Lucie Gerber

The crucian carp (Carassius carassius) is one of the most anoxia-tolerant vertebrates. While physiological underpinnings of its ability to withstand O2 deprivation are well studied, the ability to tolerate the return to normoxia is still enigmatic. Such reoxygenation is associated with detrimental oxidation damage in other organisms, where mitochondria play a central role in the damaging effects. This leads to the question whether mitochondrial adaptations play a central role in the anoxia and reoxygenation tolerance of crucian carp. We here address whether mitochondria from crucian carp circumvent the negative effects of anoxia–reoxygenation exposure, namely the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Crucian carp brain and heart mitochondria generated up to 4-fold less hydrogen peroxide (H2O2; a major ROS) compared with the closely related, anoxia-intolerant, common carp (Cyprinus carpio). The lower H2O2 emission was partly explained by higher (∼15–30%) total oxidant scavenging capacity. Complex II-mediated flux was ∼40% reduced after anoxia–reoxygenation in crucian carp heart mitochondria. Mitochondrial H2O2 generation measured in vivo was unaffected by anoxia–reoxygenation exposure in heart, brain and gill, but reduced by ∼25% in liver. There were also tissue-specific increases in protein carbonylation (∼1.8-fold in brain and gills) and mitochondrial DNA (mtDNA) damage (∼1.5-fold in liver and heart), indicating that biphasic oxidative stress responses affect tissues differently. Our data show that crucian carp avoid excessive mitochondrial ROS generation upon exposure to anoxia–reoxygenation. The tissue-specific distribution of protein and mtDNA oxidation indicate that crucian carp balance body redox signalling to secure resilience during fluctuating O2 availability.

Aim: In most vertebrates, oxygen deprivation and subsequent re‐oxygenation are associated with mitochondrial impairment and excess production of reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂). This in turn triggers a cascade of cell‐damaging events in a temperature‐dependent manner. The crucian carp (Carassius carassius) is one of few vertebrates that survives months without oxygen at cold temperatures and overcomes oxidative damage during re‐oxygenation periods. Mitochondria of this anoxia‐tolerant species therefore serve as an excellent model in translational research to study adaptation and resilience to low oxygen conditions and thermal variability.Methods: Here, we used high‐resolution respirometry on isolated mitochondria from hearts of crucian carp and the anoxia‐intolerant mouse (Mus musculus), at 37 and 8°C; two temperatures relevant for transplantation medicine (i.e., graft preservation and subsequent rewarming).Results: We find: (1) a striking difference in H₂O₂ release between the two species at 37°C despite comparable mitochondrial efficiency and capacity, (2) a massive H₂O₂ release after inhibition of complex V in mouse at 37°C that is absent in crucian carp, and prevented in mouse by incubation at 8°C or uncoupling with a protonophore at 37°C, and (3) indications that differences in mitochondrial complex I and II capacity and thermal sensitivity influence the release of mitochondrial H₂O₂ relative to respiration.Conclusion: Our findings provide comparative insights into a spectrum of mitochondrial adaptations in vertebrates and the importance of thermal variability. Furthermore, the species‐ and temperature‐related changes associated with mitochondria highlighted in this study may help identify mitochondria‐based targets for translational medicine.

We report a profound upregulation of most egln paralog mRNA levels in anoxia and upon reoxygenation, with egln3ii showing the largest, a 17-fold increase, and highest relative mRNA abundance. The relative abundance of prolyl hydroxylase domain (PHD) proteins was maintained during anoxia and increased at reoxygenation. PHD immunoreactivity was localized to axonal branches with region-specific changes during anoxia-reoxygenation. These dynamic and regional changes in crucian carp, champion of anoxia tolerance, are most likely adaptive and call for further mechanistic studies.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to −2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown if the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts ( Locusta migratoria ) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

The dynamic interactions between nitric oxide (NO) and myoglobin (Mb) in the cardiovascular system have received considerable attention. The loss of Mb, the principal O₂ carrier and a NO scavenger/producer, in the heart of some red-blooded fishes provides a unique opportunity for assessing this globin’s role in NO homeostasis and mitochondrial function. We measured Mb content, activities of enzymes of NO and aerobic metabolism [NO Synthase (NOS) and citrate synthase, respectively] and mitochondrial parameters [Complex-I and -I+II respiration, coupling efficiency, reactive oxygen species production/release rates and mitochondrial sensitivity to inhibition by NO (i.e., NO IC₅₀)] in the heart of three species of red-blooded fish. The expression of Mb correlated positively with NOS activity and NO IC₅₀, with low NOS activity and a reduced NO IC₅₀ in the Mb-lacking lumpfish (Cyclopterus lumpus) as compared to the Mb-expressing Atlantic salmon (Salmo salar) and short-horned sculpin (Myoxocephalus scorpius). Collectively, our data show that NO levels are fine-tuned so that NO homeostasis and mitochondrial function are preserved; indicate that compensatory mechanisms are in place to tightly regulate [NO] and mitochondrial function in a species without Mb; and strongly suggest that the NO IC₅₀ for oxidative phosphorylation is closely related to a fish’s hypoxia tolerance.

Mitochondrial function can provide key insights into how fish will respond to climate change, due to its important role in heart performance, energy metabolism and oxidative stress. However, whether warm acclimation can maintain or improve the energetic status of the fish heart when exposed to short-term heat stress is not well understood. We acclimated Atlantic salmon, a highly aerobic eurythermal species, to 12 and 20 °C, then measured cardiac mitochondrial functionality and integrity at 20 °C and at 24, 26 and 28 °C (this species’ critical thermal maximum ± 2 °C). Acclimation to 20 °C vs. 12 °C enhanced many aspects of mitochondrial respiratory capacity and efficiency up to 24 °C, and preserved outer mitochondrial membrane integrity up to 26 °C. Further, reactive oxygen species (ROS) production was dramatically decreased at all temperatures. These data suggest that salmon acclimated to ‘normal’ maximum summer temperatures are capable of surviving all but the most extreme ocean heat waves, and that there is no ‘tradeoff’ in heart mitochondrial function when Atlantic salmon are acclimated to high temperatures (i.e., increased oxidative phosphorylation does not result in heightened ROS production). This study suggests that fish species may show quite different acclimatory responses when exposed to prolonged high temperatures, and thus, susceptibility to climate warming.

In fishes, the capacity of thermal acclimation to preserve cardiac mitochondrial function under future warming scenarios is important to understand given the central role that cardiac energy metabolism and performance play in this taxa9s thermal tolerance. We acclimated Atlantic salmon to 12 and 20°C (for>2 months), and investigated the effects of acute and chronic warming on cardiac mitochondrial respiration and ROS production (release rate) using high-resolution fluorespirometry. Further, we compared the sensitivity of mitochondrial respiration to nitric oxide (i.e., the NO IC₅₀), and assessed the mitochondrial response to anoxia-reoxygenation (AR). Acute exposure to 20°C increased maximal mitochondrial respiration by ∼55%, however, the mitochondria9s complex I-respiratory control ratio was 17% lower and ROS production was increased by≥60%. Acclimation to 20°C: i) preserved mitochondrial coupling and aerobic capacity; ii) decreased the mitochondria9s ROS production by ∼30%; iii) increased the mitochondria9s NO IC₅₀ by ∼23%; iv) and improved mitochondrial membrane integrity at 20°C. AR did not affect mitochondrial function at 12°C, but acute exposure to 20°C and AR depressed maximal mitochondrial respiration (by ∼9%) and coupling (by ∼16%) without impacting ROS production. Finally, warm acclimation did not improve the capacity of mitochondria to recover from AR, indicating that there was no ‘cross tolerance’ between these challenges. Our findings provide compelling evidence that thermal plasticity of cardiac mitochondrial function contributes to the Atlantic salmon9s capability to survive at≥20°C for prolonged periods, but question whether this plasticity may allow them to withstand high temperatures when combined with other stressors.