Mark Adam Scott

Mitochondrial survival without oxygen

Faculty of Mathematics and Natural Sciences University of Oslo 2017

Mitochondrial survival without oxygen

By

Mark Adam Scott

Thesis presented for the degree of

PHILOSOPHIAE DOCTOR

Department of Biosciences

Faculty of Mathematics and Natural Sciences

University of Oslo

2017

© Mark Adam Scott, 2018

Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 1949

ISSN 1501-7710

All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

Acknowledgements

I would like to begin by expressing my gratitude to Göran Nilsson, Kåre-Olav Stensløkken, the Research Council of Norway, the European Research Council, and the University of Oslo for creating this opportunity for me. I am fortunate to have had supervisors that allowed me the freedom to explore the techniques that I was interested in developing. I am also grateful that my PhD enabled me not just to achieve the academic goals that I had set for myself but several other goals as well. Chief among them, being able to see as much of Europe as I’d hoped to.

I was fortunate to have a talented and supportive group of colleagues and office mates throughout my PhD. Thank you Sjannie, Christina, Marco, Anette, and especially Cathrine, thank you for teaching me everything I needed to know about the entire process of qPCR, from cloning through to analysis. Antje, thank you for welcoming me into the world of electron microscopy. Steinar, thank you for solving many of the logistical issues I had over the years. Haaken, thank you for saving me countless hours of animal husbandry.

I arrived in Norway to start my PhD without knowing anyone and I would like to express my appreciation to a few wonderful people I met over the years. Thank you Jon for taking me along on all the cabin trips and introducing me to your friends. It meant a lot to me. Ivan, it has been a privilege. Nacho, it’s a shame we didn’t meet sooner. Knut, thanks for the adventures. Siri & May-Kristin, you brightened every day. Andreas, still 4.9%. Danny, sorry for Cards Against Humanity. Lench, thanks for the Shepherd’s pie. And a heartfelt thank you to my Italian sis, I would not have managed it without your support.

Lastly, I would like to acknowledge my family and friends for their support throughout the writing process. I appreciated every home-cooked meal and the distractions of pub quizzes, beer pong, Catan, and a whole lot of pho.

Table of Contents

Abstract ------1

List of papers ------5

Abbreviations ------6

Introduction ------8 General introduction ------8 Model organisms ------9 Mitochondria ------10 Membrane potential ------12 Ultrastructure ------13 Metabolomics ------15 Anoxia ------18 Membrane potential ------19 Ultrastructure ------21 Metabolomics ------23 Concluding paragraph ------26

Aims ------28

Methods ------30 Animals ------30 Isolation of ventricular myocytes ------30 Fixed cell IA experiments ------31 Live-cell TMRM experiments ------31 Fluorescence Microscopy ------32 Anoxia exposure and tissue sampling ------32 Enzyme activities ------33 Electron microscopy ------33 Obtaining sequences for ATP synthase ------35 qPCR ------36 Metabolomics ------38

Summary of Results ------40 Paper I ------40 Paper II ------43 Paper III ------45

General Discussion ------47 Maintenance of mitochondrial membrane potential ------47 Reducing ATP demand and increasing ATP supply in anoxia ------50 Mitochondrial fusion and ultrastructure ------53 Conclusion ------56

i

Literature Cited ------57

Papers I-III

ii

Abstract

Heart research is increasingly important from both a basic and medical physiology stand point. The heart is an energetically demanding tissue and heart disease accounts for a high proportion of human disease- related mortality. Blood vessels can become clogged, oxygen and nutrients diverted, and suddenly the cells of the heart are unable to meet the energetic requirements necessary to function. Under normal conditions mitochondria are the main site of cellular energy supply, but during oxygen deprivation they switch to being a major site for cellular energy consumption. A comprehensive understanding of basic mitochondrial pathophysiology is a crucial component of addressing the current heart disease pandemic.

Once deprived of oxygen, the human heart can only function for minutes before a person succumbs to the trauma. The crucian carp, however, can maintain normal cardiac function in anoxia for days to months depending on the temperature. Research into the adaptations of the crucian carp heart is limited and investigations into crucian carp heart mitochondria have been nonexistent. It was not known how the crucian carp heart mitochondria maintained energy supply without oxidative phosphorylation nor how the mitochondria averted lethal energy consumption as is observed within minutes in humans. The following experiments were undertaken to address these two critical components of mitochondrial pathophysiology.

The experiments presented in Paper I investigate how mitochondrial membrane potential (ΔΨM) is affected by blockers of the electron transport chain (ETC). The maintenance of ΔΨM is essential to cellular survival and a loss of ΔΨM characterises an irreversible cascade towards cell death. It was not known what happens to crucian carp ΔΨM following exposure to simulated anoxia. Isolated cardiomyocytes from the anoxia-tolerant crucian carp and anoxia-intolerant brown trout were loaded with the mitochondrial fluorescent stain tetramethylrhodamine methyl ester perchlorate (TMRM) and exposed to rotenone, antimycin, cyanide, and oligomycin. Crucian carp cardiomyocytes were found to maintain ΔΨM for much longer than trout when Complex IV of the ETC was blocked with cyanide

(simulating anoxia). Additionally, inhibition of Complex V (ATP synthase) accelerated the loss of ΔΨM, + suggesting that this enzyme is acting in reverse, as an ATP consuming H pump to preserve ΔΨM during the process of acclimating to anoxia. Inhibition of complexes I and III resulted in a steady depolarization of mitochondria over 32h. When all proton transporting complexes of the ETC were inhibited the crucian

1 carp and trout mitochondria depolarized with nearly identical profiles, suggesting that the mechanisms in place for the maintenance of ΔΨM in the crucian carp mitochondria are largely associated with the ETC. Additionally, exposure of cardiomyocytes to the protonophore CCCP revealed that crucian carp cardiomyocytes were able to resist depolarization to a greater extent than the anoxia-intolerant trout. Taken together, these findings demonstrate the inherent adaptations of crucian carp mitochondria to maintain ΔΨM in anoxia and allude to a partially functioning ETC during anoxia.

In paper II electron microscopy was used to evaluate the effect of anoxia acclimation on mitochondrial volume and number in crucian carp heart and red muscle. The effects of anoxia acclimation on mitochondrial ultrastructure were not previously known. Mitochondrial volume increased in the red muscle during anoxia but anoxia had no effect on the number of mitochondria in the heart or red muscle. Mitofusin (MFN) gene expression was measured and red muscle MFN2 increased nearly fivefold in anoxia, suggesting that mitochondrial fusion may be occurring in red muscle. Furthermore, citrate synthase (CS) and cytochrome c oxidase (COX) enzyme activity was measured in heart and red muscle from anoxia-tolerant crucian carp and anoxia-intolerant brown trout. Red muscle CS and COX decreased and heart COX increased in crucian carp acclimated to anoxia for six days. The ratio of COX:CS reveals that the oxidative capacity of the crucian carp heart acclimated to anoxia is similar to that of the normoxic trout heart. The observed tissue-specific differences in oxidative capacity suggest a possible means of increasing the likelihood that scavenged oxygen is diverted to the heart rather than consumed by the red muscle. Additionally, enzyme activity assays and quantitative PCR were used to see if upon acclimation to anoxia crucian carp inhibition of ATP synthase occurs as has been observed in models of hypoxia tolerance. Inhibition of ATP synthase reversal is believed to occur in order to prevent depletion of limited anaerobic ATP supply but the underlying mechanism of ATP synthase inhibition in anoxia is not well-established. Intriguingly, both the mean reduction in ATP synthase gene expression and enzyme activity were approximately threefold. This suggests that a reduction in ATP synthase subunit gene expression is responsible for the reduced ATP synthase activity following acclimation to anoxia. More work is needed to be done to verify that mitochondrial fusion is occurring in the crucian carp red muscle. But since the fusion of damaged and healthy mitochondria is energetically less expensive than producing new mitochondria, perhaps elevated rates of mitochondrial fusion as an adaption to anoxia would not be that surprising, especially in the crucian carp.

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Very little is known about the metabolic pathways involved in mitochondrial anoxia tolerance. Paper III profiles the metabolome of the anoxia-tolerant crucian carp heart in normoxia and anoxia. The substrates of glycolysis in anoxia accumulated upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPD) and depleted downstream. This suggests an inhibition of GAPD activity, which is consistent with others’ findings in the turtle and carp brain. It is likely that this occurs in order to ration the substrates necessary for providing ATP supply but it may also be occurring to increase the levels of glycerol 3- phosphate (G3P), which increased in anoxia by a factor of 44. G3P is the electron donor in the glycerol phosphate shuttle (GPS) and when oxidized is reported to produce less reactive oxygen species (ROS) than succinate, making it beneficial to have accumulated G3P before reoxygenation. The metabolites of the malate aspartate shuttle (MAS), the primary means of transporting reducing equivalents of glycolysis to the electron transport chain, are depleted in anoxia. Alpha-ketoglutarate is reduced to below detectable levels and aspartate is likely siphoned off to the purine nucleotide cycle in order to produce fumarate. The production of fumarate in anoxia is important because in the absence of oxygen fumarate is likely serving as a terminal electron acceptor of the electron transport chain (ETC). Evidence in support of this is that all measured intermediates of the citric acid cycle decreased significantly in anoxia with the exception of succinate, which is reduced from fumarate and increased by a factor of 15. We measured an increase in the levels of hypoxanthine, xanthine, and uric acid by factors of 5, 10, and 98, respectively. These metabolites are likely accumulating as a result of AMP deamination for the purpose of preventing inhibition of adenylate kinase, as adenylate kinase plays an important role in the regeneration of ATP and shuttling of high-energy phosphates. The breakdown of AMP may also be beneficial in anoxia as ammonium is produced that combats acidification and uric acid is reported to have antioxidant properties. However, since the pool of ATP+ADP+AMP is limited and ADP is required for rephosphorylation by glycolysis, it is not clear how much AMP breakdown occurs.

The objective of this thesis was to investigate the basis of mitochondrial anoxia tolerance in the crucian carp heart. Our fascination was with the ability of the crucian carp heart to function normally in the absence of oxygen and our primary goal was to elucidate mechanisms of ΔΨM maintenance. We found evidence that shows crucian carp cardiomyocytes are able to maintain ΔΨM with an inhibited Complex IV and results that suggest in the absence of oxygen, fumarate serves as terminal electron acceptor of the electron transport chain. In all likelihood the reduction of fumarate is occurring at Complex II but the question remains of why then does inhibition of complex III result in mitochondrial depolarization. Also of importance, is that ATP synthase inhibition is orchestrated by a down-regulation of ATP synthase

3 subunit gene expression. The findings from this thesis shed light on the mechanisms underlying the remarkable adaptations of the crucian carp to survival in anoxia.

4

List of papers

I The role of the electron transport chain in maintaining mitochondrial membrane potential in anoxic crucian carp (Carassius carassius) cardiomyocytes

Scott, M.A., Nilsson, G.E. & Stensløkken, K.-O. Manuscript

II Enzymatic and morphological adjustments to anoxia in the crucian carp (Carassius carassius) mitochondria

Scott, M.A., Fagernes, C.E., Nilsson, G.E. & Stensløkken, K.-O. Manuscript

III The Metabolome of the anoxia-tolerant crucian carp (Carassius carassius) heart suggests that anoxic survival is promoted by the glycerol phosphate shuttle, purine metabolism, and fumarate as a terminal electron acceptor

Scott, M.A., Stensløkken, K.-O. & Nilsson, G.E. Manuscript

5

Abbreviations

3PG = 3-Phosphoglycerate Acetyl-CoA = Acetyl coenzyme A AK = Adenylate kinase ADP = Adenosine diphosphate AMP = Adenosine monophosphate ATP = Adenosine triphosphate BSA = Bovine serum albumin CCCP = Carbonyl cyanide m-chlorophrenylhydrazone cDNA = Complementary DNA CE = Capillary electrophoresis CK = Creatine kinase CoA = Coenzyme A Complex I = NADH dehydrogenase Complex III = Succinate dehydrogenase Complex IV = Cytochrome c oxidase Complex V = ATP synthase COX = Cytochrome c oxidase; Complex IV CS = Citrate synthase DHAP = Dihydroxyacetone phosphate DNA = Deoxyribonucleic acid ETC = Electron transport chain F16BP = Fructose 1,6-bisphosphate F6P = Fructose 6-phosphate G3P = Glycerol 3-phosphate G6P = Glucose 6-phosphate GA3P = Glyceraldehyde 3-phosphate GAPDH = Glyceraldehyde 3-phosphate dehydrogenase GMP = Guanosine monophosphate GPDH = Glycerol 3-phosphate dehydrogenase GPS = Glycerol phosphate shuttle HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMT = Human Metabolome Technologies IA = Iodoacetate IMM = Inner mitochondrial membrane IMP = Inosine monophosphate LDH = Lactate dehydrogenase L:D = Light:dark cycle MAS = Malate-aspartate shuttle MDH = Malate dehydrogenase MS = Mass spectroscopy Mw2060 = Microcystis cf. wesenbergi 2060bp, the external RNA control gene m/z = Mass-to-charge ratio NaCN = Sodium cyanide NAD = Nicotinamide adenine dinucleotide (oxidized) NADH = Nicotinamide adenine dinucleotide (reduced) OSCP = Oligomycin sensitivity-conferring protein

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PCR = Polymerase chain reaction PMF = Proton Motive Force PNC = Purine nucleotide cycle Q = Ubiquinone qPCR = quantitative real-time polymerase chain reaction QqQMS = Triple quadruple mass spectroscopy Rhod123 = [6-amino-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]azanium chloride RNA = Ribonucleic acid RM = Red muscle S.D. = Standard deviation TCA = Tricarboxylic acid cycle; Krebs cycle; Citric acid cycle TMRE = tetramethylrhodamine ethyl ester perchlorate TMRM = tetramethylrhodamine methyl ester perchlorate TOFMS = Time-of-flight mass spectroscopy Wet wt = Wet weight ΔΨM = Membrane potential, mitochondrial

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Introduction

General introduction Exposure to an environment devoid of oxygen, anoxia, is usually a lethal experience for any animal. This can be observed on an ecosystem scale when nutrient runoff from agriculture or urban areas causes algal blooms that result in the destruction of aquatic life. Anoxia can occur at night when algae shift from net oxygen producers to oxygen consumers or following the massive algae die-off when food runs out and bacterial decomposition depletes water oxygen. Some species of algae will also produce toxins that further threaten aquatic life. For example, in early 2016 it was estimated that 23 million farmed fish (salmon, coho, trout) were killed from an algal bloom off the coast of Chile with estimated economic losses worth USD $800 million (Esposito, 2016). Since warmer water holds less oxygen, it is believed that in the near future climate change will exacerbate the disastrous effects of algal blooms (Havens and Pearl, 2015; Hallengraeff, 2016; Visser et al., 2016). Anoxia-tolerant animals have evolved adaptations that enable them to transition between periods of normoxia and anoxia. Key features of anoxia-tolerant animals are the reversible differential expression of specific proteins that allows for ATP levels to remain constant in anoxia by controlling the metabolic depression of ATP demand and supply pathways (Hochachka and Lutz, 2001). Understanding how animals cope in environments with variable oxygen levels will be increasingly important as the frequency and range of oxygen-depleting events increases. Knowing how animals tolerate reduced oxygen levels will help us to make informed decisions about a whole suite of environmental conservation and remediation issues.

Harm from insufficient oxygen can also occur on a scale much smaller than an ecosystem. For example, ischemic heart disease and stroke are disorders characterized by the blockage of blood vessels and an impaired oxygen supply. During 2011 in the United States, coronary artery disease was responsible for 1 of every 7 deaths and stroke for 1 of every 20 (Mozaffarian et al., 2015). Due to the high oxidative demands of the heart and the brain, these tissues rapidly deplete energy supplies and a loss of cellular homeostasis occurs. Localized cell death will occur and unless the blockage can be removed it will lead to organ failure. The impact of these diseases is expected to rise in the future. For example, it is estimated that in the United States by 2030 40.5% of the population will have some form of cardiovascular disease and the resulting financial burden is expected to triple over that time from what it was in 2010 (Heidenreich, et al., 2011). It is clear from this that further research into the mechanisms of anoxic pathophysiology is warranted.

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Model organisms The crucian carp is well-adapted to anoxic survival. Depending on the temperature, it can tolerate months in oxygen-deprived lakes and is one of the most anoxia-tolerant vertebrates (Nilsson and Renshaw, 2004). The crucian carp is perhaps most famous for its ethanol producing pathways that alleviate the harmful effects of accumulating metabolic waste (Van Waarde, 1991). Also remarkable is its extreme oxygen binding affinity of haemoglobin and its capacity for increasing gill surface area in order to enhance oxygen uptake (Sollid et al., 2003). Furthermore, crucian carp maintain heart rate and cardiac output in anoxia (Stecyk et al., 2004), while at the same time decrease heart and skeletal muscle protein synthesis (Smith et al., 1996). Perhaps the most important adaptation of the crucian carp is its massive liver glycogen stores - largest of any known vertebrate (Hyvarinen et al., 1983; Nilsson, 1990). A key feature of anoxia-tolerant animals is an ability to reduce ATP demand and supplement a reduced ATP supply with anaerobic ATP production via glycolysis (Lutz et al., 2003). The crucian carp goes to such extreme lengths to decrease ATP demand that it has been observed going blind to save energy in anoxia (Johanssen et al., 1997). All of these adaptations make the crucian carp a true champion of anoxia tolerance and an exceptionally well-suited model for studying mitochondrial anoxia tolerance.

Similarities and differences between the crucian carp and anoxia-tolerant turtles reflect common strategies and unique adaptations for surviving periods of oxygen deprivation. For example, both animals depress metabolism in order to reduce ATP demand. However, while the crucian carp reduces locomotory behaviour, the anoxia-tolerant turtles render themselves effectively comatose (Nilsson et al., 1993; Nilsson, 2001; Nilsson and Renshaw, 2004). Furthermore, upon anoxia-exposure the turtle decreases heart rate and cardiac output (Hicks and Farrell, 2000), while in the crucian carp these parameters remain unchanged (Stecyk et al., 2004). On a molecular scale, both models of anoxia- tolerance experience a down-regulation of protein synthesis (Smith et al., 2015). However, unlike the turtle, anoxia-exposure has no effect on crucian carp brain protein synthesis (Smith et al., 1996). Both animals have developed effective methods for removing the toxic metabolic end-products of glycolysis. The turtle uses its shell and bone to buffer the lactate load (Warren and Jackson, 2006) whereas the crucian carp has developed ethanol producing pathways (Johnston and Bernard, 1983; Van Waarde, 1991; Fagernes et al., 2017). Adaptations to oxygen deprivation are varied but both models of anoxia tolerance endeavour to reduce ATP demand by restricting physical activity and overall protein synthesis while at the same time alleviating the consequences of anaerobic ATP production.

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Mitochondria Mitochondria consume the vast majority of the oxygen we breathe in order to provide a supply of cellular energy in the form of, adenosine triphosphate (ATP).This is termed oxidative phosphorylation. The high energy compound ATP is used to power essential cellular processes such as ion-pumping, protein synthesis, and muscle contraction. In mammals, heart muscle cells, cardiomyocytes, normally receive 95% of their ATP from oxidative phosphorylation (Ingwall, 2002; Opie, 2004). Cardiomyocytes are some of the most metabolically active cells in the body and without sufficient ATP supply can exhaust the high-energy-phosphate pool within seconds (Wang et al., 2010; Doenst et al., 2013). Since cardiomyocytes require such large amounts of ATP and the majority of the ATP they consume is produced from oxidative phosphorylation then oxygen deprivation is devastating to normal heart function.

Oxygen is consumed and ATP is produced at the electron transport chain (ETC; Figure 1). Electrons are transferred from electron donors such as nicotinamide adenine dinucleotide (NADH) onto electron acceptors such as NADH dehydrogenase (Complex I; NADH-ubiquinone oxidoreductase). Electrons are transferred further down the ETC from Complex I to succinate dehydrogenase (Complex II; succinate- quinone oxidoreductase), to Cytochrome c reductase (Complex III; cytochrome bc1 complex), and then finally onto the terminal electron acceptor, oxygen, at cytochrome c oxidase (Complex IV). The reactions from Complexes I, III, and IV are coupled with the transfer of protons across the inner mitochondrial membrane (as illustrated in Figure 1, for further details see Murray, 2003). The accumulation of protons in the mitochondrial matrix generates a proton gradient that provides a proton-motive force (PMF). The

PMF is utilized by the F1Fo-ATP synthase (Complex V) to produce ATP as protons flow down the electrochemical gradient through Complex V and into the mitochondrial matrix. A constant supply of oxygen to Complex IV is presumed to be necessary for the maintenance of a PMF, which enables mitochondria to provide sufficient ATP to meet cellular demand.

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Figure 1. The mitochondrial electron transport chain. The majority of electrons enter the electron transport chain (ETC) from NADH at Complex I (others enter from donors such as succinate at Complex II) and then travel down the ETC to Complex IV where they are taken up by oxygen and protons to produce water. This process leads to the transport of protons at complexes I, III, and IV from the mitochondrial matrix into the intermembrane space (IMS). The build up of a proton gradient and a mitochondrial membrane potential (ΔΨM) is utilized by Complex V to produce ATP that is either used to power energetically dependent processes in the mitochondria or is transported into the cytosol. Illustration from Sazanov (2015).

The ATP synthase consists of two regions, the Fo is embedded in the inner mitochondrial membrane

(IMM) and the F1 is in the matrix (Figure 2). The F1 is composed of one each of the gamma (γ), delta (δ), and epsilon (ε) subunits, and three each of the alpha (α) and beta (β) subunits (Jonckheere et al., 2011).

Assembly factors are proteins that are known to assist with the joining of F1 subunits during the formation of the ATP synthase (Wang et al., 2001). The Fo consists of 8 eight copies of the subunit c and one copy each of the associated proteins a, b, d, e, f, g, F6, A6L, and the oligomycin sensitivity-conferring protein (OSCP; Watt et al., 2010). The PMF causes rotation of the γ, δ, and ε subunits as well as the 8 subunits of the c-ring (Boyer and Kohlbrenner, 1981; Cox et al., 1984). For effective ATP synthesis the α and β subunits must remain stationary. The necessary structural support is provided by the a, b, d, e, f, g, F6, A6L, and OSCP subunits (Davenish et al., 2006). Mitochondrial diseases that cause faulty subunits of the Fo and F1 are often fatal because mitochondrial ATP synthesis is hindered and cellular ATP supply is insufficient to meet ATP demand.

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Figure 2. Regions and subunits of the ATP synthase. The Fo region of the ATP synthase is embedded in the inner mitochondrial membrane and the F1 region is in the matrix. The Fo consists of the rotating c- subunit ring and the associated proteins a, b, d, e, f, g, F6, A6L, and the oligomycin sensitivity-conferring protein (OSCP) that provide structural support. The F1 is composed of the stationary alpha (α) and beta (β) subunits as well as the rotating gamma (γ), delta (δ), and epsilon (ε) subunits. Illustration modified from Dabbeni-Sala et al. (2012).

Mitochondrial membrane potential The accumulation of positively charged protons in the mitochondrial intermembrane space forms a membrane potential across the IMM (see Figure 1). In addition to ATP production, the ΔΨM also plays a role in importing nuclear DNA-encoded proteins into the mitochondria and in activating hypoxia inducible factors (Martinez-Reyes et al., 2016). Around 1500 DNA-encoded proteins are imported into the mammalian mitochondria and hypoxia inducible factors allow cells to adapt and survive in low oxygen environments (Lopez et al., 2000; Benizri et al., 2008). The maintenance of ΔΨM is therefore critical for cell survival (Gottlieb et al., 2003). Mitochondria deprived of oxygen are no longer able to perform ETC proton pumping and rapidly lose ΔΨM, which initiates signaling cascades that ultimately lead to cell death.

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Cell death is dichotomised into either apoptosis or necrosis. Apoptosis is the regulated form of cell death and in an adult human is responsible for the coordinated destruction of approximately 60 billion cells per day (Alberts, 2015). An example of the utility of apoptosis is the adaptation of the anoxia-tolerant crucian carp to remove cells in the inter-lamellar space of its gills to increase the surface area available for the extraction of oxygen from its environment (Sollid et al., 2003). In contrast, necrosis is essentially uncontrolled cell death and rather than resulting in the regulated disposal of cell components, leads to cellular rupture that elicits an inflammatory response (Srivastava, 2007). The initial stages of apoptosis and necrosis are similar but an important distinguishing factor between the two pathways is that apoptosis is an energy-dependent process while necrosis is not (Morciano et al., 2015). Medical conditions involving an impaired ATP supply are therefore particularly dangerous because they involve dysregulated cell death.

Mitochondrial ultrastructure Mitochondrial ultrastructure is important for mitochondrial function. Indeed, morphological rearrangements have been shown to alter energy production (Jacobs et al., 2003). For example, changes in substrate availability during conditions of energy limitation have been shown to increase mitochondrial protein synthesis that alters ΔΨM (Leverve and Fontaine, 2001; Rossignol et al., 2004). Mitochondrial fusion (mitofusion) results in the mixing of mitochondrial substrates and merging of mitochondrial membranes (Malka et al., 2005). Mitochondria with ETC machinery damaged by reactive oxygen species (ROS) can fuse with healthy mitochondria and use the ETC machinery of the healthy mitochondria to produce ATP, enabling the cell to avoid the energetically expensive process of producing new mitochondria (Benard and Rossignol, 2008). Rearrangements in mitochondrial ultrastructure such as altering the relative rates of mitofusion and mitofision can help mitochondria preserve ΔΨM and ATP supply under conditions of resource limitation or oxygen deprivation.

Alterations to mitochondrial volume can also affect energy metabolism. It has been demonstrated that shape reconfigurations can change enzymatic reaction rates and it has been suggested that matrix volume changes play a role in metabolic control (Srere, 1980; Lizana et al., 2008). Mitochondria exposed to elevated ADP have been observed to shrink and once the ADP is phosphorylated to ATP the mitochondria swell back to normal (Packer, 1963). It is also possible that changes in mitochondrial volume affect the contractility of muscle cells (Kaasik et al., 2004). Mitochondria can change volume in response to reductions in energy supply and perhaps actively shrink or swell to affect energy

13 metabolism under stressful conditions. However, since changes in mitochondrial volume would presumably affect cardiac output then perhaps maintaining a constant mitochondrial volume is important under such conditions.

Citrate synthase (CS) catalyzes the first step of the citric acid cycle (TCA). One of the roles of the TCA is to generate the NADH that is used by the ETC to produce the PMF for making ATP. Citrate synthase is considered a pacemaker enzyme because it regulates the rate of acetyl coenzyme A (acetyl-CoA) oxidation and thus NADH production (Wiegand and Remington, 1986). Additionally, CS activity scales proportionately with the amount of mitochondria in a sample and is a common index of mitochondrial content (LaNoue et al., 1984; Kocher et al., 2015; Shoar et al., 2015). Measures of CS activity can therefore be used to investigate how mitochondrial content changes in response to stressors such as anoxia. Cytochrome c oxidase (COX) is Complex IV of the ETC. Since COX is where oxygen is consumed its enzyme activity is a commonly used index of mitochondrial respiratory capacity (Herzig et al., 2000; Brown, 2001; Larson et al., 2012). Mitochondrial respiratory capacity is informative about the ability of mitochondria to generate a PMF and thus produce ATP (Poyton et al., 1988; Porter et al., 2015). Measures of COX activity can therefore be used to investigate how the ability of mitochondria to produce ATP changes in response to stressors such low oxygen.

The mitochondria of skeletal muscle and cardiac muscle have several distinct differences. In humans, cardiac mitochondria have greater oxidative capacity than skeletal muscle mitochondrial, which is likely a result of cardiomyocytes also having greater mitochondrial content (as measured by CS activity; Park et al., 2014). Indeed, mitochondria occupy approximately 35% of the volume of a cardiomyocyte (Stride et al., 2013). This contrasts significantly with skeletal muscle (predominantly oxidative vastus lateralis) whose mitochondrial content by volume only accounts for approximately 9% (Larsen et al., 2012). In comparison, heart mitochondria occupy 22% of the cell volume in crucian carp and 45% in rainbow trout (Oncorhynchus mykiss; Vornanen, 1998), which emphasizes the importance of glycolysis in the crucian carp heart. Mitochondria in the crucian carp skeletal muscle are much more variable as they have been found to occupy 31% and 15% of fibre volume in red muscle (RM) and 6% and 2% in white muscle at acclimation to 2OC and 24OC, respectively (Johnston, 1982). Further evidence of the greater mitochondrial content in cardiomyocytes compared to skeletal muscle is that capillary density in the human heart is almost an order of magnitude higher than in skeletal muscle (Hudlicka et al., 1992). Since

14 cardiac and skeletal muscle differ in their oxidative capacity, mitochondrial content, and capillary density it makes sense that they would differ in their responses to oxidative stress.

Metabolomics Substrate-level phosphorylation via glycolysis contributes to the energy supply provided by oxidative phosphorylation. In addition to producing ATP directly (net 2 ATP per molecule of glucose; see Figure 3), glycolysis also generates NADH that passes into the mitochondria and is oxidized at the ETC (see Figure 1) for further ATP production (3 or 5 ATP; Wiley et al., 2016). In comparison, a further 25 ATP are produced from post-glycolysis substrates from oxidative phosphorylation (23 ATP) and substrate-level phosphorylation at the Krebs cycle (2 ATP; Rich, 2003). NADH produced by glycolysis serves as an inhibitor of the pathway and since NAD is required for glycolysis then the ratio of NAD:NADH regulates energy production in cells (Lehninger et al., 2013).

Figure 3. ATP production via Glycolysis. A net of 2 ATP is produced in glycolysis per molecule of glucose. The NADH generated from glycolysis is transported to the citric acid cycle to be used for ATP production via oxidative phosphorylation. Image modified from Boundless (2016).

There are ten enzyme-catalyzed reactions in glycolysis and several of the steps are entry/exit points for other substrates (see Figure 3). Glucose 6-phosphate (G6P) is the second substrate in glycolysis and is the entry point of glucose molecules stored as glycogen (Saudubray et al., 2016). Fructose can be broken down into two different intermediates of glycolysis, fructose 6-phosphate (F6P) directly or

15 glyceraldehydes 3-phosphate (GA3P) via dihydroxyacetone phosphate (DHAP; Lieberman and Marks, 2012). Glycerol 3-phosphate (G3P), which can be synthesized from amino acids and TCA intermediates, also adds to the GA3P pool via DHAP (Sledzinski et al., 2013). Glycogen, fructose, DHAP, and G3P are just a few of the metabolites that can feed the pool of glycolysis intermediates.

In addition to ATP and NADH, glycolysis also produces pyruvate (see Figure 3). The main fate of pyruvate is the mitochondria. Pyruvate to acetyl-CoA links glycolysis to the TCA and is necessary for maximizing the amount of ATP that can be produced from the complete oxidation of glucose. This process is initiated by the pyruvate dehydrogenase complex, which is regulated by the ratios of ATP to ADP, NADH to NAD, and acetyl-CoA to CoA. Pyruvate can also be directly converted to oxaloacetate and occurs under conditions of reduced ATP supply in order to replenish the pool of TCA intermediates (Westerhold and Zeczycki, 2016).

The glycerol phosphate shuttle (GPS) is a reversible reaction and is another process that regenerates NAD for glycolysis. In the forward direction, cytosolic glycerol 3-phosphate dehydrogenase (GPDH) converts DHAP into G3P and regenerates NAD (see Figure 3). In the reverse direction, inner mitochondrial membrane-embedded GPDH converts G3P back to DHAP and reduces Q of the ETC (see Figure 1). From Q electrons are passed onto Complexes III and IV where protons are pumped into the intermembrane space. In addition to regenerating NAD for glycolysis the GPS also transfers reducing equivalents to the ETC, which contributes to the maintenance of ΔΨM and production of ATP.

The malate-aspartate shuttle (MAS) is the primary means of transporting reducing equivalents from glycolysis to the ETC. Since the inner mitochondrial membrane is impermeable to NADH, malate crosses the membrane instead. Once inside the matrix malate is converted to aspartate via oxaloacetate, glutamate, and α-ketoglutarate. The conversion of malate to oxaloacetate yields one NADH. Accumulating aspartate crosses the IMM to the cytosol where it is then converted back into malate via the reverse reaction scheme, producing one NAD. In contrast to the GPS, the MAS produces 2 more ATP per molecule of glucose because the MAS includes the pumping of protons at Complex I (see Figure 1). The MAS generates NADH in the matrix that is used to fuel the ETC and NAD in the cytosol that is a necessary cofactor of glycolysis.

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Purine metabolism involves the anabolism of nucleotides such as DNA and RNA as well as the catabolism of other nitrogen-containing compounds such as adenosine monophosphate (AMP) and guanosine monophosphate (GMP). Under conditions of reduced energy supply, the breakdown of AMP stimulates ATP regeneration via ADP fusion. The interconversion of 2 ADP to ATP and AMP is carried out by Adenylate kinase (AK), an enzyme that also participates in the shuttling of high-energy phosphates from sites of ATP-production to sites of ATP-consumption (Dzeja et al. 1999; Dzeja and Terzic, 2009). The enzyme AMP deaminase is responsible for the breakdown of AMP. GMP and AMP are both broken down to the end-product uric acid but the GMP intermediates are guanosine and guanine, while the AMP intermediates are adenosine, inosine, hypoxanthine, and xanthine (Maiuolo et al., 2016).

The purine nucleotide cycle (PNC) produces fumarate from aspartate. The purpose of the PNC is to increase the concentration of TCA intermediates and make use of the AMP produced from the regeneration of ATP from 2 ADP (Salway, 2004; Arinze, 2005). It has been shown to increase the rate of oxidative phosphorylation in skeletal muscle during exercise, starvation, or when ATP supply is low (Voet and Voet, 2004). Inosine monophosphate (IMP) is formed from the deamination of AMP and reacts with aspartate and GTP to produce adenylosuccinate, which is then cleaved into fumarate, regenerating the initial AMP in the process. In order to enter the mitochondria fumarate is converted to malate via fumarase, an enzyme that is reversible and has both cytosolic and mitochondrial varieties (Bulusu et al., 2011). Once in the mitochondria, malate can be converted to other TCA intermediates.

The TCA cycle links the catabolism of sugars, fats, and proteins to the ETC. The oxidation of TCA intermediates produces NADH and FADH2, which transfer electrons onto the ETC (see Figure 1) for the generation of PMF and then ATP. Under conditions of excess ATP supply, intermediates of the TCA such as oxaloacetate can be removed to produce amino acids such as aspartate (Berg et al., 2002). Conversely, under conditions of reduced ATP supply amino acids such as aspartate can be converted back into TCA intermediates such as fumarate. For example, Aragon et al. (1981) found a fourfold increase in the concentrations of fumarate and malate in rat skeletal muscle after 10 minutes of exercise. The TCA cycle is a dynamic pathway central to cellular metabolism and oxidative phosphorylation that also provides the substrates for many other reactions.

Phosphocreatine is a creatine molecule attached to a high-energy phosphate. Under conditions of increased ATP supply the energy can be stored on creatine as phosphocreatine. Conversely, under

17 conditions of reduced ATP supply the high-energy phosphate can be transferred to ADP, yielding ATP. It is typically produced in the liver and transported to the muscle. Traditionally known for its role in building skeletal muscle, recent studies have brought to light its importance in providing energy- buffering to the heart (Balestrinto et al., 2016; Landoni et al., 2016). Phosphocreatine serves as a reservoir for high-energy phosphates and provides rapid, short term ATP supply under both normal physiological and pathophysiolocal conditions.

Anoxia Hypoxia is a physiological deficiency in oxygen, whereas anoxia is the absence of any measureable oxygen. Myocardial ischemia describes a scenario where blood supply to the heart tissue is impeded and is characterized by oxygen levels ranging from hypoxia to anoxia. Anoxia is important because following an ischemic event such as a heart attack or a stroke the available oxygen can be depleted within seconds. Shortly thereafter stored energy in the form of creatine phosphate is consumed and after several minutes ATP levels are significantly reduced (see Figure 4). By this point oxidative phosphorylation has ceased and ADP, AMP, and uric acid are accumulating. Anaerobic glycolysis is elevated to offset the reduced ATP supply resulting from the shutdown of oxidative phosphorylation. Without oxidative phosphorylation up to 18 times less ATP is produced (depending on the degree of respiratory coupling in the mitochondria) and while anaerobic glycolysis may be momentarily sufficient for the maintenance of cellular homeostasis it is not sufficient to maintain key cellular function such as contraction in a cardiomyocyte (Chien, 2013). As normal cellular function begins to cease, toxic metabolic end products such as lactate accumulate and glycolysis is inhibited (Rovetto, 1975; Choi et al., 2002; Orlav and Karkouti, 2014). Without glycolysis to produce ATP all active transport stops and ion gradients across membranes dissipate, causing cells to swell and rupture.

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Figure 4. The necrotic cell death cascade following exposure to oxygen deprivation. The inability of cells to generate sufficient ATP and maintain mitochondrial membrane potential are critical junctures in the anoxic cell death cascade. A key difference between anoxia-tolerant and anoxia-intolerant animals is the ability of anoxia-tolerant animals to regulate ATP supply and demand in order to prolong entering the cell death cascade for as long as possible. Illustration from Boutilier (2001).

Mitochondrial membrane potential Following an anoxic event, mammalian mitochondria will undergo Complex V reversal in order to maintain ΔΨM. This is only a momentary fix as ATP hydrolysis rapidly depletes ATP supply and ΔΨM can no longer be maintained. On the contrary, animals adapted to low oxygen levels such as the hypoxia- tolerant frog (skeletal muscle; St-Pierre et al., 2000) and anoxia-tolerant turtle (heart; Galli et al., 2013) have demonstrated an ability to inhibit reversal of Complex V and prevent a rapid depletion of available ATP. In addition to decreased Complex V activity the mitochondria of the anoxia-tolerant turtle brain have been shown to undergo a partial uncoupling of the proton gradient, which lessens the driving force

19 for Complex V reversal (Pamenter et al., 2016). Thus, two approaches to extending ATP supply and preventing the lethal loss of ΔΨM in anoxia are inhibition of Complex V and a controlled reduction to a lower ΔΨM.

Reductions in Complex V activity can be attributed to a down-regulation in subunit transcription (See Figure 2 for illustration of subunits). A study with hypoxia-exposed rat cardiomyocytes showed a down- regulation of ATP synthase subunit e, the regulatory subunit (Levy and Kelly, 1997). Down-regulation of subunit e expression is associated with ATP synthase dysfunction and disease (Hurtado-Lopezet al., 2015). A study with human hepatocytes found an up-regulation of subunit alpha following hypoxia- exposure (Strey et al., 2010). However, studies on mammalian systems are difficult to interpret as many changes may be pathological rather than adaptive. Recent proteomics and Western blot work by Gomez, (2016) has revealed that the anoxia-tolerant turtle undergoes a down-regulation of subunits of the peripheral stock. Expression of one of the subunits of the catalytic region was also assessed but no effect of anoxia was observed. A study with hypoxia-tolerant shrimp (Litopenaeus vannamei ) found that low-intensity oxygen deprivation challenges resulted in a down-regulation of the alpha subunit only, and following reoxygenation, significant decreases in the beta, delta, and epsilon subunits (Martinez-Cruz et al., 2015). Down-regulating ATP synthase gene expression seems critical for reducing ATP synthase activity in anoxia. However, there appears to be large variation between animals in their approach to disrupting ATP synthase activity.

In addition to Complex V, other components of the ETC are also impacted by reduced oxygen levels. Studies with mammalian models have shown an overall down-regulation in the expression levels of Complexes I-IV, which have been demonstrated to translate into a decrease in enzyme activities (Chan et al., 2009; Chen et al., 2010; Muralimanoharan, et al., 2012). It is believed that alterations to the ETC are for the purpose of reducing electron flow and limiting damage from ROS both during and following low-oxygen events (Semenza, 2007; Sirey and Ponting, 2016). In comparison, anoxia-tolerant turtles also show a significant decrease in Complex I activity. However, there appears to be no appreciable effect of anoxia on the activity of Complexes II-IV (Pamenter et al., 2016). This is consistent in both heart and brain with the exception of Complex I, which showed no decline in activity in the heart (Galli et al., 2013). It seems that the approach to surviving short term low-intensity oxygen deprivation challenges is to down-regulate the ETC for the purpose of limiting damage from ROS. In contrast, it appears that animals adapted to surviving long term high-intensity oxygen deprivation challenges do not down-

20 regulate the ETC but instead maintain ETC functioning, likely for the purpose of semi-continued proton pumping to maintain ΔΨM.

Mitochondrial ultrastructure Changes to mitochondrial ultrastructure affect oxidative capacity and the ability of mitochondria to maintain ΔΨM. Animals vary widely between species and tissues in their mitochondrial responses to oxygen deprivation. Humans acclimated to hypoxia at elevation have been found to increase skeletal muscle mitochondrial volume, whereas humans with impaired oxygen delivery to skeletal muscle as a result of disease have displayed the opposite (Jacobs et al., 2016; Baum et al., 2016). Despite opposing effects of low-oxygen challenges on skeletal muscle mitochondria, heart mitochondria from humans with end-stage heart failure had no alterations in mitochondrial ultrastructure when compared to healthy donors (Holzemet al., 2016). Similarly, heart mitochondria from rats held in chronic hypoxia had no significant change in volume, although they did increase considerably in mitochondrial number (Costa et al., 1988). In contrast to hypoxia-exposed rats, mitochondrial number did not change significantly in the pupae of 4-days anoxia-exposed flesh fly (Sarcophaga crassipalpis; Kukal et al., 1991) and decreased significantly in the skeletal muscle of hypoxia-acclimated tench (Tinca tinca; Johnston and Bernard, 1982). A decrease in mitochondrial number may be advantageous in anoxia because less energy would be required to maintain ΔΨM and there would be fewer damaged components of the ETC to produce ROS upon reoxygenation.

Dysregulation of mitochondrial fission and fusion may contribute to mitochondrial damage during anoxia. It is still not definitive if the gene that promotes mitochondrial fusion, MFN2, increases cellular survival in oxygen-deprivation challenges or participates in necrosis (Dong et al., 2016). Indeed, one study examining mitochondria from rat cardiomyocytes determined that MFN2 plays a substantial role in oxidative stress-induced apoptosis (Shen et al., 2007). Further complicating the matter, repression of

MFN2 has been seen to reduce glucose oxidation and ΔΨM (Bach et al., 2003). Consistent with this, over- expression of MFN2 causes an increase in glucose oxidation, ΔΨM, and overexpression of components of the ETC (Pich et al., 2005). Data profiling mitochondrial fission and fusion in anoxia-tolerant models is limited; however, anoxia-exposed C. elegans were observed undergoing mitochondrial fission and then fusion upon reoxygenation (Ghose et al., 2013). There is clearly no unified approach with regard to mitochondrial fission and fusion in response to oxygen-deprivation challenges. Which path an animal takes is likely based on several factors such as energy availability and preservation, ability to maintain

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ΔΨM, method for dealing with damaged mitochondria, and degree of metabolic suppression/repression of locomotion.

The heart is much less variable compared to the brain with respect to CS activity following exposure to a low-oxygen challenge. A study comparing the hearts of humans with end-stage heart failure to healthy donors found no difference in CS activity (Holzem et al., 2016). Similarly, humans acclimated to hypoxia at altitude saw no change in skeletal muscle CS activity (Jacobs et al., 2016). An increase in CS activity was observed in rat brain following 9 hours of hypoxia (Yin et al., 2008). In contrast, the brain mitochondria of anoxia-tolerant turtles decreased CS activity following acclimation to anoxia (Pamenter et al., 2016). Inconsistent with the anoxia-tolerant turtles, CS activity from anoxia-tolerant killifish embryos did not change appreciably (Wagner et al., 2016). Although there is no consistent response to low-oxygen challenges CS activity may increase in anoxia-intolerant models as a short-term strategy for increasing TCA activity and oxidative capacity, while CS activity may decrease in anoxia-tolerant models as a result of mitophagy of damaged mitochondria or to reduce ATP demand for overall maintenance of

ΔΨM.

When exposed to conditions of reduced oxygen supply, COX activity usually increases or remains unchanged. Anoxia-intolerant mammals exposed to low-oxygen challenges are known to increase COX protein expression and exhibit isoform switching in an effort to enhance aerobic capacity (Fukuda et al., 2007; Yin et al., 2008). Consistent with this, exposure to hypoxia resulted in a significant increase in mice skeletal muscle COX activity (Slot et al., 2015). There are exceptions, however, as a recent study with humans acclimated to hypoxia at altitude found no change in skeletal muscle COX protein expression or enzyme activity (Jacobs et al., 2016). Anoxia-tolerant turtles display significant increases in heart COX subunit expression when exposed to anoxia (Cai and Storey, 1996; Gomez, 2016). However, this increased subunit expression does not necessarily result in significantly increased enzyme activity (Galli et al., 2013; Warren and Jackson, 2017). Although, the African cichlid (Psuedocrenilabrusmulticolour victoriae) is known to tolerate conditions of low oxygen and has been observed to increase heart COX activity when reared in hypoxia (Crocker et al, 2013). Compared to the heart, responses of skeletal muscle to limited oxygen supply are much more variable. When exposed to hypoxia, muscle COX activity has been found to increase in the common carp (Zhou et al., 2000), decrease in the tench (Johnston and Bernard, 1982), and remain unchanged in the turtle (Warren and Jackson, 2017). There is no unified strategy for survival in low-oxygen but one approach is to increase COX activity in order to more

22 efficiently utilize oxygen. Discrepancies between animals and tissues likely arise from severity of the oxygen limitation, degree of metabolic suppression, and amount of activity while oxygen-deprived.

Metabolomics Under conditions of oxygen limitation ATP production shifts away from oxidative phosphorylation and a greater energy burden is placed on substrate-level phosphorylation. Glycolysis is an oxygen-independent metabolic pathway that animals depend on for their survival. However, it is clear by the difference in ATP production between substrate-level and oxidative phosphorylation why ATP supply is severely hindered without oxygen. In order for glycolysis to provide sufficient ATP supply throughout a low- oxygen challenge several important processes need to occur unobstructed such as a continuous supply of carbohydrates from the liver, the removal of toxic metabolic end products such as lactate in order to prevent acidosis, and inhibitors of glycolytic enzymes must remain low (Chien, 2013). One example is the NAD/NADH ratio, since NAD is a cofactor of glycolysis its continuous regeneration is necessary to prevent necrosis in ischemia (Im and Hoopes, 1989). Animals capable of metabolic depression may display a down-regulation of glycolysis in order to preserve and extend ATP supply. However, not all animals regulate glycolysis in all tissues equally, as is seen in anoxia-tolerant animals capable of metabolic depression. Thus, energy use may be shunted away from less vital tissues such as skeletal muscle and the liver in the interest of maintaining proper glycogen supply to the heart and brain (Kelly and Storey, 1988).

Glycolysis intermediates and related substrates GA3P, DHAP, G3P, and F16BP may play important roles in anoxic survival. Both the anoxia-tolerant turtle and crucian carp down-regulate brain glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in anoxia (Smith et al., 2009; Smith et al., 2015). This would lead to the accumulation of upstream intermediate of glycolysis, GA3P, and the dissipation of downstream intermediate 3-Phosphoglycerate (3PG; see Figure 3). GA3P is readily interchangeable with DHAP. The conversion of DHAP into G3P consumes NADH and regenerates NAD that is required for glycolysis. Further upstream from GA3P is fructose 1,6-bisphosphate (F16BP), which has been shown to prevent damage in hypoxia and ischemia by improving antioxidant defense (Alva et al., 2016). Under conditions of oxygen limitation intermediates of glycolysis may serve an antioxidant role or be diverted to other pathways for the regeneration of NAD or maintenance of ΔΨM.

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Lactate accumulates in anoxia because of the favourable reaction from pyruvate to lactate by lactate dehydrogenase (LDH). The reaction is favourable because it regenerates NAD that is used in glycolysis for ATP production. For this reason, lactate accumulates in both anoxia-tolerant and anoxia-intolerant models (Milligan, 1996; Warren and Jackson, 2006). To prevent lactic acidosis and impairment of glycolysis (Hartmund and Gesser, 1995), lactate must be diverted away from its site of production (Chien, 2013). In mammals, lactate is transported to other tissues to be metabolized (Brooks, 2000), in trout it can be retained in muscle (Milligan, 1996), in anoxia-tolerant turtles it is stored in the shell (Jackson, 2002), and in the crucian carp it is excreted via ethanol-producing pathways in the muscle (Nilsson, 1988). Although animals have developed very different strategies for dealing with the excess lactate produced under conditions of oxygen limitation, they all do it to ensure continued LDH function and NAD regeneration for the purpose of maintaining glycolysis and ATP production.

The GPS provides much needed NAD for glycolysis and transports reducing equivalences to the ETC. Human neutrophils are cells accustomed to hypoxia and illustrate a potential role of the GPS under conditions of oxygen limitation (Walmsley et al., 2005; Raam, et al., 2008). Neutrophils have mitochondria yet they are not used for producing ATP (Peyssonnaux and Johnson, 2004). Instead, they rely more on glycolysis than oxidative phosphorylation for ATP production and maintain ΔΨM by receiving electrons at Complex III from the GPS (Raam et al., 2008). However, since Complex III is not likely to be pumping protons in the anoxic ETC because no oxygen is available to receive electrons, it is not clear what role the GPS plays in the maintenance of ΔΨM in the anoxic crucian carp heart. The important process of delivering electrons to the ETC has yet to be examined in the anoxic crucian carp heart and should be informative about how ΔΨM is maintained in anoxia. Although studies investigating the role of the GPS in anoxia-tolerant models are limited, the central enzyme of the GPS, GPDH, has been found to decrease in activity in the heart of the freshwater turtle and hypoxia-tolerant African lungfish (Dunn et al., 1983; Willmore et al., 2001). These findings do not necessarily mean that there is no role for the GPS in a strategy for tolerating anoxia. Rather, the observed reduction in GPDH activity in these models may be a result of overall metabolic suppression or suggestive of the importance of other means of transporting reducing equivalence to the ETC such as the MAS.

The MAS seems to play an important role in anoxia tolerance. Unlike GPDH of the GPS, the enzymes malate dehydrogenase (MDH) and glutamate-oxaloacetate transaminase (GOT) of the MAS did not decrease in the turtle heart following exposure to anoxia (Willmore et al., 2001). In agreement, the

24 amount of GOT2 protein increased following anoxia exposure in the rectal gland of the anoxia-tolerant epaulette shark (Hemiscyllium ocellatum; Dowd et al., 2010). These findings suggest that NADH is being funneled to the ETC via the MAS (Dawson, 1979). However, brain glutamate and RM aspartate have been found to decrease in the anoxia-exposed turtle and goldfish, respectively (Waarde et al., 1982; Nilsson et al., 1990). One possible explanation for this is that aspartate decreases because it is broken down to succinate via GOT, MDH, and succinate dehydrogenase (SDH; Waarde et al., 1982). The apparent contradiction between maintained MAS enzymes and depleted metabolites may be explained by tissue-specific differences in ATP demand as s result of tissue-specific metabolic suppression as is seen in anoxia-tolerant models.

Under conditions of oxygen limitation, reduced ATP supply will lead to the progressive accumulation of ADP, AMP, IMP, inosine, hypoxanthine, xanthine, and uric acid. Studies of AK activity under conditions of reduced ATP supply are limited but in the skeletal muscle of exercising humans it has been demonstrated that AK activity accounts for 10% of anaerobic ATP regeneration (Normal et al., 2001; Borms et al., 2004). Furthermore, AK activity has been found to be maintained in the skeletal muscle of the hibernating (characterised by reduced ATP supply) white-tailed prairie dog (Cynomys leucurus; English and Storey, 2000). Accumulating AMP is broken down by AMP deaminase into IMP in order to prevent AMP from inhibiting AK and to combat cellular acidification by producing ammonium along with IMP (English and Storey, 2000). Anoxia-exposed turtles have shown significantly improved skeletal muscle AMP deaminase kinetic parameters (increased Vmax and decreased Km), which suggest an adaptive role for AMP deaminase in anoxia tolerance (Zhou, 2006). However, since the pool of ATP+ADP+AMP is limited, it is not clear how the removal of AMP is sustainable for long term anoxic survival. IMP is then converted to inosine, hypoxanthine, and xanthine. In the absence of oxygen the breakdown of xanthine to uric acid produces NADH (Maiuolo et al., 2016). Uric acid is the end-product of AMP breakdown and is reported to confer antioxidant features (Ames et al., 1981; Storey, 1996; Maxwell et al., 1997). Purine catabolism is a pathway that offers several benefits to an animal facing oxygen deprivation, such as the promotion of ATP regeneration and shuttling by degrading AMP that would otherwise inhibit AK activity, combat cellular acidification by producing ammonium, provide reducing equivalents in the form of NADH, and generate antioxidant defense from uric acid.

Rather than convert IMP to inosine, the PNC uses IMP along with aspartate to produce adenylosuccinate that is used to make fumarate. There is mounting evidence of the role of fumarate as a terminal

25 electron acceptor in preserving ΔΨM under conditions of oxygen limitation (Tielens and Hellemond, 1998; Buck, 2000). One such example is from Sridharan et al. (2008), who found that neonatal cardiomyocytes use fumarate as a terminal electron acceptor when Complex IV is unable to facilitate the transfer of electrons. Depletion of aspartate has been found in the skeletal muscle of goldfish (Carassius auratus) and the brain of turtles following anoxia exposure and is further evidence of fumarate production via the PNC (Arillo et al., 1972; Waarde, 1982). The accumulation of succinate in the heart of the anoxic turtle and skeletal muscle of the anoxic goldfish and common carp (Johnston, 1975; Waarde, 1982; Buck, 2000) is additional evidence in support of fumarate as a terminal electron acceptor. The PNC is a critically important pathway during conditions of oxygen limitation as it replenishes TCA intermediates, makes use of accumulating AMP, and produces fumarate that can aid in the preservation of ΔΨM.

Under conditions of oxygen limitation and reduced ATP supply phosphocreatine is rapidly consumed. This occurs because the liberation of ATP from phosphocreatine happens as soon as ATP levels drop below resting (Chien, 2013). For this reason, phosphocreatine has been shown to provide cardiac protection under conditions of reduced ATP supply (Landoni et al., 2016) and animals regularly exposed to conditions of oxygen limitation are found to have higher CK activity (Christensen et al., 1994). Indeed

CK kinetic parameters have been found to increase in Vmax and decrease in Km in the anoxic turtle heart and skeletal muscle (Birkedal and Gesser, 2004; Zhou, 2006). Consistent with this, phosphocreatine levels decrease significantly following anoxia-exposure in the skeletal muscle of several species of anoxia-tolerant fish and in the hearts of the anoxia-tolerant turtle (Waarde et al., 1990; Stecyk et al., 2009). However, there may be exceptions to this trend as oxygen-deprived crucian carp have been observed to have reduced brain CK protein levels (Smith et al., 2009). CK is not just a source of high energy phosphates under conditions of reduced ATP supply but alterations to it in anoxia-exposed animals may improve the energy buffering capacity between sites of energy production and consumption.

Concluding paragraph Research on mitochondria from animals adapted to anoxia is sorely lacking. As recently reviewed by Galli and Richards (2014), the majority of work with anoxia-tolerant models has focused on biochemical adaptations relating to glycolysis. Much of the research that has been undertaken on anoxia-adapted mitochondria has focused on the ETC, namely ΔΨM and inhibition of Complex V reversal (St-Pierre et al.,

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2000; Galli et al., 2013). Although there is agreement on general trends in strategies for anoxic survival there is hardly any consensus on mechanisms (Waarde, et al., 1982; Galli and Richards, 2014). This point is illustrated by investigations into COX activity in anoxia-tolerant models. In the skeletal muscle of oxygen-deprived animals, COX activity has been found to increase in the common carp (Zhou et al., 2000), decrease in the tench (Johnston and Bernard, 1982), and remain unchanged in the turtle (Warren and Jackson, 2017). Due to inherent species- and tissue-specific adaptations to low-oxygen challenges much more work is needed to be done before a thorough understanding can be developed. Furthermore, many questions relating to mechanisms still need to be addressed such as are there any underlying factors other than the ETC that contribute to the maintenance of ΔΨM, how is Complex V reversal inhibited, how is ΔΨM maintained in the absence of oxygen and what are the specific metabolites involved in this process?

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Aims

The overall aim of this thesis is to investigate the role of mitochondria in surviving anoxia. More specifically, the primary aim of thesis is to elucidate molecular mechanisms of anoxia tolerance in crucian carp heart mitochondria. A comparative approach is adopted and mitochondria from skeletal muscle as well as from heart muscle of the anoxia-intolerant trout are assessed alongside crucian carp heart mitochondria. A secondary aim of this thesis is to build upon the work of others and determine what adaptations are unique to the crucian carp and which are shared with other anoxia-tolerant animals. The specific aims of each manuscript are as follows:

Paper I The role of the electron transport chain in maintaining mitochondrial membrane potential in anoxic crucian carp (Carassius carassius) cardiomyocytes x To determine if inhibiting Complex IV activity affects cardiomyocyte mortality x To investigate mitochondrial adaptations to anoxia by exposing cardiomyocytes of the anoxia- tolerant crucian carp and the anoxia-intolerant brown trout to a mitochondrial uncoupler and

measuring the effect on ΔΨM

x To determine if ΔΨM is maintained in crucian carp cardiomyocytes following exposure to simulated anoxia and, if so, to see if reversal of the ATP synthase plays a role in the maintenance

of mitochondrial ΔΨM

x To see how Complex I and Complex III of the ETC contribute to the maintenance of ΔΨM as well

as to see if by blocking the ETC, other factors contribute to the maintenance of ΔΨM

Paper II Enzymatic and morphological adjustments to anoxia in the crucian carp (Carassius carassius) mitochondria x To measure mitochondrial volume following anoxia acclimation to see if mitofusion may be occurring as an adaptation to survival in anoxia x To further investigate mitofusion as an adaptation to survival in anoxia by quantifying mitofusin transcription following anoxia acclimation x To determine if mitochondrial content and oxidative capacity are reduced following anoxia acclimation

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x To test if crucian carp cardiomyocyte ATP synthase activity decreases following anoxia acclimation, likely as a means of curbing the energy-depleting process of ATP synthase reversal x To determine if the reduction in ATP synthase activity following anoxia acclimation may be attributed to a down regulation of ATP synthase subunit transcription

Paper III The Metabolome of the anoxia-tolerant crucian carp (Carassius carassius) heart suggests that anoxic survival is promoted by the glycerol phosphate shuttle, purine metabolism, and fumarate as a terminal electron acceptor x To examine the crucian carp metabolome in order to survey metabolic pathways associated with survival in anoxia x To investigate if fumarate is being used as a terminal electron acceptor for the ETC

x To make inferences about the maintenance of ΔΨM in anoxia from the levels of substrates that associate with the ETC

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Methods

Animals Crucian carp (Carassius carassius) were caught from the Tjernsrud pond, Oslo municipality. They were kept on a 12h:12h L:D regime in flow through tanks (~50 fish per 250 L) supplied with aerated and dechlorinated Oslo tap water (10oC). Fish were fed a maintenance diet daily with commercial carp food (Tetra Pond, Tetra, Melle, Germany). Trout are commonly used in comparative physiology studies and are a suitable anoxia-intolerant model for comparison with the anoxia-tolerant crucian carp (Schwarzbaum et al., 1996; Krumschnabel et al., 2000; Leveelahti et al., 2014). Brown trout were collected from Oslomarkas Fiskeadministrasjonin Sørkedalen, Oslo municipality; fed pellets from Skretting (Spirit Ørret 300 – 4.5 mm), and housed the same as the crucian carp. All animals were given at least 2 weeks for acclimatization to holding conditions and fasted for 24 h before any experiments were conducted.

Isolation of ventricular myocytes Cardiomyocytes were isolated for the purpose of viewing with fluorescence microscopy. Heart cells were isolated similarly to the protocols of Vornanen (1997). In brief, animals were sacrificed by a sharp blow to the head and spinal severance. Hearts were first gravity perfused with isolation media for 15 min to remove any excess blood cells. The Ca-free, low-Na solution contained (mM): 100 NaCl, 10 KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES that was brought to a pH of 6.9 with KOH. To begin the enzymatic digestion, 1.3 mg/mL collagenase type 1A, 1 mg/mL BSA, and 1 mg/mL trypsin were added to the perfusate. Carp cells were perfused for ~18 min and trout cells for ~6 min. Carp hearts were perfused for longer than trout hearts because a longer digestion was required to liberate individual cardiomyocytes. A reduced digestion time in carp resulted in clumping of cells, which did not occur in trout. Hearts were rinsed with isolation media before removing the bulbous arteriosus and mincing with scissors. Cardiomyocytes were passed through a pipette several times before being sedimented for 20 min on either glass slides for fixed-cell experiments or glass bottom-welled plates for live-cell experiments. Isolated cardiomyocytes could remain viable for at least 32 h, the duration of treatments.

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Fixed-cell IA experiments Chemicals were obtained from Sigma unless otherwise stated. Once sedimented and rinsed with isolation media (see above), cells were exposed to 10, 1, or 0.1 mM of NaCl (control), sodium cyanide (CN), iodoacetate (IA), or CN+IA. CN blocks Complex IV and prevents oxygen from functioning as the terminal electron acceptor of the ETC (Cooper and Brown, 2008) and is commonly used to chemically simulate anoxia (Karmazyn, 2013; Kahraman et al., 2015). IA shuts down glycolytic ATP production by inhibiting glyceraldehyde 3-phosphate dehydrogenase (Schmidt and Dringen, 2009) and is commonly used as a positive control for cell death (Leisner et al., 2012; Liao, et al., 2013; Chiu, et al., 2017). After 60 min exposure to treatments, cells were rinsed and then incubated with 1 μM Hoechst (Invitrogen), 5 nM MitoTracker (Invitrogen), and 40 μM propidium iodide (PI). Hoechst stains nuclei and MitoTracker stains mitochondria. These stains are primarily used for locating cellular and intracellular boundaries for the purpose of automated analysis of fluorescence intensity. PI stains DNA but is unable to penetrate the nucleus unless the cell is dead and the nucleus is damaged. This makes PI an effective tool for investigating cell death. 60 min later the cells were washed twice with PBS+Tween20. Cells were then fixed in 2% paraformaldehyde (diluted in PBS) for 15 min. A final wash occurred before mounting media made with gelatin and glycerol was used to adhere cover slips to slides.

Live-cell TMRM experiments Cells were stained with 12 μM Hoechst and 30 nM tetramethylrhodamine methyl ester perchlorate (TMRM) for 30 min. TMRM is a cationic membrane potential-sensitive dye that is sequestered by active mitochondria and dissipates concomitantly with a loss of ΔΨM. This provides a means of quantifying the O effects of various treatments on ΔΨM. Throughout the incubation process cells were held at 10 C and the following treatments were used (μM): 5, 0.5, 0.05, 0.005 carbonyl cyanide m- chlorophrenylhydrazone (CCCP), 1000 NaCl (control), 1000 NaCN, 0.03 Oligomycin A, 5 rotenone, and 5 antimycin A. Blockers of the ETC are routinely used to determine the contributions of specific complexes in maintaining mitochondrial homeostasis. Rotenone blocks Complex I and causes a reduction in ΔΨM as a result of impaired proton pumping (Li et al., 2003; Chan et al., 2005). Complex II does not pump protons so its blockers are often absent from studies investigating ΔΨM. Antimycin A blocks Complex III and has a similar effect to rotenone on Complex I (Won et al., 2015). CN blocks Complex IV (described above). Oligomycin impedes the flow of protons through Complex V (St-Pierre et al., 2000; Duerr and Podrabsky, 2010; Galli et al, 2013 Hawrysh and Buck, 2013). Additionally, the protonophore CCCP is a

31 common uncoupler used to dissipate ΔΨM (Dejonghe et al., 2016; Georgakopoulos et al., 2016; Rokitskaya et al, 2016).

Fluorescence Microscopy The primary reason fluorescence microscopy was used in this thesis is that it is an effective tool for quantifying changes in ΔΨM and cell death. The protocol used was similar to that of Stensløkken et al. (2014). Isolated cardiomyocytes were viewed with the ScanR fluorescence microscopy platform (Olympus IX81, inverted) equipped with a Hamamatsu C8484-05G camera. It is a highly automated system that incorporates the detection, imaging, and counting of nuclei and cells (as well as the fluorescent intensity of stained mitochondria within cells). Automated image analysis software CellR (Olympus) with an edge detection algorithm was used for analysis. For the fixed-cell IA experiments images were taken at eight automated locations in each well. For each fish and treatment cellular morphology and fluorescent intensity was averaged from readings for 139±93 cells. Controls and treatments were performed in duplicate. For the live-cell TMRM experiments images were taken at 5 min, 45 min, 2 h, 3 h, 12 h, or 32 h post-exposure. The readings at each time point were made on independent wells so as to avoid repeated measures on TMRM-loaded cells. Studies have shown that the photoexcitation of TMRM produces both oxide and hydroxide ROS (Zorov et al., 2000; Brady et al., 2004). It was important to limit ROS production in this study because free radical production could accelerate the depolarization of ΔΨM and confound results. Additionally, TMRM was selected over TMRE and Rhod123 because TMRM is less likely to inhibit the ETC (Scaduto & Grotyohann, 1999; Perry et al., 2011). Images were taken at sixteen automated locations in each well. For each fish/treatment/time point, fluorescent intensities were averaged from readings for 124±4 cells. Controls were performed in duplicate.

Anoxia exposure and tissue sampling The protocol used for acclimating crucian carp to anoxia was adapted from Nilsson et al. (2015). In brief, crucian carp were put into an opaque 25 L container and allowed to acclimate for 24 h with aerated water flowing through the container. Anoxia acclimation was achieved by bubbling nitrogen gas into the

-1 container, making the oxygen level fall below 0.01 mgO2L within 6 h. A galvanometric oxygen electrode (Oxi 340i: WTW, Weilheim, Germany) was used to verify the concentration of oxygen (detection limit

-1 0.01 mgO2L ). This should be sufficient as the crucian carp O2crit (the oxygen level below which aerobic respiration cannot be fully maintained) at 8OC is reported to be between 0.5 and 1.0 mgL-1 (Nilsson,

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1992; Sollid et al., 2003). Normoxic individuals were held in parallel with the exception that air was bubbled into the container in place of nitrogen. Over the course of the exposures temperature was maintained between 7 and 8OC by keeping the containers in a larger tank with temperature controlled water. Both groups were sampled after 6 days exposure. Reoxegenated individuals were taken from the 6 days anoxia group and held under normoxic conditions for a further 6 days before sampling. Sampling involved immediate blunt cranial trauma and spinal severance followed by dissection of the heart and RM. For the heart, the ventricle was sampled. The RM was dissected out from the lateral side between the pelvic and anal fins. All samples for gene expression and enzyme activity experiments were frozen in liquid nitrogen within 1 min and held at -80OC until analysis.

Enzyme Activities Samples were prepared and enzyme analyses conducted using protocols similar to those described by Dalziel et al., (2012) and Duerr and Podrabsky (2010) as well as Sigma-Aldrich technical bulletins for CS (CS0720) and COX (CYTOCOX1). In brief, samples were ground into a powder in a liquid nitrogen-cooled mortar and pestle. Approximately 15 mg was then sonicated in an ice-cold buffer containing 20 mM Hepes, 5 mM EDTA, and 0.1% Triton X-100, pH 7.0. Enzyme activities were assayed using a lambda double-beam cuvette spectrophotometer (Perkin Elmer, Waltham, MA) at room temperature (20OC) and analyzed with UV Kinlab software (Perkin Elmer, Waltham, MA). Enzymes assayed were citrate synthase (CS; EC 2.3.3.1, a citric acid cycle enzyme and proxy for mitochondrial content), cytochrome c oxidase (COX; EC 1.9.3.1., Complex IV of the electron transport chain and a proxy for oxidative capacity), and ATP synthase (EC 3.6.3.14, Complex V of the electron transport chain). The utility of CS and COX enzyme activities as measures of mitochondrial content and oxidative capacity are justified in Larson et al. (2012). Final cuvette concentrations for each assay were as follows: CS (0.1 mM 5,5’-dithiobis-(2- nitrobenzoic acid), DTNB, 0.3 mM Acetyl CoA, and 0.1 mM oxaloacetate in 10 mM Tris, pH 7), COX (10 μM reduced cytochrome c in 10 mM Tris, pH 7), and ATP synthase (2 mM ATP, 1.5 mM phosphoenolpyruvate, 0.17 mM NADH, 6 U pyruvate kinase, 12 U lactate dehydrogenase, and 0.6 μM oligomycin in 33 mM Tris-acetate, 83 mM sucrose, and 10 mM MgCl2, pH 7.2). All assays were optimized to ensure that substrates were not limiting. Spectrophotometers were set to 412 nm (CS), 550 nm (COX), and 340 nm (ATP synthase). A Bradford’s reagent (Sigma-Aldrich) was used to assess total protein content (not shown).

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Electron Microscopy Ultra thin sections (80 nm) were cut with a diamond knife (Diatome, Switzerland) on a Leica Ultracut UCT (Leica Microsystems, Germany) ultramicrotome and placed on 75 mesh pre-coated copper grids (Electron Microscopy Sciences). A Philips CM200 transmission electron microscope was used to image sections of heart and RM tissue at a magnification of 2750X. The resultant micrographs were used to determine mitochondrial volume and number in hearts and RM of normoxic and anoxic crucian carp. Volume was estimated using Cavalieri’s Principle, which states that “the volume of an arbitrary-shaped object can be estimated in an unbiased manner from the product of the distance between planes and the sum of the areas on systematic parallel sections through the object” (Garcia et al., 2007). In other words, it is used to overcome the challenge of describing an inner three dimensional form based on the analysis of structural slices containing only two dimensional information. For reviews, illustrations, and details of calculations see Mayhew (1992), Mandarim-de-Lacerda (2003), and West (2012). Randomly selected locations of heart or RM tissue were serial sectioned at a thickness of 0.1 μm, for a total of 6 sections. Before analysis, micrographs were assigned a random number in order to blind the researcher from knowing which were normoxic controls and which were anoxic treatment. The ‘point-counting’ method was used to profile the area of mitochondria on the first section. The method involves placing a grid atop the micrograph and the number of times the grid intersects over mitochondria is counted. The distance between each line of the grid was equivalent to 0.74 μm.The remaining 5 sections were counted for the number of disappearing profiles. These are the number of instances in a given section where mitochondria are no longer visible that were present in the preceding section. From these values, the estimated mitochondrial volume was calculated: Y = Q- / (K * t * A) Y = estimated density of mitochondria in a volume Q- = the number of disappearing profiles (K * t * A) = the estimated total mitochondrial volume K = the number of sections t = the section thickness A = profile area of mitochondria on section #1 A = ∑ (p) * d2 p = number of grid intersections in mitochondria d = distance between lines of the grid 1 / Y = estimated mitochondrial volume

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Obtaining sequences for ATP synthase In order to design primers for quantitative real-time PCR (qPCR) in the crucian carp, gene sequences needed to be obtained by cloning and the protocol used was adapted from Fagernes et al. (2017). In brief, primer pairs for each gene were designed from zebrafish (Danio rerio) sequences (retrieved from the Ensembl database [http://www.ensembl.org/index.html]) using the nucleotide sequence designer program Primer3 (http://primer3.ut.ee; Rozen and Skaletsky, [1999]). Zebrafish is the preferred model for designing crucian carp primers because it is the fully sequenced genome most similar to crucian carp (both belonging to family Cyprinidae). Sequences were aligned and annotated using GeneDoc (version 2.7; http://www.psc.edu/biomed/genedoc) and ClustalX (version 2.0.12; Chenna et al. [2003]). Primer pairs were synthesized by Thermo Scientific (Ulm, Germany).

For cloning, TRIzol (Invitrogen, Carlsbad, CA, USA) and a stand-clamp mounted rotor-stator drill homogenizer (Ultra-Turrax T8, IKA, Staufen, Germany) were used to extract total RNA from normoxic crucian carp tissues. The quantity and quality of extracted total RNA was assessed using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Rockland, DE, USA). Total RNA samples were stored at -80OC. To reduce risk of any genomic DNA contamination, extracted total RNA was treated with TURBO DNAse (Ambion Applied Biosystems, Foster City, CA, USA). The resultant purified total RNA was used to synthesize cDNA in duplicate with SuperScript III reverse transcriptase (Invitrogen) and

O oligo(dT)18. PCR was carried out using gene-specific primers and cDNA was stored at -20 C. The pGEM®- T Easy Vector System I (Promega, Madison, WI, USA) was used to transform the PCR products into Escherichia coli (E. coli) cells (TOP10 F; Invitrogen, Carlsbad, CA, USA) that were cultured on LB plates containing ampicillin and IPTG/X-gal (Promega, Madison, WI, USA). PCR was used to verify that positive colonies had inserts of the correct size. Eight colonies of each gene were cleaned with ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced (GATC, Cologne, Germany) using T7 primers (Invitrogen, Carlsbad, CA, USA). In order to increase the likelihood that paralogs were not being overlooked, several primer pairs were used and multiple products were sequenced for each primer pair (GATC, Cologne, Germany).

To confirm the ATP synthase protein that each sequenced gene belonged to, sequences were nucleotide Blasted against the zebrafish and human genome (https://blast.ncbi.nlm.nih.gov/Blast.cgi). ATPAF2 and ATPAF1 correspond with assembly factors that bind to subunits alpha and beta, respectively, and

ATPF1g is gamma of the F1 portion of the ATP synthase. Gamma forms the central shaft of the ATP

35 synthase and the assembly factors provide structural support during formation of the enzyme. ATPFoCA, ATPFoCB, ATP5g1a, ATP5g1b, and ATP5g3a are paralogs and correspond with genes encoding subunit c of the Fo portion of the ATP synthase. Subunit c forms the proton pore that is embedded in the mitochondrial membrane. ATPFoEA1, ATPFoEA2, ATPFoEB1, and ATPFoEB2 are paralogs and correspond with genes encoding subunit e, also of the Fo portion of the ATP synthase. Subunit e is anchored to the inner mitochondrial membrane and extends into the intermembrane space where it plays a role in stabilizing other subunits of the Fo, including the ATP synthase dimer complex. qPCR Quantitative real-time PCR was undertaken to investigate the expression of mitofusin and ATP synthase subunit genes in response to acclimation to anoxia and reoxygenation. It was hypothesized that mitofusin was signaling mitochondrial fusion in crucian carp exposed to anoxia and that a down- regulation of ATP synthase subunit expression was responsible for decreased ATP synthase enzyme activity. Heart and RM tissue samples from normoxic, anoxic and reoxygenated individuals were acquired as written above. Gene sequences were obtained by cloning, as described above. In order to increase the likelihood that paralogs were not being overlooked, several primer pairs were used and multiple products were sequenced for each primer pair.

The qPCR protocol used was adapted from Sandvik et al. (2012) and Lai et al. (2016). First-thawed samples from -80OC were homogenized with a stand-clamp mounted rotor-stator drill homogenizer and RNA was extracted in TRIzol (Invitrogen, Carlsbad, CA, USA). An external standard RNA control gene mw2060 (Ellefsen et al., 2008) was added to each sample on a per-mg-tissue-basis prior to tissue homogenization. Traditionally, qPCR results are presented relative to “housekeeping” genes, which perform the role of an internal standard (Hugget et al., 2005). These genes are selected based on the assumption that they are unchanging in response to the treatment applied. A problem posed by acclimation to anoxia is that reference genes are likely affected by differential regulation and a decrease in total RNA (Smith et al., 1996; Storey and Storey, 2004; Stecyk et al., 2012). For examples of the technique using mw2060 that addresses this issue, see Ellefsen et al. (2012), Stecyk et al. (2012), and Ellefsen et al. (2014). The homogenization of heart and RM was carried out in separate batches and treatments were homogenized randomly within a batch.

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The quantity and quality of extracted total RNA was assessed using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Rockland, DE, USA). To reduce risk of any genomic DNA contamination, extracted total RNA was treated with TURBO DNAse (Ambion Applied Biosystems, Foster City, CA, USA). The resultant purified total RNA was used to synthesize cDNA in duplicate with

SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18. The nucleotide sequence designer program Primer3 (http://primer3.ut.ee) was then used to design three primer pairs for each gene. The primer pairs were synthesized by Thermo Scientific (Ulm, Germany). The crossing point, primer efficiency, and melting peak were determined for each of the primer pairs. Primer specificity was evaluated by melting curve analysis and primer efficiency was estimated with LinReg (Ruijter et al., 2009). The primer pairs chosen for qPCR were those of the three with the lowest crossing point, highest efficiency, and a single melting peak curve. Additionally, primer pair products were sequenced in order to ensure a single amplicon (GATC, Cologne, Germany). cDNA amplification in qPCR involves heating-cooling cycles that cause denaturation of double-stranded cDNA and the annealing of primers to the single-stranded cDNA. The qPCR program used was: 1. 95OC, 10 min; 2. 95OC, 10 sec; 3. 60OC, 10 sec; 4. 72OC, 13 sec; 5. Repeat steps 2. to 4., 42 times. After the qPCR program finished a melting curve analysis was performed for each amplicon. A heat-stable DNA polymerase was used along with a dye (LightCycler 480 SYBR Green I Master Mix; Roche Diagnostics, Basel, Switzerland) that binds specifically to double-stranded DNA. Reaction mixtures and samples were loaded into 384 multiwell plates (Roche Diagnostics) using an Agilent Bravo robot (Agilent Technologies, USA).

For each sample, qPCR was carried out in duplicate on both cDNA syntheses. As the amount of double- stranded cDNA in the reaction mixture increases, so too does the fluorescence intensity. Based on the principle that each cycle the amount of cDNA should double, the Cp can be used to determine the initial number of transcript copies in the sample. In other words, if a low Cp signifies a high amount of mRNA then a sample that reaches its Cp earlier must have more mRNA present in the original sample. The relative mRNA expression levels were calculated using the second derivative maximum method (Roche Lightcycler 480; Rasmussen, 2001) and the LinRegPCR software (Ruijter et al., 2009) for each reaction well. The Cp was determined based on the threshold value (crossing point), which is the fluorescence value when this threshold is crossed and was obtained from the program. Primer efficiencies were obtained from the raw fluorescence data using the software (Roche) in the LinReg software. Final values

37 are presented relative to a reference gene, which is assumed to be constant between the control and treatment and in this case was the mw2060 (described above).The following formula was used to calculate relative mRNA expression of a sample:

(Emw2060^Cpmw2060)/(Etar^Cptar) E = mean primer efficiency of the primer pair Cp = mean crossing point of the two duplicate qPCR reactions tar = target gene mw2060 = external standard

Metabolomics Metabolomics is a powerful tool for understanding mechanisms of anoxia tolerance because it essentially provides a wide and instantaneous snapshot of the metabolite levels within a tissue, allowing an analysis of changes in metabolic pathways. It is named as such because it profiles the metabolome, analogous to how transcriptomics and proteomics profile the transcriptome and proteome, respectively. Metabolomics of the heart from normoxic and anoxia-exposed crucian carp was performed in order to elucidate mechanisms of anoxia tolerance. Results should complement the gene expression (Stecyk et al., 2012; Stensløkken et al., 2014; Krivoruchko and Storey, 2015) and protein (Willmore et al., 2001; Smith et al., 2009; Smith et al., 2015) work already performed by others on anoxia-tolerant models.

Tissues were sampled as described above and transported on dry ice to the Human Metabolome Technologies (HMT) facility in Yamagata, Japan. The samples arrived with sufficient dry ice remaining so as to guarantee that no thawing occurred. For examples of publications where HMT services were provided see Kami et al. (2005), Makinoshima et al. (2014), and Kim et al. (2015). Metabolomics was performed on hearts from 6 normoxic and 6 anoxic crucian carp. For specifics of the methods used see Soga and Heiger (2000), Soga et al. (2002), and Soga et al. (2003). In brief, samples were added to 50% acetonitrile in water containing internal standards (20μM for cationic metabolites and 5μM for anionic metabolites). Tissues were then homogenized (1,500 rpm, 120 sec, 3 times) and the supernatant (400 μL x 2) was filtered through a 5-kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) in order to remove macromolecules. The filtrate was concentrated by centrifugation and then resuspended in 50 μL of ultrapure water immediately before measurements.

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Results were obtained using capillary electrophoresis-time of flight mass spectroscopy (CE-TOFMS) and triple quadruple mass spectroscopy (CE-QqQMS) analysis. Capillary electrophoresis (CE) involves sorting molecules based on their ionic mobility, how they move along a medium in the presence of an electric field. Mass spectroscopy (MS) is the process of ionizing chemical species (e.g. metabolites) and sorting them based on their mass-to-charge ratio (m/z). Time-of-flight MS (TOFMS) is a variant of MS whereby the identity of an ion can be determined based on the time it takes for it to reach a detector separated by a known distance. Triple quadruple MS consists of sets of four cylindrical rods that filter ions based on their m/z when oscillating electric fields are applied to the rods. Simply put, CE-TOFMS is used to identify metabolites and CE-QqQMS is used to quantify their absolute concentrations.

Since cationic and anionic metabolites move in opposite directions in an electric field they are treated separately. For cationic measurements an Agilent CE-TOFMS system (Agilent technologies) was used. The CE voltage was positive 27kV and the MS capillary voltage was 4000V. For anionic measurements an Agilent system was used for CE and an Agilent 6460 TripleQuad LC/MS system was used for MS. The CE voltage was 30kV and the MS capillary voltage was 4000V for positive and 3500V for negative mode. Both cationic and anionic measurements used a fused silica capillary (50 μm x 80 cm). Detected peaks were analyzed with automatic integration software in CE-TOFMS (MasterHands ver. 2.17.1.11 developed at Keio University) and CE-MS/MS (MassHunter Quantitative Analysis B.06.00 Agilent Technologies, Santa Clara, CA, USA) in order to obtain m/z, migration time, and peak area. These values were used to assign metabolites from the HMT database. Absolute quantification of metabolite concentrations was determined by normalizing the peak area of each of the metabolites with respect to the area of the internal standard and by using standard curves, which were obtained by three-point calibrations.

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Summary of Results

Paper I The role of the electron transport chain in maintaining mitochondrial membrane potential in anoxic crucian carp (Carassius carassius) cardiomyocytes Mark A. Scott, Kåre-Olav Stensløkken, and Göran E. Nilsson Manuscript

The experiments presented in Paper I investigate how ΔΨM is affected by blockers of the ETC. The maintenance of ΔΨM is essential to cellular survival and a loss of ΔΨM characterises an irreversible cascade towards cell death. Isolated cardiomyocytes from the anoxia-tolerant carp and anoxia- intolerant trout were loaded withTMRM and exposed to CCCP, rotenone, antimycin, cyanide, and oligomycin. Crucian carp cardiomyocytes were found to maintain ΔΨM for much longer than trout when CCCP was administered or when Complex IV of the ETC was blocked with cyanide (Figure 5).

Additionally, inhibition of Complex V reversal with oligomycin accelerated the loss of ΔΨM, suggesting a role of the ATP synthase in preserving ΔΨM during the process of acclimating to anoxia. The loss of ΔΨM occurred in both species when complexes I and III were inhibited with rotenone and antimycin, respectively, suggesting that they contribute to the maintenance of ΔΨM in the anoxic crucian carp.

When complexes I, III, IV, and V were inhibited together the decline of ΔΨM was nearly identical between the anoxia-tolerant carp and the anoxia-intolerant trout, suggesting that specific differences in the function or utilization of these complexes are major contributors to the superior ability of crucian carp to maintain ΔΨM in anoxia. Taken together, these findings demonstrate inherent adaptations of crucian carp mitochondria to maintain ΔΨ in anoxia.

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Figure 5. Mitochondrial membrane potential in trout and crucian carp cardiomyocytes exposed to cyanide and oligomycin A. Cells were stained with the fluorescent dye TMRM for 30 min before being exposed to either cyanide (CN)(A), oligomycin A (B), or CN combined with oligomycin A (C) for 3, 12, and 32 h. Asterisks denote significant differences from 3 h (*P<0.05; Two-way ANOVA; Holm-Sidak post-hoc

41 test). Daggers denote significant differences between oligomycin A control and CN combined with oligomycin A at 32 h. (†P<0.05). Data are presented as means (±SEM) of 6 independent experiments.

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Paper II Enzymatic and morphological adjustments to anoxia in the crucian carp (Carassius carassius) mitochondria Mark A. Scott, Cathrine E. Fagernes, Göran E. Nilsson & Kåre-Olav Stensløkken Manuscript

In Paper II electron microscopy was used to evaluate the effect of anoxia exposure on mitochondrial volume and number in crucian carp heart and red muscle (RM). Mitochondrial volume increased in anoxic RM but not in heart and anoxia had no effect on mitochondrial number in the heart or RM. Mitofusin (MFN) mRNA was measured and RM MFN2 increased nearly fivefold in anoxia, suggesting that mitochondrial fusion may be occurring in RM. Additionally, enzyme activity assays and qPCR was used to see if upon exposure to anoxia crucian carp inhibition of ATP synthase occurs as has been observed in other models of anoxia tolerance. We observed a significant decrease in ATP synthase activity in the heart of anoxia-acclimated crucian carp (Figure 6). Inhibition of ATP synthase reversal is believed to occur in order to prevent depletion of limited anaerobic ATP supply but the underlying mechanism of ATP synthase inhibition in anoxia is not well-established. We observed a significant decrease in ATP synthase subunit mRNA. Specifically, reductions in gene expression occurred in the c-ring subunits of the

Fo membrane-embedded portion and the gamma subunit of the F1. Intriguingly, both the mean reduction in ATP synthase gene expression and enzyme activity were approximately threefold. This suggests that a reduction in ATP synthase subunit gene expression is responsible for the reduced ATP synthase activity following acclimation to anoxia. Furthermore, CS and COX enzyme activity was measured in heart and RM. RM CS and COX decreased and heart COX increased in crucian carp acclimated to anoxia for six days. The ratio of COX:CS reveals that the oxidative capacity of hearts of the anoxic crucian carp is similar to that of the normoxic trout heart. The observed tissue-specific differences in oxidative capacity could reflect a mechanism for prioritizing oxygen delivery to the heart rather than RM when oxygen availability is limited.

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Figure 6. ATP synthase activity in crucian carp red muscle and heart. Red muscle and heart were sampled from 6 day normoxic and 6 day anoxic crucian carp. Asterisks denote a significant difference (P<0.001; One-way ANOVA; Dunn’s post-hoc test) between treatments. Data are presented per gram wet weight of tissue and are the means (±SD) of 8 individuals.

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Paper III The Metabolome of the anoxia-tolerant crucian carp (Carassius carassius) heart suggests that anoxic survival is promoted by the glycerol phosphate shuttle, purine metabolism, and fumarate as a terminal electron acceptor Mark A. Scott, Kåre-Olav Stensløkken, and Göran E. Nilsson Manuscript

Very little is known about the metabolic pathways involved in mitochondrial anoxia tolerance. Paper III profiles the metabolome of the anoxia-tolerant crucian carp heart. The substrates of glycolysis in anoxia accumulated upstream of GAPD and were depleted downstream. This suggests an inhibition of GAPD activity, which is consistent with others’ findings in the turtle and carp brain. It is likely that this occurs in order to ration the substrates necessary for providing ATP supply but it may also be occurring to increase the levels of G3P, which increased in anoxia by a factor of 44. G3P is the electron donor in the GPS. In comparison, the metabolites of the malate aspartate shuttle, the primary means of transporting reducing equivalents of glycolysis to the electron transport chain, are depleted in anoxia. Alpha- ketoglutarate is reduced to below detectable levels and aspartate is likely directed off to the purine nucleotide cycle in order to produce fumarate. The production of fumarate in anoxia is important because in the absence of oxygen fumarate is likely serving as a terminal electron acceptor of the ETC. Evidence in support of this is that all measured intermediates of the citric acid cycle decreased significantly in anoxia with the exception of succinate, which is reduced from fumarate and increased by a factor of 15 (Figure 7). The largest effect of anoxia observed was a 98-fold increase in uric acid. Upstream metabolites of uric acid production, xanthine and hypoxanthine, also increased by factors of 10 and 5, respectively. The source of uric acid in the anoxic crucian carp is likely the breakdown of AMP. The breakdown of AMP may be beneficial in anoxia as ammonium is produced that combats acidification and uric acid is reported to have antioxidant properties.

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Figure 7. Citric acid cycle intermediates from hearts of normoxic and anoxic crucian carp. Asterisks denote a significant difference (P*<0.05. **<0.01; Welch’s t-test) between treatments of a given metabolite. Data are presented as means (±SD) of 6 individuals.

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General Discussion

Issues associated with oxygen deprivation are becoming increasingly problematic. On the scale of ecosystems down to subcellular levels, damages from anoxic events are on the rise. A better understanding of anoxia-tolerance is necessary to combat a wide range of maladies from loss of marine life to ischemic heart disease and stroke. As the site of oxygen utilization, mitochondrial function is at the centre of these issues. The collection of work in this thesis uses the anoxia-tolerant crucian carp to investigate mechanisms of anoxia-tolerance. In particular, mechanisms related to the maintenance of mitochondrial membrane potential, the anoxic ETC, alternative electron acceptors, alterations to mitochondrial ultrastructure, and the balance of ATP supply and demand.

Maintenance of mitochondrial membrane potential

The maintenance of ΔΨM is regarded to be essential for survival in anoxia. It is well established that a loss of ΔΨM rapidly progresses to cell death in all systems that have been studied (Gottlieb et al., 2003; Breukelen et al., 2010; Galli and Richards, 2014). Nevertheless, exposure to anoxia in frogs has been seen to result in a lowered baseline ΔΨM (St-Pierre et al., 2000), and it could not be taken for granted that the crucian carp heart maintained ΔΨM in anoxia. Hypothetically, it could instead have acquired means to block the lethal downstream effects of a loss of ΔΨM. However, we found that isolated cardiomyocytes from crucian carp maintained ΔΨM under conditions of simulated anoxia (cyanide exposure; Figure 5), where it did not experience significantly greater cell death (Paper I). Our results show that with respect to ΔΨM, the crucian carp heart behaves more similarly to the turtle heart than frog skeletal muscle (Galli et al., 2013). We also demonstrated with the protonophore CCCP that crucian carp cardiomyocytes have an inherent ability to maintain ΔΨM much better than that of the anoxia- intolerant brown trout. We found that Complex I and III of the ETC are necessary for the maintenance of

ΔΨM in both species. Interestingly, when inhibiting the ETC entirely, we found that the ΔΨM dissipated identically between both species. This suggests that the adaptations of crucian carp heart mitochondria to maintain ΔΨM in anoxia are associated with the ETC.

ATP synthase reversal may be playing a role in the maintenance of ΔΨM during anoxia. We measured increased ATP synthase inhibition following acclimation to anoxia (Figure 6; Paper II), which likely helps minimize consumption of ATP by ATP synthase reversal. However, some ATP synthase activity is clearly needed since inhibiting this enzyme during simulated anoxia led to a loss of ΔΨM in the cardiomyocytes

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(Paper I). Thus, ATP synthase reversal plays a role in the maintenance of ΔΨM in anoxia as has been observed in other models (St-Pierre et al., 2000; Hawrysh and Buck, 2013).

In the absence of oxygen, fumarate is likely serving as terminal electron acceptor to the ETC. Fumarate decreased by a factor of 5.9 in the anoxia-exposed crucian carp heart (Figure 7). This is likely because it is rapidly being reduced to succinate, as fumarate has been shown to preserve ΔΨM by serving as a terminal electron acceptor of the ETC (Tielens and Hellemond, 1998; Buck, 2000; Sridharan et al., 2008; see Figure 8). In support of the suggestion that fumarate serves as the terminal electron acceptor in crucian carp mitochondria deprived of oxygen, succinate levels rose by a factor of 14.8 in anoxia. Although succinate levels have not been measured in the anoxic crucian carp heart before, they have been measured in the anoxic crucian carp red and white muscle. Johnston (1975) found that in white muscle succinate levels barely changed but in the RM they increased by a factor of ~6 in anoxia, likely related to the far greater amounts of mitochondria in RM.

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Figure 8. In oxygen’s absence, fumarate serves as terminal electron acceptor to the electron transport chain also in mammals. Succinate dehydrogenase (SDH) reversal in conditions of oxygen limitation facilitates the pumping of protons through Complex I. This mechanism is apparently also at play in anoxia-tolerant animals to help maintain mitochondrial membrane potential in anoxia. It is not clear how anoxia-tolerant animals manage the accumulating succinate and following reperfusion, mitigate the harmful effects of reactive oxygen species associated with the oxidation of succinate. Illustration from Chouchani et al., (2014).

The main source of reducing equivalences to the anoxic ETC is likely NADH. However, it is not clear how NADH generated in glycolysis is being transported from the cytosol to Complex I. The most likely pathway would be the MAS but two of the main components of the MAS, malate and aspartate decreased significantly in anoxia by factors of 2.9 and 4.5, respectively (Paper III). Another central metabolite of the MAS, α-ketoglutarate, decreased from 17.9 nmol/g to below detection levels in anoxia. Metabolites of the MAS are likely decreasing in anoxia because they are being utilized in other pathways, as has been demonstrated with brain glutamate and RM aspartate in the anoxia-exposed turtle and goldfish, respectively (Arillo et al., 1972; Waarde et al., 1982; Nilsson et al., 1990). The likely destination of MAS intermediates in the anoxic crucian carp heart is the PNC, which produces fumarate from aspartate and accumulating AMP.

The GPS would normally participate substantially in the transportation of reducing equivalences from the cytosol to the ETC but this is not likely occurring because in the anoxic ETC proton pumping only occurs at Complex I (Figure 8) and the GPS transports reducing equivalences to Complex II. The metabolites of the GPS, DHAP and G3P, increased in anoxia by factors of 44 and 3.1, respectively. The significant increase in GPS intermediates can be explained by a decrease in GAPDH activity, which has been observed in the turtle and crucian carp brain (Smith et al., 2009; Smith et al., 2015), and would result in elevated levels of DHAP. The considerable increase in G3P is likely because DHAP is being reduced to G3P for the purpose of regenerating NAD for glycolysis and G3P is unable to be converted back to DHAP at the mitochondria because in the anoxic ETC no oxygen is available to facilitate electron flow from Complex II.

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Reducing ATP demand and increasing ATP supply in anoxia Maintaining energy homeostasis is essential for survival. Since cardiomyocytes normally receive 95% of their ATP from oxidative phosphorylation, at least in mammals (Ingwall, 2002; Opie, 2004), it is imperative in anoxia that energetically expensive processes be downregulated and that anaerobic ATP production be upregulated (Hochachka et al., 1996; Galli et al., 2014). In the absence of any intervention the high-energy phosphate pool can be depleted within seconds, which quickly leads to cell death (Wang et al., 2010; Doenst et al., 2013). A critical adaptation to reduce ATP demand is inhibition of ATP synthase reversal. Inhibition of ATP synthase reversal is important for long-term anoxic survival because up to 50-80% of ATP consumed during oxygen deprivation is by the reversal of the ATP synthase (Rouslin et al., 1990). Since the inhibition of ATP synthase reversal has only been observed in the hypoxia- tolerant frog skeletal muscle (St-Pierre et al., 2000), turtle heart (Galli et al., 2013), and diapausing killifish embryo (Duerr and Podrabsky, 2010) it is not known how universal ATP synthase inhibition is as a strategy for survival in anoxia.

Clear evidence has not yet been provided on the underlying mechanism of inhibition of ATP synthase reversal in anoxia (Galli et al., 2013). We found that indeed ATP synthase activity was reduced in the hearts of anoxic crucian carp (paper II). However, we did not observe any effect of anoxia on ATP synthase activity in the RM. ATP synthase activity decreased by 63% in the crucian carp heart compared to ~83% in the turtle heart (Galli et al., 2013). The greater inhibition in turtle heart likely corresponds to greater metabolic suppression. Perhaps since unlike the turtle, crucian carp maintains heart rate and cardiac output in anoxia (Hicks and Farrell, 2000; Stecyk et al., 2004), the crucian carp is unable to decrease ATP synthase activity to such an extent. Additionally, it is possible that ATP synthase activity cannot be inhibited further because reversal of the ATP synthase is necessary for the maintenance of

ΔΨM, as is observed in frog skeletal muscle (St-Pierre et al., 2000). In support of this explanation, we found that crucian carp cardiomyocytes in simulated anoxia depolarized ΔΨM when the ATP synthase was inhibited (paper I). However, it is also possible that ATP synthase reversal is a short-term solution to preventing a loss of ΔΨM during the transition into anoxia.

Proposed mechanisms for ATP synthase inhibition from mammalian studies include binding of inhibitory factors (Vander Heide et al., 1996) and S-nitrosylation (Sun et al., 2007) but there is not yet any evidence in support of these mechanisms from an anoxia-tolerant model. Our comprehensive findings on the gene expression of ATP synthase subunits reveal a significant down-regulation (Paper II; See Figure 2 for

50 an illustration of subunits). Interestingly, the mean decrease in ATP synthase subunit expression was 67%, which is similar to the observed 63% decrease in ATP synthase enzyme activity, suggesting that a decrease in ATP synthase subunit expression causes a decrease in ATP synthase activity in anoxia. Of the genes measured, mRNA levels decreased 3-4 times for c, gamma, and two of the four paralogues for e, but no changes were observed in the assembly factors that bind to alpha and beta. The main transmembrane subunit, c, is where protons traverse the inner mitochondrial membrane. Recently subunit c has been identified as a critical component in mitochondrial permeability transition (Bonora et al., 2013; Beutner et al., 2016; Biasutto et al., 2016). Since mitochondrial permeability transition leads to cell death upon reoxygenation (Griffiths and Halestrap, 1995; Yellon and Hausenloy, 2007; Boyman et al., 2016) then perhaps crucian carp down-regulate subunit c expression in anoxia so as to mitigate the effects of such reperfusion injury.

Subunit gamma forms the central shaft that connects the Fo rotary motor to the F1 catalytic core. Presumably a decrease in gamma would result in reduced activity but it is not readily apparent why reducing gamma would be preferred over other subunits of the ATP synthase. Levels of gamma have been observed to increase in a study of mammalian heart failure but no explanation is offered why (Liu et al., 2014). In light of this evidence, perhaps reducing gamma expression is an adaptation of anoxia- tolerant animals to reduced oxygen. However, expression of gamma was unchanged in hypoxia-exposed whiteleg shrimp (Martinez-Cruz et al., 2015) so perhaps this adaptation is unique to the crucian carp or other truly anoxia-tolerant models.

Neither RM ATP synthase subunit expression nor ATP synthase enzyme activity decreased significantly in anoxia (Paper II). This evidence supports the hypothesis that a reduction of ATP synthase subunit gene expression is required for inhibition of ATP synthase activity, as is observed in the anoxic crucian carp heart. This finding is in contrast with the observed decrease in skeletal muscle ATP synthase activity in the anoxia-tolerant frog (St-Pierre et al., 2000). However, an explanation for this could be that the frogs hibernate (Donohoe and Boutilier, 1998) and do not require their skeletal muscle to be functional whereas the crucian carp still maintain some locomotory behaviour in anoxia (Nilsson et al., 1993) and must therefore retain some degree of skeletal muscle function. Moreover, crucian carp RM is highly active in ethanol production during anoxia (Fagernes et al., 2017). It seems reasonable that there is some minimal level of ATP synthase activity that is required for cellular function and even though the heart activity decreases significantly, it does not decrease below the muscle ATP synthase activity.

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The metabolomics results from Paper III gave suggestions for strategies for maintaining energy homeostasis in anoxia. In glycolysis, intermediates upstream of GAPDH accumulated (mean of 4.4x) and intermediates downstream were depleted (by a mean factor of 5). This would be expected to occur if GAPDH activity decreased as has been observed in the turtle and crucian carp brain (Smith et al., 2009; Smith et al., 2015). It has been suggested that GAPDH activity decreases in anoxia in order to extend the use of glycolytic reserves (Kelly and Storey, 1988). GA3P increased by a factor of 11 in anoxia. GA3P is readily interchangeable with DHAP, which increased by a factor of 3. The conversion of DHAP into G3P regenerates NAD that is necessary for glycolysis. As evidence for the conversion of DHAP into G3P as a means of maintaining ATP production via glycolysis in anoxia, G3P rose by a factor of 44. Additionally, G3P is reported to produce less ROS than succinate when oxidized (Kikusato and Toyomizu, 2015; see Figure 8) so perhaps having excess G3P in anoxia is beneficial during reoxygenation. Another intermediate of glycolysis to increase in anoxia was F16BP, by a factor of 3.5. F16BP may provide some benefit to the crucian carp heart in anoxia as it has been shown to prevent damage in hypoxia and ischemia by improving antioxidant defence (Alva et al., 2016).

Adenylate kinase (AK), which interconverts ADP with ATP and AMP may be playing an important role in anoxia. AK is believed to shuttle high-energy phosphates from sites of ATP-production to sites of ATP- consumption (Dzeja et al. 1999; Dzeja and Terzic, 2009), particularly from glycolysis (Zeleznikar et al., 1990; Wyss and Kaddurah-Daouk, 2000), which is the predominant form of ATP production in anoxia. Indeed, hypoxic AK-knockout hearts from mice have been shown to have depressed ATP levels (Pucar et al., 2000) and AK has been shown to stabilize energy charge in the skeletal muscle of the hibernating prairie dog (English and Storey, 2000). We observed in the anoxic crucian carp heart that ATP levels decreased by a factor of 2, and ADP and AMP increased by factors of 3.5 and 13.8, respectively. This demonstrates that as ATP is being consumed, AMP is accumulated. In order to prevent the inhibition of AK, AMP must be disposed of, as demonstrated in trout and turtle myocardial tissue (Hartmund and Gesser, 1996). However, the extent to which AMP catabolism occurs in the anoxic crucian carp is unclear, since the pool of AMP+ADP+ATP is limited and ADP is required for rephosphorylation to ATP by glycolysis.

The pathway that removes accumulating AMP likely begins with AMP deaminase and ends with uric acid. AMP deaminase has been observed to have significantly increased kinetic parameters (increased

Vmax, decreased Km) following anoxia exposure in the skeletal muscle of the anoxia-tolerant turtle (Zhou,

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2006), which suggest an adaptive role for AMP deaminase in anoxia tolerance (Zhou, 2006). A benefit of removing accumulating AMP by this pathway is that AMP deaminase produces ammonium, which combats cellular acidification in anoxia (English and Storey, 2000). However, IMP and inosine levels did not change significantly in the anoxia-exposed crucian carp heart (Paper III). This is likely because they were further being degraded to hypoxanthine, xanthine, and finally uric acid, which increased by factors of 4.5, 10.3, and 98.1, respectively. Normally this process would produce ROS but in the absence of oxygen NAD is reduced instead (Maiuolo et al., 2016). Furthermore, uric acid is the readily excreted end- product of AMP breakdown and it is not clear why it is being retained. A possible explanation is that uric acid accumulation is beneficial upon reoxygenation as it has been reported to confer antioxidant features (Ames et al., 1981; Storey, 1996; Maxwell et al., 1997).

Two other process that increase ATP supply in anoxia are the regeneration of NAD by LDH and the liberation of high energy phosphates by CK. The NAD regenerated from LDH is a cofactor for ATP production by glycolysis. For this reason, lactate accumulates in both anoxia-tolerant and anoxia- intolerant models (Milligan, 1996; Warren and Jackson, 2006). We observed both a significant decrease in pyruvate (by a factor of 2.1) and a significant increase in lactate (by a factor of 8.5) in the anoxia- exposed crucian carp heart (Paper III). These findings are consistent with the investigations from others (Johnston, 1975; Johnston and Bernard, 1983; Johanssen et al., 1995). Unique to the crucian carp and goldfish, accumulating lactate is transformed to ethanol in RM and white muscle and released to the water over the gills (Nilsson, 2001). Under conditions of oxygen limitation and reduced ATP supply phosphocreatine is rapidly consumed. This occurs because the liberation of ATP from phosphocreatine occurs as soon as ATP levels drop below resting (Chien, 2013). We observed a decrease in phosphocreatine levels by a factor of 7.2 in the hearts of anoxia-exposed crucian carp (Paper III). Phosphocreatine has been shown to provide cardiac protection under conditions of reduced ATP supply (Landoni et al., 2016) and our findings are consistent with others (Johnston and Bernard, 1983; Waarde et al., 1990; Stecyk et al., 2009).

Mitochondrial fusion and ultrastructure It is not clear how changes in mitochondrial ultrastructure contribute to survival in anoxia. We observed a significant increase in RM mitochondrial volume but did not measure any change in volume in the heart mitochondria of anoxia exposed crucian carp (Paper II). The absence of any effect of oxygen limitation on heart mitochondrial volume is also seen in rats held in chronic hypoxia and humans with

53 end-stage heart failure (Costa et al., 1988; Holzem et al., 2016). Perhaps no difference is observed between the anoxia-tolerant crucian carp and anoxia-intolerant models because maintaining constant mitochondrial volume is necessary for normal function of the heart. Since changes in mitochondrial volume may affect the contractility of muscle cells (Kaasik et al., 2004), and the crucian carp maintains cardiac output in anoxia (Stecyk et al., 2004), then the crucian carp may have to maintain mitochondrial volume in the heart.

The observed increase in RM but not heart mitochondrial volume may be related to how crucian carp locomotory behaviour changes in anoxia. Upon acclimation to anoxia the crucian carp is known to reduce swimming (Nilsson and Renshaw, 2004). If the crucian carp is not using its RM as much in anoxia then perhaps it can afford to compromise muscle function in exchange for reducing the overall energy burden of maintaining ΔΨM. The increase in mitochondrial volume may be a result of mitochondrial fusion. Presumably a lower surface area-to-volume ratio of mitochondria would result in less ΔΨM lost to proton leak and therefore mitochondria that are less energetically expensive to maintain. In contrast, mitochondria from skeletal muscle of humans with impaired oxygen delivery as a result of disease have been observed to decrease in volume (Baum et al., 2016). An increase in mitochondrial volume in crucian carp but not human skeletal muscle would be consistent with the hypothesis that mitofusion is an adaptation to tolerating low oxygen conditions.

Mitofusion, which involves the mixing of mitochondrial substrates and merging of mitochondrial membranes, would be advantageous in anoxia because damaged mitochondria could be rescued by healthy ones. Consistent with this hypothesis, we observed a significant increase in MFN2 expression in both the RM and heart of anoxia-exposed crucian carp. However, the increase was to a greater extent in the RM. Mitochondrial fusion is associated with protection from apoptotic stress (Tondera et al., 2009). However, it is still not definitive if MFN2 increases cellular survival in oxygen-deprivation challenges or participates in necrosis (Dong et al., 2016). One study examining mitochondria from rat cardiomyocytes determined that MFN2 plays a substantial role in oxidative stress-induced apoptosis (Shen et al., 2007). Further complicating the matter, repression of MFN2 has been seen to reduce glucose oxidation and

ΔΨM (Bach et al., 2003). Consistent with this, over-expression of MFN2 causes an increase in glucose oxidation, ΔΨM, and overexpression of components of the ETC (Pich et al., 2005). Our findings are evidence in support of mitofusion occurring in the RM of anoxic crucian carp but the role of increased MFN2 expression in the heart is not clear.

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Elevated levels of MFN2 in the crucian carp heart may be playing a role in glucose oxidation and the maintenance of ΔΨM. In addition to initiating mitofusion, MFN2 is also involved in the expression of components of the ETC (Chen et al., 2005; Pich et al., 2005; Soriano et al., 2006). Complex IV specifically, has been observed to increase in intermediate hypobaric hypoxia preconditioning (Chitra and Boopathy, 2014) and is associated with having antioxidant effects by reducing the generation of ROS from complexes I and II and by reducing electron leakage (Chen et al., 2003; Parise et al., 2005). We observed a significant increase in anoxia-exposed crucian carp heart Complex IV activity. Our finding is comparable to the non-significant increase in Complex IV activity observed in the anoxic turtle heart (Galli et al., 2013) and is consistent with the significant increases in turtle heart COX subunit expression following exposure to anoxia (Cai and Storey, 1996; Gomez, 2016). Taken together, although small, the increase in Complex IV activity may be beneficial to the anoxic heart.

In contrast with the heart, we observed a significant decrease in crucian carp RM COX activity. At first this seemed peculiar as having higher COX activity would increase aerobic capacity, as has been observed in the skeletal muscle of hypoxia-exposed common carp (Zhou et al., 2000). But perhaps the discrepancy between RM and heart COX activity in the anoxia-exposed crucian carp reflects any oxygen available during hypoxia is prioritized for use by the heart. We also observed a significant decrease in CS activity in anoxia-exposed crucian carp RM. The effect of anoxia on CS activity is similarly observed in the anoxia-tolerant turtle brain (Pamenter et al., 2016). The decrease in CS activity may reflect metabolic suppression, mitophagy following mitochondrial damage, or a targeted down-regulation of mitochondrial content in order to decrease the ATP demand required to maintain ΔΨM. It seems reasonable that if tissues are no longer operating at capacity then it will not be necessary to maintain the same pre-anoxia compliment of mitochondria.

However, we measured no significant change in mitochondrial number in crucian carp heart or RM. This would suggest that mitochondrial fusion may not be occurring and that elevated levels of MFN are for the aforementioned purpose of effecting glucose oxidation or the maintenance of ΔΨM. An increase in mitochondrial volume without a decrease in mitochondrial number would result in more mitochondrial membrane in anoxia, which would enhance ethanol production in crucian carp RM. The ability of crucian carp to avoid lactic acidosis by converting lactate to ethanol via acetaldehyde in the skeletal muscle is a critical adaptation for survival in anoxia (Mourik et al., 1982; Johnston and Bernard, 1983; Nilsson,

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1988). More inner mitochondrial membrane would enhance ethanol production because pyruvate decarboxylase and the pyruvate dehydrogenase complex associate with the inner mitochondrial membrane (Stanley and Perham, 1980; Shimoda, 2014). In light of the recent work by Fagernes et al. (2017), future studies investigating the effect of anoxia on skeletal muscle mitochondrial ultrastructure should consider the possibility of membrane changes to enhance ethanol production and survival.

Conclusion This thesis adds evidence to the mounting number of studies describing the importance of energy homeostasis and maintaining ΔΨM as adaptations to surviving oxygen deprivation. However, the main contributions of this thesis are with respect to the mechanisms underpinning the adaptations of the crucian carp heart to anoxia. Specifically, that a reduction in ATP synthase subunit transcription is the likely cause of inhibition of ATP synthase reversal in anoxia and that fumarate is likely serving as terminal electron acceptor to the anoxic ETC. These mechanisms appear to constitute important means by which the crucian carp is able to reduce ATP demand and maintain ΔΨM in anoxia. Other findings from this thesis are that an inhibited crucian carp ETC depolarises identically to that of a brown trout, suggesting that the ETC is needed for the maintenance of ΔΨM in the anoxic crucian carp heart. Additionally, elevated levels of MFN2 may be responsible for the observed increase in RM mitochondrial volume, which may be linked to ethanol production, and may also play a role in anoxia tolerance of the heart.

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