In an article published in the FASEB Journal, RIDC Redoxoma researchers led by Alicia Kowaltowski, a professor at Instituto de Química at Universidade de São Paulo (USP), demonstrated that changes in mitochondrial morphology alter mitochondrial calcium uptake and retention properties, changing cellular calcium homeostasis and promoting endoplasmic reticulum stress. They showed that larger mitochondria exhibit faster and larger calcium uptake than fragmented mitochondria.

The primary function of mitochondria is generation of energy in the form of adenosine triphosphate (ATP). But they also perform other crucial activities, including calcium uptake and storage (Ca²⁺, calcium ion). Calcium is fundamental for the body's function. In addition to forming our bones and teeth, it is a central regulator of cellular functions, controlling metabolism in many ways, for example by regulating ATP production, glycogen breakdown, and the glycolytic pathway. Also, calcium is an important intracellular signal in processes such as muscle contraction, cell differentiation, inflammation, among others.

“Our group had already discovered, in the first phase of Redoxoma, that different diets affect calcium transport in animal mitochondria. Calorie-restricted animals have improved calcium transport. On the other hand, studies in the literature show that different caloric inputs change the shape of mitochondria. We were seeing two different things happening in mitochondria, but they had not been interconnected. Our work was to investigate whether the shape of mitochondria affects calcium uptake. And our results show that there is a tight link between mitochondrial shape and calcium homeostasis in mitochondria and cells. We have discovered a new role for mitochondrial morphology in the control of calcium uptake,” Kowaltowski said.

Mitochondria can fuse to produce larger and more elongated organelles and can divide and produce smaller, rounded mitochondria. They not only vary in size and shape in different cell types but also rapidly reshape their morphology in response to environmental changes such as nutrient availability. Increased mitochondrial fusion is often associated with increased bioenergetic efficiency. Mitochondrial fission, on the other hand, is associated with low bioenergetic efficiency. Another interesting aspect, according to the researchers, is that caloric restriction and nutrient deprivation also modulate mitochondrial morphology, stimulating mitochondrial fusion, while nutrient overload is often associated with mitochondrial fission.

Several major biological events involve simultaneous changes in mitochondrial morphology and calcium homeostasis, including immune activation, cell differentiation, insulin secretion, and fatty acid metabolism, among others. According to Kowaltowski, “it is tempting to speculate that at least part of the regulatory mechanisms in these processes involve changes in mitochondrial and cellular calcium homeostasis promoted by the modulation of mitochondrial morphology that we describe in this paper.”

Calcium Homeostasis

Calcium impacts almost every aspect of cell life. Calcium ions are found in a 10,000-fold greater concentrations outside of the cell compared to inside. Eukaryotic cells have created different mechanisms to maintain calcium homeostasis, that is, to maintain intracellular calcium stocks actively over time. In cells, most calcium is sequestered in the endoplasmic reticulum (ER) and mitochondria. The ER captures calcium with more affinity than mitochondria, absorbing ions when concentrations are lower. But mitochondria have greater storage capacity.

In mitochondria, calcium uptake into the mitochondrial matrix occurs through a mitochondrial Ca²⁺ uniporter (MCU) and is driven by the inner mitochondrial membrane potential, which attracts positively charged species. Within mitochondria, calcium ions act as regulators of important metabolic pathways, determining the rate of ATP synthesis. Excessive mitochondrial calcium uptake, however, is disruptive to cellular integrity under several pathological conditions, including stroke, ischemic heart disease, and inflammatory liver conditions.

To study the effects of modulating mitochondrial morphology on calcium uptake, the researchers used the C2C12 myoblasts, which are cells with a functional and dynamic mitochondrial network. In some cells, they inhibited mitofusin 2 (MTF2), a protein needed for mitochondrial fusion, obtaining fragmented mitochondria. In others, they inhibited DRP1, the protein responsible for fission, thus obtaining larger mitochondria. “We inhibit proteins moderately. With this, we obtained changes in mitochondrial shape like those that occur in the real life of cells,” the researcher said. Mitochondrial morphology studies were performed by Kowaltowski using super-resolution confocal microscopy in the laboratory of Professor Orian Shirihai, co-author of the study, at the University of California Los Angeles (UCLA).

The researchers then quantified calcium uptake by mitochondria of different sizes and shapes using fluorescent calcium indicators that do not cross the organelle membrane. With these assays, it was possible to calculate the maximum amount of calcium taken up by mitochondria and calcium uptake rates.

One possible reason for the observed differences in calcium uptake could be related to changes in mitochondrial inner membrane potentials, which are the driving force for calcium uptake. After calibrating the fluorophores used to measure membrane potential according to mitochondrial size, they observed potential changes only in fragmented mitochondria, which does not explain the increase in calcium uptake capacity and rates in larger mitochondria. The results showed that changes in mitochondrial morphology and dynamics are sufficient to alter calcium homeostasis in the organelle.

The researchers also evaluated the implications of mitochondrial morphology modulation on cellular calcium homeostasis. Measuring cytosolic calcium levels in intact cells, they observed that cells in which fusion was impaired had lower cytosolic and ER calcium concentrations.

In these cells, lower basal cellular calcium levels and lower ER calcium stores were accompanied by ER stress, which is related to various pathologies such as diabetes and neurodegenerative diseases. These studies were performed in collaboration with the group of Francisco Laurindo, a professor at the Instituto do Coração at FMUSP and a member of the RIDC Redoxoma, co-author of the article. Cells with fragmented mitochondria also show less ER calcium reuptake.

Mitochondria and the endoplasmic reticulum are physically linked and their interaction is important for calcium signaling and redox signaling. “What we show in this article is that not only is calcium transported from the reticulum to the mitochondria, but the opposite also happens: calcium leaves the mitochondria and repopulates the reticulum. If mitochondria are not capturing calcium well, the reticulum runs out of calcium,” the researcher explained.

The article Mitochondrial Morphology Regulates Organellar Ca²⁺ Uptake and Changes Cellular Ca²⁺ Homeostasis, by Alicia J. Kowaltowski, Sergio L. Menezes-Filho, Essam Assali, Isabela G. Gonçalves, Nathaniel Miller, Patricia Nolasco, Francisco RM Laurindo, Alexandre Bruni Cardoso and Orian Shirihai, can be accessed at https://www.fasebj.org/doi/10.1096/fj.201901136R