcium concentrations on the rate of oxygen consumption, comparing isolated mouse liver mitochondria and intact cultured hepatocytes - liver cells. The liver is one of the most important tissues for metabolism.

Vilas-Boas explains that to study isolated mitochondria, they used several substrates and different calcium concentrations. The analysis was carried out using an equipment called Oroboros, a high-resolution respirometer that quantifies oxygen consumption by mitochondria according to time. In intact cells, they used modulators, both a chelator to remove intracellular calcium, which led to a decrease in respiration, and strategies to increase calcium concentration. Intact cells were studied using the Seahorse analyzer, which measures oxygen consumption rates of living cells. “Both devices have high resolution and evaluate similar things. With them, it is possible to measure small variations of oxygen.”

Increased calcium levels in mitochondria of hepatocyte cells: on the left in the control condition, in the center just after the addition of thapsigargin (TG), a drug that increases calcium, and on the right at the final time. – Image: Eloisa Vilas-Boas

The results showed that calcium concentrations greatly impact mitochondrial respiration, with a Goldilocks-like effect, in which both too much and too little calcium limit oxidative phosphorylation, but the ‘just right' concentration promotes significant activation.

In all situations, calcium levels were quantified. “In addition to measuring cytosolic calcium, I did an experiment that showed calcium inside mitochondria. With this, I demonstrated that it really needs to enter mitochondria to cause the effect,” the researcher said.

According to Kowaltowski, the interesting thing was to observe that the cell remains in the ideal range of calcium to optimize respiration. “The cell normally does not maintain maximum production of ATP. It can increase this production and maintain the necessary calcium levels for this.”

The article Goldilocks calcium concentrations and the regulation of oxidative phosphorylation: too much, too little, or just right, by Eloisa A. Vilas-Boas, João Victor Cabral-Costa, Vitor M. Ramos, Camille C. Caldeira da Silva, and Alicia J Kowaltowski, can be accessed here.