Thermal tolerance of Antarctic notothenioid fishes correlates with level of circulating hemoglobin.

The West Antarctic Peninsula region is experiencing some of the most rapid elevations in temperature of any marine environment. We assessed thermal tolerance of white- and red-blooded Antarctic notothenioid fishes inhabiting these waters, using a modified critical thermal maximum (CT(max)) design. Temperature was elevated acutely from ambient at a constant rate of 3.6°C h(-1), and CT(max) was defined as the temperature where animals lost righting response. CT(max) temperatures of white-blooded icefishes Chionodraco rastrospinosus (13.3° ± 0.2°C) and Chaenocephalus aceratus (13.9° ± 0.4°C) were significantly lower than those of red-blooded fishes Gobionotothen gibberifrons (15.5° ± 0.2°C) and Notothenia coriiceps (17.1° ± 0.2°C). Lepidonotothen squamifrons, a red-blooded species with low hematocrit, exhibited a CT(max) (14.2° ± 0.4°C) that was significantly lower than that of the other red-blooded animals and similar to that of icefishes. A strong relationship between CT(max) and hematocrit (r(2) = 0.76) suggests that the oxygen-carrying capacity of blood may partially dictate acute lethal temperature. Despite a short treatment duration, we detected a rise in the mRNA level of hypoxia response gene HIF-1α in N. coriiceps heart tissue. One-week exposure to 4°C had no effect on the CT(max) of N. coriiceps, indicating an inability to compensate for rising temperature under these experimental conditions. Our results suggest that icefishes are particularly sensitive to temperature elevation because of a lack of hemoglobin and may be a sentinel taxon for climate change.

Phenylhydrazine-induced anemia causes nitric-oxide-mediated upregulation of the angiogenic pathway in Notothenia coriiceps.

Antarctic icefishes possess several cardiovascular characteristics that enable them to deliver oxygen adequately in the absence of hemoglobin (Hb). To gain insight into mechanisms driving development of these cardiovascular characteristics of icefish, we chemically induced severe anemia in a red-blooded notothenioid, Notothenia coriiceps. After 10 days of treatment with phenylhydrazine HCl, the hematocrit and Hb concentration of N. coriiceps decreased by >90% and >70%, respectively. Anemic fish exhibited a significantly higher concentration of nitric oxide (NO) metabolites in their plasma compared with that of control animals, indicating that corporeal levels of NO are higher in anemic animals than in control fish. The activity of nitric oxide synthase (NOS) was measured in brain, retina, pectoral muscle and ventricle of control and anemic animals. With the exception of retina, no significant differences in NOS activities were observed, indicating that the increase in plasma NO metabolites is due to loss of Hb, which normally plays a major role in the degradation of NO, and not due to an overall increase in the capacity for NO production. To determine whether loss of Hb can stimulate remodeling of the cardiovascular system, we measured expression of HIF-1alpha, PHD2 and VEGF mRNA in retinae of control and anemic fish. Expression of all three genes was higher in anemic animals compared with control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that probably is mediated through nitric oxide signaling.

Relationship among circulating hemoglobin, nitric oxide synthase activities and angiogenic poise in red- and white-blooded Antarctic notothenioid fishes.

Nitric oxide (NO)-mediated angiogenesis may play a role in establishing dense retinal vasculatures of Antarctic hemoglobinless icefishes (suborder: Notothenioidei). We hypothesized that loss of hemoglobin (Hb) leads to elevation in [NO] due to decreased degradation of the compound when the NO-scavenger Hb is absent, thereby inducing vascular growth. We found that total mass of NO metabolites, nitrite plus nitrate (NO(x)), in plasma is greater in icefishes than in red-blooded notothenioids [e.g. C. aceratus (Hb-), 22.7+/-2.9 microM; N. coriiceps (Hb+), 14.7+/-1.7 microM], suggesting a higher NO load in hemoglobinless animals. High NO levels do not appear to be a result of greater NO synthesis; we consistently measured lower activities of the enzyme catalyzing NO production, nitric oxide synthase, in tissues of icefishes than in Hb-expressing notothenioids [e.g. 96+/-10 and 216+/-39 pmol(min g wet wt)(-1) in brain tissue of C. aceratus (Hb-) and G. gibberifrons (Hb+), respectively]. Levels of mRNA for hypoxia-induced (HIF-1alpha and PHD2) and angiogenic genes (VEGF) were similar in red- and white-blooded species, indicating that vascular maintenance in adult animals does not require differences in angiogenic tone. This does not preclude a cause-and-effect relationship between absence of Hb and NO-mediated angiogenesis during earlier ontogenetic stages of icefishes.

Morphological and physiological study of the cardiac NOS/NO system in the Antarctic (Hb-/Mb-) icefish Chaenocephalus aceratus and in the red-blooded Trematomus bernacchii.

The nitric oxide synthase (NOS)/nitric oxide (NO) system integrates cellular biochemical machinery and energetics. In heart microenvironment, dynamic NO behaviour depends upon the presence of superoxide anions, haemoglobin (Hb), and myoglobin (Mb), being hemoproteins are major players disarming NO bioactivity. The Antarctic icefish, which lack Hb and, in some species, also cardiac Mb, represent a unique model for exploring Hb and Mb impact on NOS/NO function. We report in the (Hb(-)/Mb(-)) icefish Chaenocephalus aceratus the presence of cardiac NOSs activity (NADPH-diaphorase) and endothelial NOS (eNOS)/inducible NOS (iNOS) zonal immuno-localization in the myocardium. eNOS is localized on endocardium and, to a lesser extent, in myocardiocytes, while iNOS is localized exclusively in myocardiocytes. Confronting eNOS and iNOS expression in Trematomus bernacchii (Hb(+)/Mb(+)), C. hamatus (Hb(-)/Mb(+)) and C. aceratus (Hb(-)/Mb(-)) is evident a lower expression in the Mb-less icefish. NO signaling was analyzed using isolated working heart preparations. In T. bernacchii, L-arginine and exogenous (SIN-1) NO donor dose-dependently decreased stroke volume, indicating decreased inotropism. L-arginine-induced inotropism was NOSs-dependent, being abolished by NOSs-inhibitor NG-monomethyl-L-arginine (L-NMMA). A SIN-1-induced negative inotropism was found in presence of SOD. NOS inhibition by L-N5-N-iminoethyl-L-ornithine (L-NIO) and L-NMMA confirmed the NO-mediated negative inotropic influence on cardiac performance. In contrast, in C. aceratus, L-arginine elicited a positive inotropism. SIN-1 induced a negative inotropism, which disappeared in presence of SOD, indicating peroxynitrite involvement. Cardiac performance was unaffected by L-NIO and L-NIL. NO signaling acted via a cGMP-independent mechanism. This high conservation degree of NOS localization pattern and signaling highlights its importance for cardiac biology.

Morphometry of retinal vasculature in Antarctic fishes is dependent upon the level of hemoglobin in circulation.

We quantitatively assessed ocular vascular patterns of six Antarctic notothenioid fishes that vary in their expression of the circulating oxygen-binding protein, hemoglobin (Hb). Digital image analyses revealed marked differences in vessel morphometries among notothenioid species. Hemoglobinless (-Hb) icefishes display mean vessel length densities that are greater (Chaenocephalus aceratus, 5.51+/-0.32 mm mm(-2); Champsocephalus gunnari, 5.15+/-0.50 mm mm(-2)) than those observed in red-blooded (+Hb) species (Gymnodraco acuticeps, 5.20+/-0.46 mm mm(-2); Parachaenichthyes charcoti, 4.40+/-0.30 mm mm(-2); Trematomus hansoni, 3.94+/-0.08 mm mm(-2); Notothenia coriiceps, 2.48+/-0.21 mm mm(-2)). -Hb fishes also have mean vessel diameters that are approximately 1.5 times greater than vessel diameters of +Hb species (-Hb, 0.193+/-0.006 mm; +Hb, 0.125+/-0.005 mm). Vascular density index (VDI), a stereological index that is affected by both vessel number and length, is greatest in -Hb C. aceratus (3.51+/-0.20) and lowest in +Hb N. coriiceps (1.58+/-0.14). Among four +Hb species, there is a direct relationship between red blood cell content and retinal vasculature. Hematocrit (Hct) is inversely correlated to vascular density (r(2)=0.934) and positively correlated to intervessel distance (r(2)= 0.898) over a >2.3-fold range of Hct. These results indicate that anatomical capacity to supply blood to the retina increases to compensate for decreases in oxygen-carrying capacity of the blood.

When bad things happen to good fish: the loss of hemoglobin and myoglobin expression in Antarctic icefishes.

The Antarctic icefishes (Family Channichthyidae) provide excellent examples of unique traits that can arise in a chronically cold and isolated environment. Their loss of hemoglobin (Hb) expression, and in some cases, loss of myoglobin (Mb) expression, has taught us much about the function of these proteins. Although absences of the proteins are fixed traits in icefishes, the losses do not appear to be of adaptive value. Contrary to some suggestions, loss of Hb has led to higher energetic costs for circulating blood, and losses of Mb have reduced cardiac performance. Moreover, losses of Hb and Mb have resulted in extensive modifications to the cardiovascular system to ensure adequate oxygen delivery to working muscles. Recent studies suggest that losses of Hb and Mb, and their associated nitric oxide (NO)-oxygenase activities, may have accelerated the development and evolution of these cardiovascular modifications. The high levels of NO that should occur in the absence of Hb and Mb have been shown in other animal groups to lead to an increase in tissue vascularization, an increase in the lumenal diameter of blood vessels, and an increase in mitochondrial densities. These characteristics are all hallmark traits of Antarctic icefishes. Homeostatic feedback mechanisms thus may have accelerated evolution of the pronounced cardiovascular traits of Antarctic icefishes.

Temperature sensitivity of calcium binding for parvalbumins from Antarctic and temperate zone teleost fishes.

Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants (KD) were measured as a function of temperature (0-25 degrees C) for PV from two Antarctic (Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species (Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that KD values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of KD are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on KD. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 degrees C, the Gibbs free energy change (deltaG) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (-43.4+/-1.5 kJ mol(-1) and -46.6+/-3.0 kJ mol(-1), respectively), and the relative contribution of entropy to deltaG for calcium binding to PV from the Antarctic species was about twice that of carp (deltaS=16.0+/-0.8 J degrees C(-1) mol(-1) for G. gibberifrons; deltaS=7.5+/-0.8 J degrees C(-1) mol(-1) for C. carpio).

Fatty acyl CoA synthetase from Antarctic notothenioid fishes may influence substrate specificity of fat oxidation.

Antarctic notothenioid fishes possess large lipid stores that are important fuels for aerobic metabolism. Oxidative muscle tissues of these animals oxidize long-chain mono-unsaturated fatty acids more readily than saturated fatty acids. The mechanistic basis(es) for the substrate specificity of their fatty acid-oxidizing pathway is unknown. We examined the substrate specificity of fatty acyl coenzyme A synthetase (FACS) to determine whether the enzyme contributes to targeting unsaturated fatty acids for preferential transport into mitochondria as fuels for beta-oxidation. Maximal activities of FACS were measured in isolated mitochondria from Notothenia coriiceps and Chaenocephalus aceratus oxidative skeletal muscles in the presence of fatty acids differing in chain lengths and degrees of unsaturation. With the exception of C(22:6), maximal activities were greater with unsaturated substrates than with C(16:0), a saturated fatty acid. Monoenoic fatty acids did not produce the highest activities. Predicted amino acid sequences of FACS from Antarctic C. aceratus, Gobionotothen gibberifrons, and N. coriiceps and sub-Antarctic Notothenia angustata and Eleginops maclovinus were determined to identify amino acid candidates that may be important for determining the substrate specificity of FACS. Substitutions cysteine548 and polar threonine552 within the putative fatty acid binding pocket may contribute to preference for unsaturated fatty acyl substrates compared to saturated fatty acids.

Isolation, characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus.

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K(m) at 0.5 degrees C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K(m) values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6-7.7 at 0.5 degrees C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 degrees C. The apparent V(max) of C. aceratus MMCK at 0.5 degrees C is 94+/-4 S.D. (n=9) micromol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 degrees C and 8-fold greater than rabbit MMCK measured at 0.5 degrees C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.

The myoglobin gene of the Antarctic icefish, Chaenocephalus aceratus, contains a duplicated TATAAAA sequence that interferes with transcription.

Six of the 16 known species of Antarctic icefish (family Channichthyidae) have lost the ability to express cardiac myoglobin (Mb) via at least four independent events during radiation of these species. We report here that the lesion in Chaenocephalus aceratus Mb is a duplicated TATAAAA element that blocks transcription. This lesion is distinct from those of other icefish species that do not express cardiac Mb. The C. aceratus Mb gene is nearly identical to that of Chionodraco rastrospinosus, a closely related Mb-expressing icefish species, with one exception. A 15-bp segment is present in C. aceratus but absent from C. rastrospinosus; this insertion is located 648 bp upstream from the reference transcription start site of C. rastrospinosus and includes the sequence TATAAAA, which bound HeLa cell transcription factor IID (TFIID) and icefish nuclear proteins in gel-retardation assays. Reporter constructs containing the 'full-length' C. aceratus Mb promoter were not expressed in transient expression assays in oxidative skeletal muscle of live icefish. By contrast, constructs employing the nearly identical 'full-length' C. rastrospinosus Mb promoter were efficiently expressed in parallel assays in the same tissue. Truncated constructs of C. aceratus Mb that did not contain the 15-bp duplication were expressed at very low levels. These data confirm a third independent mechanism of Mb loss among channichthyid species, indicate that C. aceratus aerobic muscle is capable of expressing functional Mb genes and demonstrate that duplication of the muscle-specific TATAAAA sequence in an inappropriate context can result in loss of a gene's expression, resulting in significant physiological consequences.

Notothenioid fish, krill and phytoplankton from Antarctica contain a vitamin E constituent (alpha-tocomonoenol) functionally associated with cold-water adaptation.

The vitamin E (VE) content of tissues from the Antarctic notothenioid fish, Chaenocephalus aceratus, Champsocephalus gunnari and Gobionotothen gibberifrons, and extracts of Antarctic krill Euphausia superba and phytoplankton collected from the Antarctic Peninsula was examined. Included in the VE composition was a newly described 'marine-derived' tocopherol (MDT), an unsaturated-isoprenoid derivative of alpha-tocopherol, that is attributed to enhancing antioxidant protection of cellular lipids at low temperature. MDT was found to co-exist with alpha-tocopherol in all Antarctic samples, ranging from 2.8 to 22.3% of the total VE composition. The highest level of VE was found in the liver of G. gibberifrons (VE=416.7 pmol/mg wet tissue) although this tissue had a low MDT composition (7.7%), whereas the greatest MDT composition was measured in the liver of C. gunnari (MDT=22.3%). In notothenioids, the pectoral adductor muscle, which has a high density of mitochondria, contained higher levels of VE than white myotomal muscle, but differences in MDT composition were small. Phytoplankton and krill also contained MDT, which supports the contention that MDT is obtained directly from the primary food chain. Our finding of MDT in Antarctic organisms is consistent with its putatively adaptive function to enhance antioxidant protection in coldwater metabolism.

Teleost introns are characterized by a high A+T content.

We previously observed that Antarctic fish genes contain intron sequences of high A+T content (60-70% average A+T) which are in stark contrast with adjacent protein coding-sequences. Here, we report that this disparity in intron/exon base composition is a common feature among teleosts. We analyzed 483 teleost genomic DNA sequences, containing 2583 introns, from 80 teleost genera that populate polar, temperate, or tropical habitats. Eighty-nine percent of teleost introns display an A+T content between 50-84% A+T with a mean of 60% A+T. In contrast, only 37% of teleost exons have an A+T content greater-than 50% with a mean of 48% A+T. A comparison to homologous mammalian genes showed a striking difference; in this case, introns and exons have similar base compositions, averaging 45-47% A+T. This indicates that most teleost genes exhibit a large difference in base composition between their introns and exons. There was no correlation of teleost intron A+T content to intron length or habitat temperature range. Thus, teleost intron sequences tend to show the common feature of being much higher in A+T content then neighboring exons.

Cold acclimation induces proliferation of sarcoplasmic reticulum without increase in Ca2+-ATPase activity in white axial muscle of striped bass (Morone saxatilis).

The effects of acclimation of striped bass to cold (5 degrees C) and warm (25 degrees C) temperatures upon ultrastructural features of white axial skeletal muscle are quantified. Surface density of sarcoplasmic reticulum (SR) increased by almost 30%, and SR volume density increased by about 20% during cold acclimation. Proliferation of SR suggests an increase in available SR surface for re-sequestration of Ca2+ and a decrease in diffusion path length for Ca2+ during cold acclimation. Average cross-sectional areas and cross-sectional perimeters of myofibrils situated in the center of muscle fibers decreased during cold acclimation by approximately 20% and 11%, respectively. Additionally, average major and minor axes of ellipses fit to central myofibrillar cross-sections decreased by approximately 12% and 8%, respectively, during cold acclimation. These measurements define a decrease in average myofibrillar diameter and suggest a decrease in diffusion path length for Ca2+ to and from myofibrillar activation sites. Measurements of peripheral myofibrils that had elongated profiles in cross-sections indicate that maximum profile length of these myofibrils decreases by about 17%. Peripheral myofibrils may break up into smaller myofibrils with more rounded cross-sectional profiles during cold acclimation. SR Ca2+-ATPase of white axial muscle was also measured in unfractionated homogenates and in crude SR-enriched subcellular fractions from cold- and warm-acclimated striped bass. No difference in SR Ca2+-ATPase activity per g wet weight was observed between cold- and warm-acclimated animals. Lack of increase in SR Ca2+-ATPase per g wet weight, despite a significant proliferation of SR, probably results in a decrease in average Ca2+-ATPase pump density within the SR membrane during cold acclimation. Thus, compensation for decreased diffusion coefficient of Ca2+ during cold acclimation appears due to the combined effects of proliferation of SR surface density and a decrease in average myofibrillar diameter.