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First published online May 1, 2006
Journal of Experimental Biology 209, 1791-1802 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02091
Commentary |
When bad things happen to good fish: the loss of hemoglobin and myoglobin expression in Antarctic icefishes
1 School of Marine Sciences, University of Maine, 5751 Murray Hall, Orono,
ME 04469-5751, USA
2 Institute of Arctic Biology, University of Alaska, Fairbanks, PO Box
757000, Fairbanks, AK 99775, USA
* Author for correspondence (e-mail: bsidell{at}maine.edu)
Accepted 12 January 2006
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Summary |
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 TOP Summary IntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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Key words: hemoglobin, myoglobin, Antarctic icefish, nitric oxide, heart, circulation
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Introduction |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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). Although we may
take issue with the grammar of this sentence, the concept is unassailable. In
the realm of comparative cardiovascular physiology, few groups of animals can
rival the Antarctic icefishes in meeting the criterion described by Krogh. It
is upon this group that we will focus our Commentary.
The Antarctic icefishes (Family Channichthyidae) are one of eight families
of the single perciform suborder, Notothenioidei, which dominate the fish
fauna surrounding Antarctica (for excellent reviews, see
Eastman, 2005;
Kock, 2005). They occupy the
coldest, most thermally stable marine environment on earth. Sea temperatures
near the Ross Ice Shelf at McMurdo Station, Antarctica, are nearly constant at
–1.9°C (Littlepage,
1965) and even those in the more northerly Antarctic Peninsula
range only between summer temperatures of +1.5°C to winter temperatures of
–1.8°C (DeWitt,
1971). The water column south of the Antarctic Polar Front is
exceedingly well mixed vertically, and all depths are close to complete oxygen
saturation. Because oxygen solubility in seawater is inversely proportional to
temperature, the cold Antarctic seas thus are an exceptionally oxygen-rich
aquatic habitat.
Notothenioids account for approximately 35% of fish species and 90% of fish
biomass found south of the Antarctic Polar Front
(Ekau, 1990). Radiation of
closely related notothenioid species has occurred rapidly (within the last 12
million years, MY) (Bargelloni et al.,
1994) and under a very unusual set of conditions. First,
notothenioids have evolved in relative oceanographic isolation from other
faunas due to circumpolar currents and deep ocean trenches surrounding the
continent. Second, the Southern Ocean has been characterized by severely cold
water temperatures for the last 10–14 MY
(Kennett, 1977). Finally,
evolution of these fishes has progressed under conditions of very low levels
of niche competition because a dramatic crash in fish diversity occurred in
the Southern ocean sometime between the mid-Tertiary and present (see
Eastman, 1993;
Eastman, 2005). These features
make Antarctic notothenioid fishes an uniquely attractive group for the study
of physiological and biochemical adaptations to cold body temperature. Today's
notothenioids are arguably the end result of an extraordinary natural
experiment. They provide a window into the exceptional physiological
characteristics that can arise in animals living at chronically cold body
temperatures. Some of these characteristics clearly are adaptive (e.g.
development of antifreeze glycoproteins). Others would be deleterious, if not
lethal, in warmer and more competitive environments.
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Patterns of oxygen-binding protein loss among the Channichthyidae |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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).
Icefishes are the only known vertebrate animals to lack Hb as adults
(Fig. 1). Oxygen is found
solely in physical solution in icefish blood, which has an oxygen carrying
capacity of <10% of that seen in red-blooded notothenioid fishes
(Holeton, 1970). Several
fairly draconian modifications of the cardiovascular system of icefishes
compensate for their lack of a circulating oxygen-carrier. Icefishes possess
very large hearts compared to red-blooded fishes of equivalent body size,
resulting in a weight-specific cardiac output that is four- to fivefold
greater than that of red-blooded species
(Hemmingsen et al., 1972;
Fig. 2). The blood volumes of
icefishes are up to fourfold those of red-blooded teleosts, and the diameter
of their capillaries is unusually large
(Fitch et al., 1984). These
features collectively permit a large volume of blood to circulate throughout
the bodies of icefishes at high flow rate, yet, at low vascular pressure
because of decreased peripheral resistance. Combined with the very high oxygen
content of Antarctic waters and relatively low absolute metabolic rates, these
unusual cardiovascular attributes ensure that adequate oxygen is delivered to
tissues to support the obligately aerobic mode of metabolism of these animals
(Hemmingsen, 1991).
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The intracellular oxygen-binding protein, myoglobin (Mb), is also not
uniformly expressed in species of Family Channichthyidae. Myoglobin is widely
distributed in aerobically poised tissues of animals, and has been ascribed
critical roles in both intracellular storage and diffusion of oxygen
(Wittenberg and Wittenberg,
2003). Indeed, The Journal of Experimental Biology
recently carried a Commentary (Ordway and
Garry, 2004), the title of which indicated that myoglobin was an
`essential hemoprotein in striated muscle'. It would appear that the
channichthyid icefishes confirm the old adage that there are exceptions to
every rule. Among the 16 known species of the family, ten icefish species do
express Mb in their heart muscle, while six others do not produce the protein
(Grove et al., 2004).
The very close phylogenetic relationship among families of Hb-expressing
and Hb-less notothenioids and among Mb-expressing and Mb-lacking icefishes
presents an unique matrix of `naturally occurring genetic knockouts' that can
be exploited to probe and understand the myriad of processes that regulate
both oxygen delivery and utilization in aerobic tissues. Because these
`knockouts' have withstood the tests of real-world biology, they offer
advantages over experimentally produced genetic knockouts for Mb expression in
mice (Garry et al., 1998;
Gödecke et al.,
1999).
The pattern of Hb and Mb expression in icefishes leads us to a series of intriguing questions. How and when did the losses of expression of these important oxygen-binding proteins come about? Was loss of expression of either hemoglobin or myoglobin of adaptive value? If `yes', what advantage was conferred? If `no', why have the traits persisted in populations of these animals? How has the suite of physiological characteristics, which appear to be aimed at compensating for loss of each of these proteins, come about? Obviously, many of the `answers' to these questions must, of necessity, fall within the realm of speculation. However, recent findings are pointing toward a series of provocative explanations.
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When and how were hemoglobin and myoglobin lost in the icefishes? |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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; Ruud, 1965).
These conditions are thought to have been attained ca. 10–14 MYA
(Kennett, 1977). This timing
is consistent with the emergence of most notothenioid lineages, which is
thought to have occurred 12–5 MYA, based on genetic distances
(Bargelloni et al., 2000). These same genetic data suggest that the
channichthyid icefishes diverged only 5.5–2 MYA. By this time, the
Southern Ocean had become a stable, cold environment, favoring the survival of
this group. Despite their descent from an ancestral red-blooded stock, genomic
DNA of today's icefishes completely lacks any detectable vestiges of the gene
for ß-globin and contains only remnants of the gene encoding
-globin, the two subunits of which Hb is composed
(Cocca et al., 1995;
Cocca et al., 1997).
The pattern of Mb loss among the channichthyid icefishes is more puzzling.
We have been unable to detect expression of Mb in oxidative skeletal muscle of
any icefish or red-blooded notothenioid species that we have examined to date,
suggesting that this phenotype is even more ancient than absence of Hb
(Moylan and Sidell, 2000). A
`myoglobin-like' protein has been detected in glycolytic skeletal muscle of
icefishes based upon immunochemical methods
(Morlá et al., 2003).
This observation seems perplexing, given that Mb expression is typically
restricted to aerobically poised oxidative muscle, and is not expressed in
anaerobically poised glycolytic, skeletal muscles; we have been unable to
duplicate their results.
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;
Small et al., 2003). The most
parsimonious interpretation of this pattern is that Mb is nonfunctional at the
very cold body temperature of these animals and its loss, by whatever
mechanism, might be advantageous, or selectively neutral at worst
(Sidell et al., 1997). All
available data, however, point toward rejection of this hypothesis.
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Was loss of Hb and/or Mb of adaptive value? |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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) and polar fishes
generally display lower hematocrits than warmer-bodied fishes (e.g.
Scholander and Van Dam, 1957;
Wells, 1990). These
observations have prompted some to conclude that the hemoglobinless condition
of channichthyids is at the extreme end of a general trend toward a reduction
in hematocrit at cold body temperature (e.g.
di Prisco et al., 1991). In
fact, Egginton reports that hematocrit values of red-blooded notothenioids
show considerable overlap with the range of values observed in fishes from
warmer waters (Egginton, 1996)
and that the major difference between these groups is a lower mean corpuscular
hemoglobin concentration (MCHC) in red-blooded notothenioids, not lower
hematocrit. These arguments further highlight the divergent mechanisms that
underlie differences in Hb expression between icefishes and cold-bodied but
red-blooded fishes. The loss of Hb expression in icefishes is the result of a
wholesale gene deletion. In contrast, the decrease in Hb content that occurs
in response to a decrease in temperature in red-blooded fishes, is brought
about by the downregulation of an existing gene. Clearly, these are
fundamentally different processes. Many physiologists, nonetheless, have
deduced that loss of Hb and red cells in icefishes results in an energetic
advantage because of reduction in the intrinsic viscosity of blood (e.g. see
di Prisco et al., 1991;
Zhao et al., 1998;
Cocca et al., 1997). These
arguments are sound only if one considers the work necessary to pump an
identical volume of blood. However, energetic arguments ultimately must work
at the organismal level, and there are vast differences in heart sizes, blood
volumes and cardiac outputs between red-blooded and white-blooded
notothenioids.
When taking into account published mean ventral aortic pressures and
cardiac output values normalized to body mass, it is possible to calculate the
cardiac power development necessary to support an identical body mass of fish
for both Hb-containing notothenioids and Hb-lacking icefishes
(Table 2). [Calculation of
actual cardiac work would require subtraction of venous return pressure to the
heart from mean ventral aortic pressure. Few estimates of the former are
available, but the consensus is that pressures in the sinus venosus of these
animals are very low, approaching zero. For comparative purposes, body
weight-specific cardiac power output thus is an excellent proxy for actual
cardiac work.] The results are both instructive and initially surprising. On
average, icefishes expend approximately twice the cardiac energy per unit time
than do red-blooded notothenioids of equivalent body mass. Although each ml of
blood can be moved by icefish at lower energetic cost, they pump a far greater
volume per unit time to support an equivalent body mass. This emphasis on high
volume circulation in icefishes prompted Tota and Gattuso to cite the very
large hearts of icefishes as exemplifying pumps with Type I or spongy cardiac
morphology (Tota and Gattuso,
1996), which achieve a high throughput of fluid predominantly by
high stroke volume, despite being capable of attaining only modest output
pressures (ca. 3 kPa). [For an excellent overview of the functional morphology
of fish hearts, see Tota et al. (Tota et
al., 1991).] As a consequence, loss of Hb and red cells ultimately
is correlated with a substantially higher expenditure of cardiac energy at the
organismal level, and definitely does not result in energetic savings! In
fact, it has been estimated that 22% of resting metabolic rate in icefishes is
devoted to cardiac work (Hemmingsen and
Douglas, 1977). Cardiac work thus represents a far greater
fraction of total energetic expenditure in icefishes than the range of 0.5% to
5.0% of total metabolism reported for temperate zone fishes and 2.3% of total
metabolism for even such athletic fishes as skipjack tuna
(Farrell and Jones, 1992). In
contrast to hearts of icefish, tuna hearts have been cited as clear examples
of mammalian-like pumps, possessing a well developed compact epicardium and
predominantly relying upon pressure development to elevate cardiac work
(Tota and Gattuso, 1996). In
light of these energetic considerations and the rather draconian compensatory
alterations in cardiovascular anatomy and physiology seen in icefishes, it
seems reasonable to conclude, as suggested originally
(Wells, 1990), that loss of Hb
and red cells did not confer an adaptive advantage to the channichthyids.
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The multiple occurrences of Mb loss observed among the icefishes are even more perplexing. This is particularly evident when one realizes that each clade characterized by lack of Mb expression is more closely related to clades that produce the protein than to those that do not (cf. Fig. 3). The ability to produce Mb has thus been lost through several completely independent mutational events. This pattern suggests that Mb might be a `vestigial' protein that may not work well at the severely cold body temperature of icefishes. We have pursued several independent lines of investigation to evaluate this possibility.
We used stopped-flow spectroscopy to establish that oxygen binds and
dissociates from icefish Mb more rapidly than from mammalian Mbs at all
temperatures (Cashon et al.,
1997). When measurements are compared at respective physiological
temperatures, however, Mbs of these two groups show very similar kinetics of
binding and dissociation. In short, icefish Mb appears to function at 0°C
as well as mammalian Mb does at 37°C. Our results also showed that
cold-temperature function of Mb is not unique to icefishes, but is a trait
shared with other teleosts. The enhanced activity of teleost Mbs at cold
temperature appears to be due to the replacement of the common D-helix found
in mammalian Mbs with a random-coil. This substitution undoubtedly confers
greater conformational flexibility to the protein, enhancing oxygen's entry
and exit from the heme binding-pocket
(Cashon et al., 1997).
Oxygen-binding kinetics, therefore, demonstrate that icefish Mb is functional
at cold temperature.
Even more compelling evidence of Mb function in icefishes has been obtained
from isolated, perfused heart studies
(Acierno et al., 1997). Hearts
from icefish species that possess Mb can maintain cardiac output at higher
afterload pressures than closely related species that lack Mb
(Fig. 4). We were further able
to substantiate that these differences in cardiac performance were
unequivocally due to the presence or absence of Mb, by using sodium nitrite, a
selective poison of Mb oxygen-binding function. When 5 mmol
l–1 NaNO2 is incorporated into the perfusate,
mechanical performance of hearts from species that express the protein is
significantly impaired, while those naturally lacking the protein are
refractory to this treatment (Fig.
4). The unexpected result of these experiments, however, was that
hearts that naturally lack Mb performed better than Mb-expressing hearts in
which the function of the protein had been chemically ablated. This result
suggested that physiological features have developed to compensate for the
lack of Mb in those hearts that normally do not express the protein. Several
lines of evidence indicate that NaNO2 is an Mb-specific inhibitor
and does not release NO into the perfusate, which would confound these
conclusions. This point is essential because nitric oxide can affect cardiac
function in several ways. First, at high concentrations, NO inhibits the
activity of cytochrome c oxidase (COX)
(Antunes et al., 2004). This
does not appear to occur in the experiments. We have previously shown that the
activity of cytochrome c oxidase per g ventricular tissue is
equivalent between C. aceratus and C. rastrospinosus
(O'Brien and Sidell, 2000).
Thus, if NO is present at sufficient levels to inhibit cytochrome oxidase, it
should inhibit COX activity to the same extent in hearts from both species.
This is clearly not the case; hearts from C. aceratus are refractory
to the treatment of NaNO2, whereas cardiac output in hearts from
C. rastrospinosus significantly declines. Second, NO has a positive
inotropic effect on hearts of icefish
(Pellegrino et al., 2004).
Thus, if NO were being released from NaNO2, one would anticipate an
increase in cardiac output, particularly in hearts of C. aceratus
lacking Mb, and not a decrease, as was seen in hearts from C.
rastrospinosus.
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All of the evidence cited above strongly indicates that the losses of the
ability to express Hb and Mb were not advantageous. Indeed, available
information clearly suggests that each of these losses must have resulted in a
decrement in physiological performance of the fishes. Under the nomenclature
of Baum and Larsen, this would qualify both of these traits as `disaptations'
(Baum and Larsen, 1991).
Indeed, multiple losses of myoglobin during evolution of the icefishes have
been cited as a prime example of a disaptation among Antarctic fishes
(Montgomery and Clements,
2000). Such a conclusion appears at odds with modern evolutionary
theory, which suggests that selective pressure should lead to the retention of
Hb and Mb expression, and that mutations causing their loss should be subject
to negative selection and eliminated from the population. Regardless of the
specific nomenclature employed to describe them, persistence of these traits
appears to be a conundrum.
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Why have the hemoglobinless and myoglobinless traits persisted in icefishes? |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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Both environmental and physiological characteristics help explain why Hb and Mb losses are not lethal at the level of the individual organism. We have seen that the very cold temperature and extensive vertical mixing of the Southern Ocean results in highly oxygenated water. Moreover, the absolute metabolic rates of Antarctic fishes are relatively low because of cold body temperature and their modest locomotory activity, as a result of their descent from a common sluggish demersal ancestor. Convergence of these features likely ensured sufficient tissue oxygenation to sustain life in early channichthyids, despite the loss of oxygen-binding proteins. Although providing an explanation of why losses of oxygen-binding proteins may not have been lethal, this line of reasoning still does not address the more difficult question of why such apparently `disadvantageous' traits were maintained at the population level.
It is essential to remember that, if a trait is sublethal, then it is
`disadvantageous' only within the context of competition with other organisms.
In other words, if competition is relaxed sufficiently and environmental
resources (e.g. caloric resources) are not limiting, sublethal traits may have
no real effect on the fitness of organisms. The unusual evolutionary history
of the Antarctic fish fauna suggests that they may have radiated under
conditions of little or no niche competition. The massive crash of species
diversity among fishes in the Southern Ocean occurring between the
mid-Tertiary and present left an ancestral stock of demersal notothenioids to
colonize approximately 10% of the world's ocean volume. The prevailing view is
that this event explains the ultimate dominance of notothenioid species in
Antarctic seas (Eastman,
1993).
The final piece of the puzzle comes with recognition that the climatic
cooling of Antarctica during the last 25 MY has not been smoothly monotonic.
Indeed, evidence exists to suggest that deep (>100 m) ice-free marine
embayments developed during periodic recessions of the glacial shield of the
continent on several occasions during even the last 5 MY (e.g. see
Webb, 1990). Periodic
availability of these deep fjords to colonization by sparsely distributed
notothenioids provides the final ingredient of refugia that may have
contributed to both the exceptionally rapid radiation of notothenioid species
in general, and the fixation of the unusual Hb-less and Mb-less traits of some
icefish species.
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What other apparently compensatory mechanisms for oxygen delivery are expressed in icefishes and how have they come about? |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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Enhanced vascular densities
Reflecting their very large blood volumes, Antarctic icefish display large
lumenal diameters of the microvasculature
(Fitch et al., 1984). Although
we have long known that the capillary bore of Hb-less icefishes is two- to
threefold greater than that of red-blooded relatives
(Fitch et al., 1984), the
majority of vascular adaptations that have been described for these fish are
at the gross systemic level. In collaboration with Dr Joseph Eastman of Ohio
University, we recently performed a series of preliminary vascular perfusions
of Hb-producing and Hb-lacking Antarctic notothenioids that reveal stunning
differences in the vascular densities of a highly aerobic tissue, the retina
of the eye (Fig. 5). Vascular
densities of the Hb-less icefish are dramatically greater than those of
Hb-expressing notothenioid species. This has the effect of reducing the
diffusion distance for oxygen and ensuring greater oxygenation of the highly
aerobic retinal tissue in animals whose blood has an oxygen carrying capacity
per unit volume far below that of their red-blooded counterparts.
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Altered structural and ultrastructural features of heart muscle
We have compared structural features of hearts from three species of
Antarctic notothenioid fishes that differ in expression of oxygen-binding
proteins: Gobionotothen gibberifrons (a red-blooded species whose
heart also contains Mb), Chionodraco rastrospinosus (an Hb-lacking
icefish that does express cardiac Mb) and, Chaenocephalus aceratus
(an Hb- and Mb-lacking icefish). At the tissue-level, we found that hearts of
both Hb-lacking icefishes were more spongy (i.e. the average diffusion
distance that oxygen would have to traverse between lumenal blood and the
tissue was shorter) than were hearts from red-blooded species
(O'Brien et al., 2000)
(Table 3). Hearts of both
Mb-expressing and Mb-lacking icefishes, however, showed no significant
difference in this feature. It appears that icefish hearts have developed a
more pervasive system of blood-filled lacunae within their spongy myocardium
to ensure adequate oxygen delivery from their comparatively oxygen deficient
blood.
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At the level of fine cellular structure, mitochondrial densities in the
oxidative muscle of Antarctic fishes are also correlated with the presence or
absence of Hb and Mb (O'Brien and Sidell,
2000). Mitchondria (1) account for approximately 16% of cell
volume in hearts of red-blooded Gobionotothen gibberifrons, which
also contain Mb; (2) account for 
20% of cell volume of cardiomyocytes in
Chionodraco rastrospinosus, which lack Hb but express Mb, and (3)
displace 36% of cell volume in cardiac myocytes from Chaenocephalus
aceratus, which are devoid of both Hb and Mb
(Table 3;
O'Brien and Sidell, 2000).
Thus, loss of Hb results in only a modest (4%) expansion of the
mitochondrial population in heart muscle, as long as the tissue continues to
express Mb. However, when both Hb and Mb are absent, expansion of the
mitochondrial population is dramatic (i.e. a further 16% increase in cell
volume). Surprisingly, the high densities of mitochondria in the hearts of
fishes lacking Hb and Mb do not increase aerobic metabolic capacity. In fact,
the activity of aerobically poised enzymes (per g cardiac tissue) such as
cytochrome oxidase and citrate synthase, are equivalent among all three
species, despite the dramatic differences in mitochondrial number. The high
densities of enlarged mitochondria in icefishes lacking Hb and Mb, result in
the formation of an interwoven network of membranes. This lipid highway likely
serves as an important pathway for oxygen, enhancing its delivery in the
absence of oxygen-binding proteins
(Sidell, 1998).
The suite of anatomical and physiological characteristics of icefishes that appear to be linked to the loss of Hb and Mb expression is extensive. How then could these apparently adaptive traits have evolved under conditions of relaxed competition? One possibility is that the loss of Hb and Mb triggered immediate, ameliorating modifications in icefish physiology, which became fixed traits over time. In other words, the initial loss of Hb and Mb accelerated the evolution of secondary cardiovascular traits. This idea is supported by recent studies illuminating the novel role of Hb and Mb in the metabolism of the potent signaling molecule, nitric oxide.
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Hemoglobin and myoglobin as nitric oxide-oxygenases |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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;
Minning et al., 1999;
Gardner et al., 1998). These
ancestral globins function as NO oxygenases, using oxygen to convert NO to
nitrate. Studies of Hb–NO oxygenase activity in unicellular organisms
provided precedence for later work on vertebrate hemoglobins, which showed
that in addition to binding oxygen, both Hb and Mb also break down NO to
nitrate (Gardner, 2005;
Flögel et al., 2001).
Moreover, this function is integral to an animal's physiology. The absence of
Mb in the much-studied myoglobinless mice leads to enhanced sensitivity to NO
and the activation of downstream pathways regulated by NO
(Flögel et al., 2001;
Grange et al., 2001). These findings prompt us to widen the scope of our questions related to the physiology of icefish and ask: What are the potential effects of the loss of Hb and Mb as NO-oxygenases? The answers are enticing, and suggest that the loss of NO-oxygenase activity, and subsequent elevation of NO levels, could explain many, if not all, of the unique cardiovascular and physiological traits that have evolved in icefishes.
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The production of nitric oxide |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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). In
higher vertebrates, NO is the product of a reaction catalyzed by three
distinct isoforms of the enzyme nitric oxide synthase (NOS; E.C. 1.14.13.39):
endothelial NOS (eNOS, NOS-III), neuronal NOS (nNOS, ncNOS, NOS-I) and
inducible NOS (iNOS, mNOS, NOS-II). All NOS isoforms catalyze a 5-electron
oxidation of arginine to produce NO and L-citrulline using NADPH as
an electron source (Moncada and Higgs,
1993). Two of the isoforms, eNOS and nNOS, are constitutively
expressed in mammals, while iNOS is inducible by cytokines and other stimuli,
including hypoxia (Kerwin, Jr et al.,
1995). Even the constitutively expressed NOS isoforms, however,
are upregulated in response to physiological stimuli such as hypoxia and
hemodynamic shear stress (Shaul,
2002).
All three isoforms of NOS are present in fish. Endothelial NOS (eNOS) has
been reported in vascular endothelium and heart muscle of developing zebrafish
(Fritsche et al., 2000), and
the presence of nNOS and iNOS clearly has been established in a variety of
tissues from several fish species (e.g.
Holmqvist et al., 1998;
Holmqvist et al., 2004;
Morlá et al., 2003,
Pellegrino et al., 2002;
Pellegrino et al., 2004). A
recent study shows that nNOS is expressed at a higher level in skeletal muscle
from icefishes than in the tissue from red-blooded species
(Morlá et al., 2003).
Both eNOS and iNOS have been identified in ventricular cardiomyocytes of white
and red-blooded nototheniods, and eNOS is also found in the endothelium and
epicardium in heart ventricles from these fishes
(Tota et al., 2005). NO has
been shown to regulate cardiovascular activities in icefish, including
dilation of branchial vasculature, cardiac stroke volume and power output
(Pellegrino et al., 2003).
Interestingly, NO has a positive inotropic effect on cardiovascular function
in icefish, whereas in other fish and mammals, NO has a negative inotropic
effect (Tota et al., 2005). At
this point it is unknown if this difference is related to the absence of
hemoglobin, or some other species-specific difference in the expression of
signaling molecules operating downstream of NO. Regardless, it seems likely
that if NO is present at sufficient levels to control these processes, then it
likely also contributes to the regulation of additional features of the
cardiovascular and muscular system.
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What pathways may be upregulated in response to high levels of nitric oxide? |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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). In
addition, NO-mediated pathways have been implicated in promoting the growth of
capillary networks (angiogenesis) (reviewed by
Conway et al., 2001).
Angiogenesis involves the expansion, growth and remodeling of blood vessels
into a mature network. This process occurs via sprouting of new
vessels from the ends and sides of existing vessels or by longitudinal
splitting of existing vessels. NO induces upregulation of one of the most
potent factors influencing blood vessel proliferation, vascular endothelial
growth factor (VEGF) (Kimura et al.,
2000). Nitric oxide appears to play a role in both hypoxia- and
exercise-induced stimulation of VEGF and angiogenesis in muscle
(Milkiewicz et al., 2005;
Kimura and Esumi, 2003). It is
also noteworthy that VEGF, along with Angiopoietin-1 (Ang-1), promote
enlargement of the lumenal diameter of the microvasculature
(Suri et al., 1998).
Nitric oxide and mitochondrial biogenesis
Only within the last couple of years we have learned that another very
important role of NO is to stimulate and maintain high densities of
mitochondria in a variety of tissues
(Nisoli et al., 2003;
Nisoli et al., 2004). NO
induces mitochondrial biogenesis via a guanylate cyclase and
cGMP-dependent pathway (Fig.
6). It also plays a role in maintaining constitutive levels of
mitochondrial densities. Null-mutant mice, lacking eNOS, have lower levels of
mtDNA, as well as mRNA levels of subunit IV of cytochrome oxidase (COXIV) and
cytochrome c, compared to wild-type mice in brain, liver and heart
tissue (Nisoli et al.,
2003).
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Nitric oxide activates mitochondrial biogenesis through the transcriptional
coactivator, peroxisome proliferator-activated receptor 
coactivator
1 (PGC-1) (Wu et al.,
1999). PGC-1 is a member of the PPAR family of
transcriptional coactivators, which modulate the activity of transcription
factors through protein–protein interactions with transcription factors,
proteins with histone acetyl transferase activity, and RNA processing proteins
(reviewed in Puigserver and Spiegelman,
2003). PGC-1 stimulates and modulates the expression of two
nuclear transcription factors, nuclear respiratory factors 1 and 2 (NRF-1, -2)
(Wu et al., 1999). Together,
these transcription factors regulate the expression of a number of
nuclear-encoded mitochondrial genes, including cytochrome c, subunits
of both ATP synthase and cytochrome c oxidase, and enzymes of heme
biosynthesis (Scarpulla,
1997). NRFs also induce expression of mitochondrial
transcriptional factor-A (mtTFA), which translocates to the mitochondrion and
controls mtDNA transcription (Shadel and
Clayton, 1993; Wu et al.,
1999). In concert, these factors coordinate the upregulation of
genes required for mitochondrial biogenesis.
Nitric oxide and muscle hypertrophy
High levels of NO can induce cardiac hypertrophy in animals lacking
myoglobin. The heart-to-body-mass index increases by 33% in myoglobinless mice
over-expressing iNOS, compared to wild-type animals
(Gödecke et al., 2003).
The details of the pathway regulating these changes are unknown.
Nitric oxide also induces muscle hypertrophy and activates satellite cells
in the skeletal muscle of mammals (Smith
et al., 2002; Anderson,
2000). Neuronal nitric oxide synthase is part of the dystrophin
glycoprotein protein complex, which links actin to components of the basal
lamina. Neuronal NOS is activated by both mechanical force and pressure. The
subsequent production of NO induces the expression of the two cytoskeletal
proteins, talin and vinculin, resulting in muscle hypertrophy
(Tidball et al., 1999). NO
produced by nNOS also activates muscle satellite cells, although the molecular
components of this pathway are unknown
(Anderson, 2000).
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Nitric oxide as a unifying trigger for traits of icefishes |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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When mutations leading to the loss of expression of Hb and Mb occurred
during icefish evolution, the primary degradative pathways for NO were
eliminated. NO constitutively produced by the NOS system in these animals
would then have a much longer biological half-life and the end result should
have been elevation of steady-state levels of NO in icefish tissues compared
to those of Hb- and Mb-expressers. In fact, preliminary data suggest that
circulating levels of NO in icefish are >2-fold those observed in
red-blooded notothenioids (B.D.S., unpublished). We would predict that
elevated levels of NO would lead to virtually all of the hallmark
characteristics that we have described for icefishes. Elevated vascular
densities in retinal tissue, and increased lumenal diameters of blood vessels
in the oxidative muscle of icefishes, are consistent with a marked
upregulation of angiogenic processes, that would be expected in response to
constitutively higher NO levels. Likewise, elevated mitochondrial densities
and modified mitochondrial morphologies are exactly the patterns that we would
predict if NO levels regulate densities of mitochondria in the tissues. In
fact, NO may also play a role in regulating the size difference in
mitochondria among species that vary in their expression of oxygen-binding
proteins. We find that mitochondria from Mb/Hb-less fishes are significantly
larger than those from red-blooded fishes
(O'Brien and Sidell, 2000).
Higher NO levels in icefish tissue would be consistent with findings that show
a decrease in NO production reduces mitochondrial size
(Nisoli et al., 2004).
Increases in nitric oxide could also account for the enlarged heart size of
icefishes. We find that the heart-to-body-mass of notothenioids correlates
with Mb and Hb expression, with values from red-blooded species being the
smallest, those from icefish expressing Mb intermediate, and from icefishes
lacking Mb, the largest. These observations are in accordance with the role of
NO in inducing cardiac hypertrophy in myoglobinless mice.
Intriguingly, elevated NO concentration may even help clarify the unknown
origin of another striking feature of muscles in Antarctic fishes. Muscle
fiber size of fishes generally increases as body temperature decreases
(Egginton and Johnston, 1984;
Egginton and Sidell, 1989;
Rodnick and Sidell, 1997;
Johnston et al., 1998). This
trend correlates with the inverse relationship between hematocrit and body
temperature mentioned previously. Muscle fibers from Antarctic fishes are even
larger than those of temperate species and fibers of Antarctic icefish lie at
the extreme of this continuum, with oxidative muscle fibers that are
approximately twofold greater in cross-sectional area compared to red-blooded
Antarctic species (O'Brien et al.,
2003; Egginton et al.,
2002). All oxidative pectoral muscle from Antarctic species lack
Mb. Thus, higher NO levels as a result of the loss of Mb and Hb, may also
influence maintenance of unusually large fiber size in the pectoral muscle of
icefish.
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Conclusion |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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The recent discovery of Hb and Mb as NO-oxygenases prompts us to return to the icefishes and examine their physiology from a new perspective. We now have the opportunity to ask: What are the potential adaptations that might occur in the absence of NO-oxygenase activity? The answers are exciting. Current research in mammalian systems suggests that nearly all of the hallmark traits of channichthyid may be a result of high levels of nitric oxide, an outcome that one would expect in the absence of Hb and Mb. These results, coupled with molecular biological techniques, will help us to uncover the genetic underpinnings that have led to establishment of many of the distinctive traits in icefish including: high vascular and mitochondrial densities, large capillary lumenal diameters and even, perhaps, enlarged muscle fibers.
The novel globin proteins, cytoglobin and neuroglobin, have recently been
identified in several vertebrate species
(Pesce et al., 2002). This
latter protein is expressed in brain and neuronal tissue, and has been
implicated in combating nitrosative stress, caused by high levels of nitric
oxide (Herold et al., 2004).
Intriguingly, we have observed a distinct reddish tint in the retinal tissue
C. aceratus, hinting that this may be a heme-containing globin
protein that is expressed in this animal. The channichthyids, or `blodlaus
fisk' (Ruud, 1954) will
undoubtedly help us to learn more about the function of these proteins in the
future, and provide us with at least another 50 years of fruitful studies on
the fascinating physiology of these animals.
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List of abbreviations |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric...What pathways may be...Nitric oxide as a...ConclusionList of abbreviationsReferences |
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peroxisome proliferator-activated receptor coactivator
1
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Acknowledgments |
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References |
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TOPSummaryIntroductionPatterns of oxygen-binding...When and how were...Was loss of Hb...Why have the hemoglobinless...What other apparently...Hemoglobin and myoglobin as...The production of nitric... |
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