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CELL FEATURE: PROBLEMS IN THE POWERHOUSE
PROBLEMS IN THE POWERHOUSE
BY ALEX EVANS
Energy is often called the fuel of life, responsible for driving biological growth, reproduction and survival in the face of a changing world. Almost all eukaryotic organisms rely on mitochondria to generate this energy and yet mitochondrial activity can be heavily affected by environmental factors – both in wild organisms and in laboratory cultures. Plenty of research is taking place from the cellular level and upwards to investigate mitochondrial function. Let’s hear from some of those researchers.
SOME (DON’T) LIKE IT HOT
Temperature is a major environmental factor at play for all life on Earth, and is currently on the rise worldwide. Nicolas Pichaud, Associate Professor in the Department of Chemistry and Biochemistry at the University of Moncton in Canada, is working to understand how mitochondria respond to environmental stress, including temperature, dietary resources and oxygen availability. Nicolas’s career started at the opposite end of the biological scale, looking at population ecology and whole ecosystems, before he worked his way inwards towards the cell. ‘During my MSc in oceanography, I became really intrigued by how organisms can live and survive in changing environmental conditions,’ he explains. ‘This led me to do my PhD on Drosophila, trying to link variations of the mitochondrial genotype in related populations to the mitochondrial functions at different temperatures to see which population had adapted to better deal with temperature than the other.’
Nicolas points out that as well as producing ATP, mitochondria are responsible for many other processes and there are enzymatic complexes within the electron transport chain that tend to get completely overlooked. ‘We found that the capacity of these complexes is usually increased to compensate for a problem in one of the “classical” complexes induced by a highfat diet,1 an incompatibility between nuclear and mitochondrial genomes2 or at high temperatures,3’
he explains. ‘So instead of looking at the classical mitochondrial complexes, it is important to look at these alternative complexes to really understand what’s going on and how they can help maintain (or not) cellular homeostasis when needed.’
As with many researchers interested in mitochondrial function within the cell, Nicolas measures the oxygen consumed by the organelle with a high-resolution respirometer, but first the mitochondria has to be extracted. ‘When I started my PhD, I had to isolate mitochondria from Drosophila thorax to measure mitochondrial respiration, which was a pain,’ he says. ‘At that time, I was dissecting 90–120 Drosophila of the same age and sex to barely have enough mitochondria to run my experiment!’4 Thankfully, Nicolas developed an improved method by permeabilising tissue with a high-resolution respirometer that allowed him to run the experiment with only three thoraxes.
Metabolic demand rises as temperatures go up and the rate of mitochondrial aerobic respiration must keep pace – or else. ‘It has been suggested that mitochondrial dysfunction may explain organismal failure at critically high temperatures and thus mitochondria have been under scrutiny in the past few years as an important keystone explaining thermal limits of ectotherms,’ explains Nicolas. ‘With Lisa Bjerregaard Jørgensen and Johannes Overgaard, we recently showed in a comparative model of Drosophila with different critical temperature maximums (CTmax) that alternative complex respiration is decreased at temperatures close to CTmax and that above CTmax, an increased capacity to oxidise succinate and glycerol-3-phosphate is observed.’
Interestingly, this adaptation was not unique to the mitochondria of Drosophila, as was found by Nicolas and his team. ‘We then showed that this switch of substrate oxidation is partially conserved in other insect species such as the honeybee (Apis mellifera) and the Colorado potato beetle (Leptinotarsa decemlineata),’ he says.5 ‘Altogether, these results suggest that mitochondrial substrate oxidation capacity is important for the thermal physiology of insects and might be involved in specific metabolic processes involved in temperature adaptation.‘
Further exploring how these alternative complexes are used in other species, Nicolas expands on some recent and surprising findings. ‘We actually thought that winter honeybees had an increased complex I capacity to sustain shivering thermogenesis and that their immune system would be decreased because there are fewer risks of infection during winter,’ he says. ‘We actually demonstrated that it is the complete opposite and I would like to check how the different things we saw in summer versus winter honeybees are regulated in terms of signalisation and if temperature is the only factor at play here!’6
STRESS UPON STRESS
Individually, high temperature and hypoxia may be a challenge for mitochondria of some species, but when combined, the interactions become more complex and potentially more catastrophic. Inna Sokolova, Professor of Marine Biology at the University of Rostock in Germany, leads a laboratory that is focused on understanding the impact of multiple environmental stressors on marine organisms. ‘We want to determine the physiological mechanisms that set limits to organisms’ tolerance under the multiple stressor pressure and find ways to mechanistically link changes at the lower levels of biological organisation to the fitness consequences at the whole organism level,’ she says.
‘I was studying the impacts of extremely low salinity on molluscs and realised that extremely low salinity was not so much an osmotic problem, but an oxygen problem for the molluscs,’ she explains. ‘I knew that in terrestrial mammals, depriving a tissue of oxygen and then re-oxygenating it again as might happen during recovery from stroke or heart attack is extremely damaging, and this damage is associated with the mitochondrial damage and malfunction.’
Above Nicholas Pichaud with honeybees Photo credit: Nicolas Pichaud
However, there are organisms that are able to go through these hypoxia–reoxygenation cycles twice a day without any apparent ill effects, raising the question of how the mitochondria are able to function this way – especially at the wide range of temperatures experienced by intertidal species. ‘Molluscs are a very successful group in many environments and their metabolic plasticity, and the ability of their mitochondria to withstand and recover from environmental stressors, contributes to this success,’ she explains. ‘Furthermore, understanding how the mitochondria of the tolerant species withstand these stressors might allow us to find interventions to help less tolerant mitochondria (such as ours) cope with stress and perhaps limit tissue damage during pathologies such as ischaemia–reperfusion.’
As well as providing answers to long-standing questions about mitochondria, this line of research has also been able to produce some more unexpected results. ‘The molluscan mitochondria are full of surprises,’ says Inna. ‘One very interesting thing that we found in our studies is that, unlike the mitochondria of terrestrial mammals that become damaged and lose the respiratory and ATP synthesis capacity after even short-term hypoxic stress, molluscan mitochondria can withstand several bouts of hypoxia–reoxygenation without loss of function and even enhance their oxygen consumption after hypoxic stress.’
Inna’s proteomic work with molluscan mitochondria also made another unexpected discovery that may help to explain how these animals resist the dangers of reactive oxygen species (ROS) generation. ‘Tolerant mitochondria do not seem to upregulate antioxidants during hypoxia–reoxygenation stress to protect against the excessive ROS during reoxygenation,’ she says. ‘Instead, they upregulate the mitochondrial quality control mechanisms that degrade damaged mitochondrial proteins and remodel the metabolic pathways, which might help the tolerant mitochondria keep ROS production under the tight control.’
The next steps for Inna’s team lie in assessing the role of post-translational modifications of proteins, such as phosphorylation and glycation, in fine-tuning the mitochondrial metabolism during environmental stress. ‘We also want to explore how the mitochondrial metabolism and anaerobic glycolysis are coordinated during hypoxia and post-hypoxic recovery.’
Inna graciously concludes the interview by acknowledging the hard work of her colleagues. ‘I would like to thank my graduate students and postdocs, without whom none of this work would have been possible and who are a constant source of motivation and inspiration to me,’ she says. ‘A lot of my best ideas come from our discussions of their data, interesting literature finds and ideas, and their energy and enthusiasm are contagious – I feel privileged and grateful for being able to serve as their mentor.’
NO OXYGEN, NO WORRIES?
As well as temperature, the availability of oxygen is a major environmental factor in physiological stress at the cellular level. Amanda Bundgård, a postdoctoral researcher at CECAD, Cologne University in Germany, and Section for Zoophysiology, Department of Biology, Aarhus University in Denmark, is researching how mitochondria have adapted to hypoxic conditions in animals such as freshwater turtles and naked-mole rats. ‘I’ve always been interested in physiology and biochemical adaptations,’ says Amanda. ‘The reason I studied biology in the first place was a newspaper article about haemoglobin in high-flying geese, and how a few mutations in the gene have ensured that the geese can take up enough oxygen to fly over the Himalayas.’
‘When I was looking for a master’s project, I came across this mystery of how hypoxia-tolerant turtles avoid oxidative damage after the hypoxia and anoxia they encounter when they overwinter,’ she says. ‘Mitochondria, as the main cellular oxygen consumers and producers of ROS that cause oxidative damage, are naturally really central to hypoxia tolerance, so I was lucky enough to get to do first a master’s project and then a PhD about the role of mitochondria in the anoxia tolerance of turtles.’
‘Mitochondria are important in almost all aspects of animal physiology,’ says Amanda. ‘They produce most of the cell’s ATP, and so any changes that affect mitochondrial ATP production are central to the rest of the cell and thereby the whole organism and the ability of an animal to adapt to their environment.’ ‘My research has shown that hypoxia-tolerant animals such as turtles are able to maintain mitochondrial homeostasis even without oxygen,’ she says. ‘In hypoxia-intolerant mammals, such as mice and humans, oxygen deprivation causes all kinds of mitochondrial disruptions, including reversal of ATP synthase, loss of ATP, loss of membrane potential and induction of apoptosis and production of ROS, which cause oxidative damage upon the return of oxygen.’ However, turtles appear not to suffer from these issues and Amanda’s research is helping us to understand why. ‘We’ve been able to show that the turtle mitochondria don’t produce excess ROS upon reoxygenation after anoxia because they are able to maintain ATP and avoid excessive accumulation of succinate,’ she explains.
Amanda is no stranger to technology, relying on a wide range of interesting techniques that assess how changes in oxygen can affect mitochondrial function all the way down to the protein level. ‘This includes exposing whole animals, isolated organs or just tissue to hypoxia or anoxia and then assessing the effect on mitochondrial function with high-resolution respirometry, fluorometric and mass-spectrometric measurement of ROS production, microscopy and histochemistry of tissues, and biochemical analysis of proteins and oxidative status,’ she says. ‘I’m also using enzyme assays to assess effects on protein activity and mass-spectrometric metabolomics to assess how oxygen deprivation affects the metabolic pathways.’
‘To me, it’s surprising that turtles completely avoid excess ROS production in their hearts after anoxia,’ she explains.7 ‘We know from mammals like humans, mice and rats that their hearts are very damaged with reoxygenation after anoxia/ischaemia and that much of this damage comes from excess production of ROS in the mitochondria.’ This kind of issue is of particular significance in the fields of heart disease treatment and organ transplantation. When it comes to future avenues of research, Amanda relishes
the opportunities opening up ahead of her. ‘I’m still intrigued by the difference in stability of mitochondrial supercomplexes across animal species that I’ve found, and I’d like to investigate that in more detail and whether it might be linked to fatty acid composition of mitochondrial membranes,’ she concludes. ‘There’s a recent report that supercomplex interactions change with hibernation status in ground squirrels, which I think is really cool, but the function of these interactions is still not really clear.’8
THE CULTURE CLUB
While many researchers with an interest in mitochondrial function set out to investigate the effects of a changing world by experimenting with laboratory-cultured mitochondria, there are some that are committed to making sure those laboratory cultures are as robust and reliable as they can be. Jeff Stuart, Professor in the Department of Biological Sciences at Brock University in Canada, is one such researcher working towards better technology and protocols for understanding mitochondria.
Jeff’s initial interest in mitochondria stems from his PhD studying metabolic rate suppression in snails. ‘This ultimately meant understanding how mitochondria work and how their activity can be regulated,’ he explains. ‘This interest was later developed when I studied the functions of uncoupling proteins as a postdoctoral fellow’. Since then, Jeff has focused on developing new computational methods to recognise and quantify aspects of mitochondrial network morphology. ‘Within the past several years, I have also spent considerable time developing improved cell culture methods that preserve more “normal” mitochondrial functions in vitro,’ he says. ‘Thus, my role at this point in my career has become providing tools, equipment and protocols that improve the study of mitochondria in live cultured cells.
One of Jeff’s main focuses is on the improvement of maintaining physioxia, or physiologically realistic oxygen partial pressures, in cell culture. ‘Since human cells were first routinely cultured almost seven decades ago, there has been little attention focused on the regulation of oxygen levels,’ explains Jeff. ‘This has occurred despite our longstanding awareness that cells in vivo experience the equivalent of 2–6% oxygen in most tissues.’
The impact of incorrectly prepared and maintained mitochondrial cultures can have serious consequences on both fundamental research and healthcare applications, as Jeff explains. ‘Our published studies over the past several years, as well as some yet-to-be-published work, show how non-physiologically elevated oxygen levels in culture affect gene expression, cellular energy
metabolism and mitochondrial network morphology,’ he says. ‘We have helped to show that the failure to maintain physioxia in cell culture alters the effects of drugs, hormones, toxins and perhaps almost any cellular perturbation – the consequences are considerable and wide-ranging.’
Jeff and his team have also increasingly incorporated ‘omics’ approaches to help them identify new processes that might be affected by culture conditions, including oxygen saturation. ‘We have been using transcriptomics, proteomics, metabolomics and recently lipidomics to this end,’ he says. ‘These combine with our more familiar use of Seahorse extracellular flux assays to assess metabolism and live cell confocal fluorescence imaging to understand mitochondrial form and function.’
Alongside his drive to improve laboratory cultures for mitochondrial research, Jeff’s most recent research direction takes me somewhat by surprise. ‘We have been using the game design software Unity to create simulations of mitochondrial dynamics and are almost at a point where we have a usable program,’ he says. ‘I am excited to explore this space in the future, but we also have lots of plans for improving the culture workflow of our cell physiology experiments in ways that we think will be useful for other laboratories as well.’
Reference:
1. Cormier RP, Champigny CM, Simard CJ, et al. Dynamic mitochondrial responses to a high-fat diet in Drosophila melanogaster. Sci Rep 2019; 9: 4531. 2. Pichaud N, Bérubé R, Côté G, et al. Age dependent dysfunction of mitochondrial and ROS metabolism induced by mitonuclear mismatch. Front Genet 2019; 10: 130. 3. Jørgensen LB, Overgaard J, Hunter-Manseau F, et al. Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila. J Exp Bio 2021; 224: jeb240960. 4. Pichaud N, Chatelain EH, Ballard JWO, et al. Thermal sensitivity of mitochondrial metabolism in two distinct mitotypes of Drosophila simulans: evaluation of mitochondrial plasticity. J Exp Biol 2010; 213: 1665–1675. 5. Menail HA, Cormier SB, Ben Youssef M, et al. Flexible thermal sensitivity of mitochondrial oxygen consumption and substrate oxidation in flying insect species. Front Physiol 2022; 13: 897174. 6. Cormier SB, Léger A, Boudreau LH, et al. Overwintering in
North American domesticated honeybees (Apis mellifera) causes mitochondrial reprogramming while enhancing cellular immunity. J Exp Biol 2022; 225: jeb244440. 7. Bundgaard A, James AM, Joyce W, et al. Suppression of reactive oxygen species generation in heart mitochondria from anoxic turtles: the role of complex I S-nitrosation. J Exp
Biol 2018; 221: jeb174391. 8. Hutchinson AJ, Duffy BM, Staples JF. Hibernation is super complex: distribution, dynamics, and stability of electron transport system supercomplexes in Ictidomys tridecemlineatus. Am J Physiol Regul Integr Comp Physiol 2022; 323: R28–R42.
Above Screenshot of Jeff Stuart’s ‘Mitochondrio’ mitochondrial dynamics simulation game built using the Unity game engine Photo credit Jeff Stuart
Top Left A transmission electron microscope image of mitochondria from turtle hearts Photo credit Amanda Bundgård
