Re-oxygenation resilience - the overlooked element of anoxic survival

It is well known that the crucian carp (Carassius carassius) has a unique ability to survive without oxygen. This ability is used by the fish during the winter, when the small lakes they live in gets covered by a thick layer of ice that limits the entry of oxygen from the air to the water, and the formation of oxygen from plants. Crucian carp balances its energy needs to the amount of energy it can produce, and has a molecular adaptation that allows it to convert lactic acid into alcohol, which it can get rid of over the gills. Therefore, it can survive longer by using anaerobic metabolism, without accumulating lactic acid that would otherwise acidify the blood. Total lack of oxygen can still be expected to disturb the equilibrium of the mitochondria (the organelles responsible for respiration and the formation of energy in the form of ATP), and this disturbance can lead to the cell emitting a "death signal", which causes the cell itself to die. As the crucian carp survives repeated periods of anoxia and subsequent re-oxygenation, it must have properties that make it possible to repair and perhaps even limit the extent of damage, which makes it special compared to most other animals. When you get a blood clot in your brain or heart, diseases that affects many people every year, a lack of blood supply leads to a lack of oxygen, and when the blood supply is restored, it leads to long-term damage to the tissue. In this project, we wanted to investigate which processes contribute to the crucian carp?s ability to survive especially re-oxygenation, something that has been less in focus. The knowledge we can gain from studies on the crucian carp can expand our understanding of the physiological mechanisms, an understanding that can even be useful in biomedical research. In the project, we have developed a protocol to examine the nucleotide sequences (mRNA) that will be translated into protein in the cells in relation to how much of the mRNA is present at a given time for a gene. We have identified several genes that may be of interest for further research. For example, there is a sharp increase in genes encoding proteins that regulate the activity of HIF (hypoxia inducible factor), a factor that is usually activated when there is no oxygen. But the activity of HIF is not necessarily an advantage if there is no oxygen to take up from the water at all, and this may be the explanation why the crucian carp is trying to regulate the activity of HIF. We have also observed that gene expression of molecules that bind oxygen, such as myoglobin, is reduced in the brain. This is something that needs to be investigated further, but one can speculate that it is beneficial to reduce the amount of myoglobin to avoid a too rapid increase in oxygen in the cells, which can lead to the formation of reactive oxygen species (ROS). The results show that in addition to specific genes being switched on and off, and translated into protein, the dynamics between gene expression and further translation into protein during anoxia and reoxygenation itself can change, and thus have a mechanistic role for the crucian carp's ability to survive anoxia and reoxygenation. Using mass spectrometry, we have measured metabolites involved in energy metabolism, such as ATP, succinate and lactate in several tissues, and how the levels are affected by anoxia and reoxygenation. The results show that there is a big difference in how fast changes occur, especially in reoxygenation, where the heart seems to return more slowly to normal levels compared to the brain. In addition, it seems that succinate, which inside the mitochondria is the molecule that can give rise to the formation of ROS, is transported from different tissues and into the blood. We speculate that this indicates that succinate is transported, for example, to the liver, which then metabolizes it, or possibly that some of the succinate is transported to the gills and diffuses into the water. Both can be a mechanism to protect more sensitive and critical organs such as the brain and heart. In addition to a possible active handling of succinate as protection against the formation of ROS, measurements on isolated mitochondria have shown that the crucian carp, compared to the closely related common carp (Cyprinus carpio), forms less ROS per oxygen molecule used. This observation is also supported by the fact that ROS does not accumulate to a great extent and there is no increase in oxidative damage in the tissues to any particular degree during reoxygenation. Overall, the project has shown that the crucian carp may have developed a number of specific and hitherto unknown mechanisms and adaptations for dealing with reoxygenation. By studying these mechanisms in more detail, we can gain knowledge that is not only important for the biological understanding of anoxia tolerance, but also provide ideas that can be used in interdisciplinary projects in biomedical research.

The information obtained in this project has furthered our understanding of one of the important models in comparative physiology - one of the few vertebrate animals that can survive without oxygen. The project has shown the power of using a multi-omics approach to discover important mechanisms that one cannot extrapolate from the pathological conditions induced by anoxia in mammals. The sequencing data will serve as a resource also for other researchers and for future work with the genome and evolutionary questions. The findings are particularly useful for further developing cross-disciplinary projects in collaboration with biomedical researchers, due to the role that lack of oxygen and reoxygenation plays in some diseases. The data will continue to inspire new questions, as further details are uncovered and followed up upon.

The ability of the crucian carp (Carassius carassius) to survive anoxia for several months makes this animal a unique model. This anoxia tolerance is gained by matching ATP demand with ATP production. Nevertheless, without oxygen, the respiratory chain may be at a full stop, resulting in mitochondrial depolarization - a death signal in other animals. Moreover, it is often overlooked that when oxygen is restored, free oxygen radicals are bound to be produced, damaging mitochondria and DNA. Indeed, preliminary data reveal an increased occurrence of apoptotic cells in the crucian carp brain after re-oxygenation, but not in anoxia. Thus, the crucian carp must posses effective repair mechanisms, as it evidently survives many years of repeated re-oxygenation episodes in nature. This makes crucian carp interesting even from a biomedical perspective, as reperfusion after the ischemia associated with stroke and heart attack can be as detrimental as the ischemia itself. Furthermore, recent analyses reveals that a large proportion of the crucian carp brain transcriptome is differentially regulated in anoxia and re-oxygenation, revealing very active molecular responses rather than shutting down and waiting for 'better times'. At the same time, post-transcriptional mechanisms may be at work to modulate or even dampen the transcriptional response. Using an array of methods, from real-time quantitative PCR and western blotting, to the recently developed ribosome footprint profiling, I will examine and identify the pathways that are keys to the re-oxygenation response. I will look at the damage of the brain in more detail, to determine if the processes are restricted to certain areas, and also look at different stages of recovery. Lastly, to be able to quantify the functional consequences of anoxia/re-oxygenation damage, I will investigate if increased cell death also occurs in the heart, another sensitive organ, and measure its effects on cardiac performance.