Mitochondrial morphology regulates organellar Ca²⁺ uptake and changes cellular Ca²⁺ homeostasis

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca²⁺ uptake regulation and overall cellular Ca²⁺ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin-related protein 1 (DRP1)] markedly increased both mitochondrial Ca²⁺ retention capacity and Ca²⁺ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)-2 strongly reduced the mitochondrial Ca²⁺ uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1-DN-promoted fusion resulted in faster cytosolic Ca²⁺ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca²⁺ uptake, which impacts cellular Ca²⁺ homeostasis.-Kowaltowski, A. J., Menezes-Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral-Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni-Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca²⁺ uptake and changes cellular Ca²⁺ homeostasis.