Mitochondrial survival without oxygen

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Mark Adam Scott

Mitochondrial survival without oxygen

Faculty of Mathematics and Natural Sciences University of Oslo

2017

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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

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© Mark Adam Scott, 2018

Series of dissertations submitted to the

Faculty of Mathematics and Natural Sciences, University of Oslo No. 1949

ISSN 1501-7710

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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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.

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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

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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

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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.

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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

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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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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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9 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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10 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 Foconsists 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 Foconsists 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

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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 2^OC and 24^OC, 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

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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

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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

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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

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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-

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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, overexpression 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

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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

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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

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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 (10^oC). 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.

Mitochondrial survival without oxygen

Mark Adam Scott

Mitochondrial survival without oxygen

Faculty of Mathematics and Natural Sciences University of Oslo

2017

Mitochondrial survival without oxygen

Acknowledgements

Table of Contents

Abstract

List of papers

Abbreviations

Introduction

Aims

Methods