Mitochondrial Morphology Regulates Organellar Ca²⁺Uptake and Changes Cellular Ca²⁺Homeostasis

Abstract

Changes in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca 2+ uptake regulation and overall cellular Ca 2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1-DN) markedly increased both mitochondrial Ca 2+ retention capacity and Ca 2+ 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 2 (MFN2) strongly reduced mitochondrial Ca 2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in 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 measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1-DN-promoted fusion resulted in faster cytosolic Ca 2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca 2+ uptake, which impacts on cellular Ca 2+ homeostasis.

Increased glycolysis is an early consequence of palmitate lipotoxicity mediated by redox signaling

“…Indeed, changes in the predominant shape of the mitochondrial network in response to their microenvironment have been associated with modifications in bioenergetic function [ 13 , 20 ]. To study maximized oxidative phosphorylation (known as state 3 respiration) in the presence of specific substrates, cell plasma membranes were permeabilized in order to promote oxidative phosphorylation by adding excess ADP, while preserving cell and mitochondrial architecture [ 25 , 26 ]. Under these conditions, we found no differences in respiration in cells that had undergone 6 h of palmitate lipotoxicity using a combination of respiratory substrates (labelled as “all” in Fig.…”

Metabolic and functional specialisations of the pancreatic beta cell: gene disallowance, mitochondrial metabolism and intercellular connectivity

“…Therefore, ablation of both mitofusins may have a greater deleterious impact on beta cell function and survival rather than targeting and inactivating a single mitofusin gene. Interestingly, in studies from Shirihai and colleagues [ 52 ], promotion of a fragmented phenotype in cardiomyocyte-derived C2C12 cells resulted in a marked reduction in mitochondrial Ca 2+ accumulation, hinting that similar changes may impair the uptake of these ions into mitochondria in beta cells, with consequences for glucose metabolism and insulin secretion. Nonetheless, the role and regulation of mitochondrial fission and fusion factors in the beta cell in diabetes mellitus remain to be fully elucidated.…”

Increased glycolysis is an early outcome of palmitate-mediated lipotoxicity

“…Prior studies in hepatic cells (Bao et al, 2010;Egnatchik et al, 2014;Geng et al, 2020) suggested that palmitate promoted a late decrease in inner mitochondrial membrane potentials and increased oxygen consumption, secondary to calcium efflux from the ER and increased glutamine catabolism (Egnatchik et al, 2014). We should note, however, that while mitochondrial function in intact cells is reliably evaluated by oxygen consumption rates (Brand and Nicholls, 2011), uncalibrated mitochondrial inner membrane potential estimates are much more artifact-prone (Gerencser et al, 2012), particularly when overt changes in morphology occur (Kowaltowski et al, 2002, Kowaltowski et al, 2019. Our experiments here show that, surprisingly, within 1 hour of incubation, palmitate can strongly increase glycolytic flux.…”