Cardiac survival in anoxia-tolerant vertebrates: An electrophysiological perspective

Certain vertebrates, such as freshwater turtles of the genus Chrysemys and Trachemys and crucian carp (Carassius carassius), have anoxia-tolerant hearts that continue to function throughout prolonged periods of anoxia (up to many months) due to successful balancing of cellular ATP supply and demand. In the present review, we summarize the current and limited understanding of the cellular mechanisms underlying this cardiac anoxia tolerance. What emerges is that cold temperature substantially modifies cardiac electrophysiology to precondition the heart for winter anoxia. Intrinsic heart rate is slowed and density of sarcolemmal ion currents substantially modified to alter cardiac action potential (AP) characteristics. These changes depress cardiac activity and reduce the energetic costs associated with ion pumping. In contrast, anoxia per se results in limited changes to cardiac AP shape or ion current densities in turtle and crucian carp, suggesting that anoxic modifications of cardiac electrophysiology to reduce ATP demand are not extensive. Additionally, as knowledge of cellular physiology in non-mammalian vertebrates is still in its infancy, we briefly discuss the cellular defense mechanisms towards the acidosis that accompanies anoxia as well as mammalian cardiac models of hypoxia/ischemia tolerance. By examining if fundamental cellular mechanisms have been conserved during the evolution of anoxia tolerance we hope to have provided a framework for the design of future experiments investigating cardiac cellular mechanisms of anoxia survival.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro , while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.     

Calcium and protein phosphatase 1/2A attenuate N-methyl-D-aspartate receptor activity in the anoxic turtle cortex

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.     

Adventures in oxygen metabolism

Peter W. Hochachka led a grand life of science adventure and left as his legacy a whole new field--biochemical adaptation. Oxygen was at the core of Peter's career and his laboratory made major contributions to our understanding of how animals deal with variation in oxygen availability in many forms. He analyzed the molecular mechanisms that support facultative anaerobiosis, studied muscle exercise metabolism for high speed flight, swimming and running, investigated mammalian diving on many trips to the Antarctic to study Weddell seals, and probed the metabolic and genetic adaptations that provide optimal hypoxia tolerance for humans residing at high altitudes. His work illuminated both biochemical and physiological mechanisms that are used to optimize aerobic metabolism, to compensate for hypoxic insults, and to conserve energy by strong metabolic rate depression under anoxia. His articles, books and lectures galvanized the field with leading-edge insights and theories and he consistently challenged comparative biochemists to use their unique model systems to explore the range and breadth of animal strategies of biochemical adaptation. Lessons drawn from my training in Peter's laboratory have led me on continuing explorations of adaptations in enzyme function, signal transduction, gene expression, and antioxidant defenses ranging over systems of anoxia tolerance, freezing survival, estivation, and mammalian hibernation.     

Adenosine as a signal for ion channel arrest in anoxia-tolerant organisms

Certain freshwater turtles and fish are extremely anoxia-tolerant, capable of surviving hours of anoxia at high temperatures and weeks to months at low temperatures. There is great interest in understanding the cellular mechanisms underlying anoxia-tolerance in these groups because they are anoxia-tolerant vertebrates and because of the far-reaching medical benefits that would be gained. It has become clear that a pre-condition of prolonged anoxic survival must involve the matching of ATP production with ATP utilization to maintain stable ATP levels during anoxia. In most vertebrates, anoxia leads to a severe decrease in ATP production without a concomitant reduction in utilization, which inevitably leads to the catastrophic events associated with cell death or necrosis. Anoxia-tolerant organisms do not increase ATP production when faced with anoxia, but rather decrease utilization to a level that can be met by anaerobic glycolysis alone. Protein synthesis and ion movement across the plasma membrane are the two main targets of regulatory processes that reduce ATP utilization and promote anoxic survival. However, the oxygen sensing and biochemical signaling mechanisms that achieve a coordinated reduction in ATP production and utilization remain unclear. One candidate-signaling compound whose extracellular concentration increases in concert with decreasing oxygen availability is adenosine. Adenosine is known to have profound effects on various aspects of tissue metabolism, including protein synthesis, ion pumping and permeability of ion channels. In this review, I will investigate the role of adenosine in the naturally anoxia-tolerant freshwater turtle and goldfish and give an overview of pathways by which adenosine concentrations are regulated.     

AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.     

The role of Bax/Bcl-2 and pro-caspase peptides in hypoxia/reperfusion-dependent regulation of MAPK(ERK): discordant proteomic effect of MAPK(p38)

BACKGROUND: The molecular regulation of MAPKs and apoptosis was investigated in a model of hypoxia-tolerance. Survival of neurons in Chrysemys picta bellii, an anoxia-tolerant turtle, involves a reduction in energy metabolism. The biochemical/physiological mechanisms of anoxia tolerance have been examined at the level of ion transport and ATP turnover. However, changes in the phosphorylation state of key enzymes and kinases, mainly, MAPKs, may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis. METHODS: If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain was then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing brain in a buffer, followed by threonine and tyrosine phosphorylation determination of MAPKs, and caspase activity. RESULTS: MAPK(p38) was decreased after reoxygenation following 1 day and 1 week hypoxia. The effect of hypoxia on the phosphorylation of MAPK(ERK) was biphasic: Enhancement at 5h and inhibition at 6 weeks. Pro-caspases 8/9 were unchanged by hypoxia until increasing at 6 weeks. Both pro-caspases were upregulated by reoxygenation at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation induced the cleavage of pro-caspases 8/9 into p20 and p10, respectively. Furthermore, hypoxia induced Bax at 3 days and 1 week, and reoxygenation increased Bax #8776; 4-fold at 1 day. Although the expression of Bcl-2 was slightly increased by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with Bax. CONCLUSION: These results indicate that hypoxia up-regulates MAPK(ERK) but not MAPK(p38;) hypoxia/reperfusion increases the expression of caspases and pro-apoptotic cofactors. The patterns of MAPK regulation suggest the significance of these kinases in cellular adaptation to oxygen deprivation with biomedical correlations, and thereby identify novel natural responsive signaling cofactors in Chrysemys picta bellii with potential pharmacologic and clinical applications.     

Mechanism, origin, and evolution of anoxia tolerance in animals

Organisms vary widely in their tolerance to conditions of limiting oxygen supply to their cells and tissues. A unifying framework of hypoxia tolerance is now available that is based on information from cell-level models from highly anoxia-tolerant species, such as the aquatic turtle, and from other more hypoxia-sensitive systems. The response of hypoxia-tolerant systems to oxygen lack occurs in two (defense and rescue) phases. The first lines of defense against hypoxia include a drastic, if balanced, suppression of ATP demand and supply pathways; this regulation allows ATP levels to remain constant, even while ATP turnover rates greatly decline. The ATP requirements of ion pumping are down-regulated by generalized ‘channel’ arrest in hepatocytes and by the arrest of specific ion channels in neurons. In hepatocytes, the ATP demands of protein synthesis are down-regulated on exposure to hypoxia by an immediate global blockade of the process (probably through translational arrest caused by complexing between polysomes and elongation factors). In hypoxia-sensitive cells, this translational arrest seems irreversible, but hypoxia-tolerant systems activate ‘rescue’ mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal transduction pathway that leads to the specific activation of some genes (increased expression of several proteins) and to specific down-regulation of other genes (decreased expression of several other proteins). The functional roles of the oxygen-sensing and signal-transduction system include significant gene-based metabolic reprogramming — the rescue process — with maintained down-regulation of energy demand and supply pathways in metabolism throughout the hypoxic period. We consider that, through this recent work, it is becoming evident how normoxic-maintenance ATP turnover rates can be down-regulated by an order of magnitude or more — to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. Because the phylogenies of the turtles and of fishes are well known, we are now in an excellent position to assess conservative vs. adaptable features in the evolution of the above hypoxia-response physiology in these two specific animal lineages.     

Metabolic adaptation to prolonged anoxia in leaves of American cranberry (Vaccinium macrocarpon)

The indigenous North American Cranberry ( Vaccinium macrocarpon ), when cultivated in specially constructed cranberry bogs, is normally flooded in winter to prevent frost injury. This protection under ice can give rise to prolonged periods of anoxia, which depending on the state of the vines and environmental conditions, can cause severe oxygen-deprivation injury. An experimental study of the tolerance of cranberry vines to controlled total anoxia reveals that mature dark-green perennating leaves with high carbohydrate levels are able to survive prolonged periods of total oxygen-deprivation while younger newly formed leaves are readily damaged. During the anoxic treatment the mature leaves exhibit a marked downregulation of metabolism. Carbohydrate consumption and energy metabolism stabilize at low levels soon after the switch from aerobic to anaerobic pathways. Pathways such as TCA cycle or photosynthesis, which are non-operating during the anoxia treatment, are severely affected but still measurable after 28 days anoxia. In the post-anoxic period the perennating leaves rapidly re-establish their capacity for aerobic respiration and photosynthesis.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H₂O₂ release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H₂O₂ release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.     

Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain

Climate cooling over the past one hundred thousand years has resulted in seasonal ice cover at northern and southern latitudes that has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range, North American freshwater turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds. From a comparative perspective this gives us the opportunity to understand how an extremely oxygen-sensitive organ, such as the vertebrate brain, can function without oxygen for long periods. Brain function is based on complex excitatory (on) and inhibitory (off) circuits involving the major neurotransmitters glutamate and, γ-aminobutyric acid (GABA) respectively. When a mammalian brain becomes anoxic, glutamate levels rise within minutes resulting in excitotoxic cell death which does not occur in anoxic turtle brain. The response in turtle brain has been remodelled – GABA levels rise rapidly resulting in large inhibitory GABA receptor currents and inhibition of glutamate receptor function that together depress neuronal activity.     

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).