Regulation of the cardiorespiratory system of common carp (Cyprinus carpio) during severe hypoxia at three seasonal acclimation temperatures

Little is known of the cardiorespiratory control mechanisms utilized by hypoxia-tolerant teleost fish to tolerate prolonged periods (h) of near anoxic exposure. Here, we report on the cardiorespiratory control mechanisms of the common carp Cyprinus carpio L. during normoxia and prolonged, severe hypoxic (<0.3 mg O(2) L(-1)) exposure at acclimation temperatures of 5 degrees C, 10 degrees C, and 15 degrees C. Through serial intra-arterial injections of alpha - and beta -adrenergic, cholinergic, and purinergic antagonists while measuring cardiac output (Q), heart rate (f(H)), ventral aortic blood pressure, and respiration rate, we established that autonomic cardiovascular and respiratory control was preserved during severe hypoxia at all three acclimation temperatures and contributed to downregulation of cardiorespiratory activity. Specifically, inhibitory cholinergic tone mediated up to 76% reductions in f(H) and Q during hypoxia, whereas the accompanying arterial hypotension was attenuated by an upregulation of an alpha -adrenergically mediated peripheral vasoconstriction. Despite the overall cardiac downregulation, a large, stimulatory cardiac beta -adrenergic tone was present during prolonged, severe hypoxia, possibly to protect the heart from attendant acidotic conditions. Purinergic blockade, following alpha -adrenergic and cholinergic antagonists, showed that the hypoxic ventilatory depression, which reversed the 2.3- to 7.7-fold increases in respiration rate that occurred with the onset of hypoxia, was a result of purinergic inhibition at all three acclimation temperatures. In contrast, purinergic inhibition of cardiac activity during hypoxia might be important only at 5 degrees C. Finally, given that cardiac power output was reduced 72%-87% during prolonged, severe hypoxia and that glycolysis yields approximately 94% less ATP per mole glucose than oxidative phosphorylation, it seems unlikely that the common carp sufficiently reduces its cardiac energy demand to a level to preclude activation of a partial Pasteur effect. This means that glycogen stores will be used and waste products will accumulate at faster rates, a finding that may help explain why the common carp cannot tolerate such extended periods of severe hypoxia (weeks to months) at cold acclimation temperatures as the freshwater turtle, which is able to reduce its cardiac energy demand to a level that does not require a Pasteur effect and also blunts autonomic cardiovascular control.     

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.     

Intrinsic contractile properties of the crucian carp (Carassius carassius) heart during anoxic and acidotic stress

The crucian carp (Carassius carassius) seems unique among vertebrates in its ability to maintain cardiac performance during prolonged anoxia. We investigated whether this phenomenon arises in part from a myocardium tolerant to severe acidosis or because the anoxic crucian carp heart may not experience a severe extracellular acidosis due to the fish's ability to convert lactate to ethanol. Spontaneously contracting heart preparations from cold-acclimated (6-8°C) carp were exposed (at 6.5°C) to graded or ungraded levels of acidosis under normoxic or anoxic conditions and intrinsic contractile performance was assessed. Our results clearly show that the carp heart is tolerant of acidosis as long as oxygen is available. However, heart rate and contraction kinetics of anoxic hearts were severely impaired when extracellular pH was decreased below 7.4. Nevertheless, the crucian carp heart was capable of recovering intrinsic contractile performance upon reoxygenation regardless of the severity of the anoxic + acidotic insult. Finally, we show that increased adrenergic stimulation can ameliorate, to a degree, the negative effects of severe acidosis on the intrinsic contractile properties of the anoxic crucian carp heart. Combined, these findings indicate an avoidance of severe extracellular acidosis and adrenergic stimulation are two important factors protecting the intrinsic contractile properties of the crucian carp heart during prolonged anoxia, and thus likely facilitate the ability of the anoxic crucian carp to maintain cardiac pumping.     

Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.     

1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish, the common carp (Cyprinus carpio)

All 20.000 different fish species vary greatly in their ability to tolerate and survive fluctuating oxygen concentrations in the water. Especially fish of the genus Carassius, e.g. the crucian carp and the goldfish, exhibit a remarkable tolerance to limited/absent oxygen concentrations. The metabolic changes of anoxia-tolerant crucian carp were recently studied and published. Contrary to crucian carp, the hypoxia-tolerant common carp cannot survive a complete lack of oxygen (anoxia). Therefore, we studied the ¹H-NMR-based metabolomics of brain, heart, liver and white muscle extracts of common carp, subjected to anoxia (0 mg O₂ l^?1) and hypoxia (0.9 mg O₂ l^?1) at 5 °C. Specifically, fish were exposed to normoxia (i.e. 9 mg O₂ l^?1; controls 24 h, 1 week and 2 weeks), acute hypoxia (24 h), chronic hypoxia (1 week) and chronic hypoxia (1 week) with normoxic reoxygenation (1 week). Additionally, we also investigated the metabolic responses of fish to anoxia for 2 h. Both anoxia and hypoxia significantly changed the tissue levels of standard energy metabolites as lactate, glycogen, ATP/ADP and phosphocreatine. Remarkably, anoxia induced increased lactate levels in all tissues except for the heart whereas hypoxia resulted in decreased lactate concentrations in all tissues except for brains. Furthermore, hypoxia and anoxia influenced amino acids (alanine, valine/(iso)leucine) and neurotransmitters levels (GABA, glutamate). Lastly, we also detected ‘other’ i.e. previously not reported compounds to play a role in the present context. Scyllo-inositol levels changed significantly in heart, liver and muscle, providing novel insights into the anoxia/hypoxic responses of the common carp.     

Studies of ribonucleotide reductase in crucian carp-an oxygen dependent enzyme in an anoxia tolerant vertebrate

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O(2)) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.     

The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum

The epaulette shark (Hemiscyllium ocellatum) is among the few vertebrates that can tolerate extreme hypoxia for prolonged periods and, as shown here, anoxia. We examined how anoxia affected this shark's level of responsiveness, concentration of brain ATP and adenosine -- an endogenous neuronal depressant. In addition, we investigated how these variables were affected by aminophylline, an adenosine receptor antagonist. Epaulette sharks placed in an anoxic environment (<0.02 mg O2 l(-1)) lost their righting reflex after 46.3 +/- 2.8 min, but immediately regained vigilance upon return to normoxia. Then 24 h later, the same sharks were injected with either saline or aminophylline (30 mg kg(-1)) in saline and re-exposed to anoxia. In this second anoxic episode, controls sharks showed a 56% decrease in the time taken to lose their righting reflex but maintained their brain ATP levels; conversely, aminophylline-treated epaulette sharks displayed a 46% increase in the time to loss of righting reflex and had significantly lower brain ATP levels. Since anoxia also caused a 3.5-fold increase in brain adenosine levels, these results suggest that adenosine receptor activation had a pre-emptive role in maintaining brain ATP levels during anoxia. Perhaps because adenosine receptor activation initiates metabolic depression, indicated by the early loss of responsiveness (righting reflex), such a mechanism would serve to reduce ATP consumption and maintain brain ATP levels.     

Distribution of aldehyde dehydrogenase and alcohol dehydrogenase in summer-acclimatized crucian carp, Carassius carassius L.

The tissue distribution of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) in summer-acclimatized crucian carp showed almost the same exceptional pattern as previously found in winter-acclimatized specimens. There was a nearly complete spatial separation of ALDH and ADH; in other vertebrates these enzymes occur together. This exceptional enzyme distribution is probably an adaptation to the extraordinary ability of Carassius to produce ethanol as the major metabolic end product during anoxia. Since the crucian carp is less likely to encounter anoxia during the summer, the present results suggest that the crucian carp is unable to switch over to a 'normal' ALDH and ADH distribution in the summer. However, it is also possible that there is an advantage for the summer-acclimatized crucian carp in keeping ALDH and ADH separate, because of occasional anoxic periods.     

Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9 degrees C). In contrast, anoxia exposure (0.01 mg O2/l at 8 degrees C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is alpha2, beta1B, gamma2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory gamma2-subunit increased in the heart during anoxia. In the brain, expression of the alpha1-, alpha2-, and gamma1-subunits decreased with anoxia exposure, but expression of the gamma2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates

Most vertebrates die within minutes when deprived of molecular oxygen (anoxia), in part because of cardiac failure, which can be traced to an inadequate matching of cardiac ATP supply to ATP demand. Cardiac power output (PO; estimated from the product of cardiac output and central arterial pressure and an indirect measure of cardiac ATP demand) is directly related to cardiac ATP supply up to some maximal level during both normoxia (ATP supply estimated from myocardial O(2) consumption) and anoxia (ATP supply estimated from lactate production rates). Thus, steady state PO provides an excellent means to examine anoxia tolerance strategies among ectothermic vertebrates by indicating a matching of cardiac glycolytic ATP supply and demand. Here, we summarize in vitro measurements of PO data from rainbow trout, freshwater turtles and hagfishes to provide a reasonable benchmark PO of 0.7 mW g(-1) for maximum glycolytic potential of ectothermic hearts at 15 degrees C, which corresponds to a glycolytic ATP turnover rate of about 70 nmol ATP g(-1) s(-1). Using this benchmark to evaluate in vivo PO data for hagfishes, carps and turtles, we identify two cardiac survival strategies, which in conjunction with creative waste management techniques to reduce waste accumulation, allow for long-term cardiac survival during anoxia in these anoxia-tolerant species. Hagfish and crucian carp exemplify a strategy of evolving such a low routine PO that routine cardiac ATP demand lies within the range of the maximum cardiac glycolytic potential. Common carp and freshwater turtles exemplify an active strategy of temporarily and substantially decreasing cardiac and whole body metabolism so that PO is below maximum cardiac glycolytic potential during chronic anoxia despite being quite close to this potential under normoxia.     

盐度对异育银鲫呼吸和氨氮排泄生理的影响

研究了异育银鲫(Carassius auratus gibeliovar. E′erqisi)从淡水向盐度1.5‰、3‰、6‰、9‰、12‰突变与适应过程中的呼吸和氨氮排泄生理的变化规律。结果表明:盐度突变开始时,异育银鲫的耗氧率和排氨率随外界盐度的升高而增大,随盐度作用时间延长,耗氧率和排氨率升到最大值后又开始下降并最终维持在稳定水平,但开始下降的时间和下降的幅度以及最终的稳定水平与外界盐度有关。盐度1.5‰和3‰处理组在作用12h时耗氧率升到最大值,此后下降,于第3天后保持在比淡水对照组略低的稳定水平,但二者差异不显著(P0.05);盐度6‰和9‰处理组在作用到第3天后才开始缓慢下降,并分别于第10、15天时保持在显著高于淡水对照的稳定水平(P0.05);盐度12‰处理组也在第3天后下降,到第10天后保持在比淡水对照组略低的稳定水平,但二者差异不显著(P0.05)。各盐度处理组的排氨率均在盐度作用24h时达到最大值,其中盐度1.5‰处理组的变化不显著(P0.05),其他各组均显著升高(P0.05),并都于第10天时下降到稳定水平,其中盐度3‰处理组的稳定水平略低于对照组(P0.05),盐度6‰、9‰、12‰处理组的稳定水平显著高于对照组(P0.05)。     

三种鲫鱼品系同工酶比较研究

采用聚丙烯酰胺凝胶垂直板电泳技术,对彭泽鲫、银鲫D系以及野鲫三种鲫鱼品系的心、肝和肾脏组织的乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、苹果酸酶(ME)、酯酶(EST)和超氧化物歧化酶(SOD)同工酶表型进行了比较研究.结果表明彭泽鲫乳酸脱氢酶同工酶比银鲫D系在肝组织多出二条酶带(LDH7'和LDH8');超氧化物歧化酶在心和肾组织中分别多出一条酶带(SOD12'),表明彭泽鲫和银鲫已在生化水平产生明显的分化,推测它们可能起源于不同的地区,由不同的祖先,独立演化而形成.此外,彭泽鲫和银鲫D系的同工酶电泳图谱都包含野鲫的基本酶带,而彭泽鲫和野鲫的酯酶同工酶电泳图谱尤为相似,推测彭泽鲫和银鲫可能起源于野鲫,而彭泽鲫和野鲫的关系较近,银鲫和野鲫的关系较远.     

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.     

Role of adenosine in the hypoxia-induced hypothermia of toads

The concept that hypoxia elicits a drop in body temperature (T(b)) in a wide variety of animals is not new, but the mechanisms remain unclear. We tested the hypothesis that adenosine mediates hypoxia-induced hypothermia in toads. Measurements of selected T(b) were performed using a thermal gradient. Animals were injected (into the lymph sac or intracerebroventricularly) with aminophylline (an adenosine receptor antagonist) followed by an 11-h period of hypoxia (7% O(2)) or normoxia exposure. Control animals received saline injections. Hypoxia elicited a drop in T(b) from 24.8 +/- 0.3 to 19. 5 +/- 1.1 degrees C (P < 0.05). Systemically applied aminophylline (25 mg/kg) did not change T(b) during normoxia, indicating that adenosine does not alter normal thermoregulatory function. However, aminophylline (25 mg/kg) significantly blunted hypoxia-induced hypothermia (P < 0.05). To assess the role of central thermoregulatory mechanisms, a smaller dose of aminophylline (0.25 mg/kg), which did not alter hypoxia-induced hypothermia systemically, was injected into the fourth cerebral ventricle. Intracerebroventricular injection of aminophylline (0.25 mg/kg) caused no significant change in T(b) under normoxia, but it abolished hypoxia-induced hypothermia. The present data indicate that adenosine is a central and possibly peripheral mediator of hypoxia-induced hypothermia.     

Anaerobic wintering of crucian carp (Carassius carassius L.)--II. Metabolic products

In long-term experimental anoxia (up to 140 days) crucian carp excreted ethanol and acetic acid. The maximum concentration of ethanol found in fish blood was ca 0.1%. The excretion rate of ethanol at temperatures from -0.5 to +5 degrees C was ca 25 micrograms/g fish wet wt/hr, but increased rapidly with increasing temperature. At 2 and 5 degrees C the ethanol excretion rate was independent of fish size, but at 12 and 18 degrees C a higher rate was observed in smaller fish. The decrease of water pH below 5 in the experimental vial during long anoxia was assumed to be due to excretion of acid compounds by the fish.     

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca²⁺. In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase‐like protein, t‐complex protein, and guanine nucleotide protein G_(o) subunit alpha (G_o alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia‐tolerant species), there are species‐specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.