Low temperatures and high oxygen solubility characterize the Southern Ocean surrounding Antarctica. Mean annual temperature in McMurdo Sound is −1.86 °C (Littlepage, 1965), while the temperatures of waters surrounding the Antarctic Peninsula range between +0.3 °C during the austral summer to −1.1 °C during the winter months (DeWitt, 1971). Despite these chronically cold temperatures, coastal Antarctica supports an abundant fish fauna dominated by species of the perciform suborder Notothenioidei, a group that has been evolving since the formation of the Antarctic Circumpolar Current, between 14 and 25 million years ago (Eastman and Grande, 1989).
Channichthyid icefishes are thought to have diverged from other notothenioid families approximately 1–3 million years ago (Bargelloni et al., 1994). The 15 species of the Channichthyidae are unique among adult vertebrates in their complete lack of expression of hemoglobin, a characteristic first described in the scientific literature by Ruud (1954). These fishes show profound cardiovascular modifications (large hearts and blood vessels, high cardiac output, increased blood volume) that apparently ensure adequate delivery of oxygen to the tissues despite the lack of circulating hemoglobin (Hemmingsen et al., 1972; Hemmingsen, 1991). In addition to
the absence of a circulating oxygen-transport protein, many investigators have characterized icefishes as also lacking the 16–17 kDa intracellular oxygen-binding protein myoglobin. Myoglobin is a monomeric protein containing a single coordinated heme group that binds oxygen reversibly with a 1:1 molecular stoichiometry. It has an important role in the storage and transport of oxygen from capillaries to mitochondria in oxidative muscle tissues of vertebrates (Covell and Jacquez, 1987; Wittenberg and Wittenberg, 1989). While the cardiovascular alterations described above may help offset the loss of hemoglobin, it is difficult to envisage how these features could compensate for the reported absence of myoglobin in highly aerobic heart tissues (Hamoir, 1988; Eastman, 1990).
Although the consensus view has been that icefishes lack myoglobin, Douglas et al. (1985) reported that myoglobin was expressed in heart tissue of two icefish species, Pseudochaenichthys georgianus and Chaenocephalus aceratus. The technical basis for this report, however, was the detection of the formation of pyridine hemochromagen in crude supernatant extracts from hearts, a method that could easily lead to false positive results in tissues containing high concentrations of mitochondrial cytochromes. To resolve the JEB2492
We used a combined immunochemical and molecular approach to ascertain the presence and concentrations of both the intracellular oxygen-binding hemoprotein myoglobin (Mb) and its messenger RNA (mRNA) in 13 of 15 known species of Antarctic channichthyid icefishes. Mb protein is present in the hearts of eight species of icefishes: Chionodraco rastrospinosus, Chionodraco hamatus, Chionodraco myersi, Chaenodraco wilsoni, Pseudochaenichthys georgianus, Cryodraco antarcticus, Chionobathyscus dewitti and Neopagetopsis ionah. Five icefish species lack detectable Mb protein: Chaenocephalus aceratus, Pagetopsis macropterus, Pagetopsis maculatus, Champsocephalus gunnari and Dacodraco hunteri. Mb concentrations range from 0.44±0.02 to 0.71±0.08 mg Mb g−−1wet mass in heart ventricle of species expressing the protein. A Mb-mRNA-specific cDNA probe
was used to quantify mRNA in five Mb-expressing icefishes. Mb mRNA was found in low but detectable amounts in Champsocephalus gunnari, one of the species lacking detectable Mb. Mb mRNA concentrations in heart ventricle from Mb-expressing species ranged from 0.78±0.02 to 16.22±2.17 pg Mb mRNAµµg−−1total RNA). Mb protein and Mb mRNA are absent from the oxidative skeletal muscle of all icefishes. Steady-state concentrations of Mb protein do not parallel steady-state concentrations of Mb mRNA within and among icefishes, indicating that the concentration of Mb protein is not determined by the size of its mRNA pool.
Key words: myoglobin, mRNA, Antarctic fish, Channichthyidae, Nototheniidae, heart, cardiac muscle, mRNA.
Summary
Introduction
CONCENTRATIONS OF MYOGLOBIN AND MYOGLOBIN mRNA IN HEART
VENTRICLES FROM ANTARCTIC FISHES
THOMAS J. MOYLAN ANDBRUCE D. SIDELL*
School of Marine Sciences, University of Maine, 5741 Libby Hall, Orono, ME 04469-5741, USA
*Author for correspondence (e-mail: Bsidell@Maine.edu)
question of myoglobin expression in the icefishes, our laboratory has recently used more definitive immunochemical and molecular techniques to establish that myoglobin is expressed in heart ventricles of some icefish species while being completely absent from the same tissue in others (Sidell et al., 1997). Furthermore, the disparate positions of myoglobin non-expressers within the phylogeny of icefishes suggested that multiple independent mutational events led to the loss of myoglobin expression during the evolution of the family. The establishment of discretely different mutational mechanisms among these myoglobin non-expressers has corroborated this conclusion (Small et al., 1998). The seemingly random pattern of loss of myoglobin among icefish species appeared to suggest that the protein may not be of functional significance at the severely cold body temperature of Antarctic icefishes, thus relaxing all selective pressure on the retention of its expression and/or structure.
The provocative suggestion that myoglobin may not function at the body temperature of Antarctic fishes led our laboratory and collaborators to examine the functional characteristics of oxygen-binding by myoglobin from these animals and its potential physiological role in those Antarctic icefishes that do express the protein. Using stopped-flow kinetics measurements, we found that myoglobins from both Antarctic and temperate-zone teleost fishes show more rapid binding and release of oxygen at cold temperature than those from mammals (Cashon et al., 1997). These results indicate that fish myoglobins, including those from Antarctic species, possess alterations in protein structure/sequence that increase the speed of binding and release of oxygen at low temperatures. Additional experiments with isolated, perfused hearts from two icefishes, one lacking (Chaenocephalus
aceratus) and one containing (Chionodraco rastrospinosus)
myoglobin protein, demonstrated that hearts possessing myoglobin were capable of greater mechanical performance than those lacking it (Acierno et al., 1997). Both lines of evidence strongly indicate that myoglobin is functional at the normal body temperature of icefish and that it does play a physiological role in the delivery of oxygen to working muscle. These conclusions make the observation that myoglobin is absent from oxidative skeletal muscle of all notothenioid fishes examined to date even more perplexing in the light of the highly aerobic metabolism of this tissue (Sidell et al., 1987).
The confirmation of extremely variable expression of myoglobin in the notothenioid suborder (Fig. 1) is therefore at odds with strong evidence indicating a functional role for the protein and raises questions about the molecular mechanisms responsible for myoglobin loss from aerobically poised muscles. To shed further light on this conundrum, we undertook experiments (i) to estimate the intracellular concentrations of myoglobin in heart ventricle of channichthyid and nototheniid species expressing the protein, to help assess its potential physiological relevance, and (ii) to quantify the concentrations of myoglobin mRNA in channichthyid and nototheniid species as an essential step
towards deciphering the molecular basis for the unusual pattern of myoglobin expression. These measurements also permitted us to evaluate any correlation between pools of myoglobin mRNA and the concentration of myoglobin protein in the heart ventricle of notothenioid species.
Materials and methods
Animal and tissue collection
Chionodraco rastrospinosus, Pseudochaenichthys georgianus, Chaenodraco wilsoni, Chaenocephalus aceratus, Champsocephalus gunnari, Gobionotothen gibberifrons, Trematomus newnesi and Notothenia coriiceps were collected
by 18 foot otter trawl net deployed from the R/V Polar Duke while fishing off the Antarctic Peninsula in Dallman Bay near Astrolabe Needle (64°10′S, 62°35′W) in March–May 1993 and 1996. Fish were transported live to the US Antarctic research station, Palmer Station, and maintained there in running seawater tanks (−1.5 to +1.0 °C). Animals were killed by a sharp blow to the head followed by severing the spinal cord immediately posterior to the head. The heart ventricle and pectoral adductor profundus tissues were rapidly dissected on a chilled stage, weighed and frozen in liquid nitrogen prior to storage at −80 °C.
Tissues from Pagetopsis macropterus, Pagetopsis
maculatus, Dacodraco hunteri, Chionodraco myersi and Cryodraco antarcticus were generously provided by Dr A.
DeVries (University of Illinois) and were collected in McMurdo Sound, Antarctica. Drs R. Acierno and G. di Prisco (Italian National Antarctic Program) kindly supplied samples of Chionodraco hamatus, collected in the vicinity of Terra Nova Bay, Antarctica. Dr T. Iwami (Tokyo Kasei Gakuin University, Japan) supplied Chionobathyscus dewitti and
Neopagetopsis ionah collected in the Weddell Sea. Tissues
were dissected and maintained frozen (liquid nitrogen, dry ice or −80 °C storage) until use.
Purification of myoglobin standard
To determine myoglobin concentrations in icefishes, a myoglobin standard was purified from the related nototheniid species Notothenia coriiceps. Frozen Notothenia coriiceps heart ventricle (2.4 g) was weighed and homogenized [40 % (w/v)] in filtered 100 mmol l−1 potassium phosphate buffer,
pH 7.8, with a glass tissue grinder (Tenbroeck). The homogenate was centrifuged at 23 000 g for 30 min at 4 °C. The resulting supernatant was collected and centrifuged at 23 000 g for another 30 min. The final supernatant was applied to a 2.5 cm×100 cm Bio-Rad P-100 gel permeation column that had been equilibrated with 100 mmol l−1 potassium phosphate
buffer, pH 7.8, containing 0.02 % sodium azide. The column was maintained at 4 °C. Fractions (3 ml) were collected at an elution rate of 13 ml h−1. Fractions containing myoglobin were
DE-52 anion-exchange column (5 ml bed volume) and eluted with a 60 ml gradient of 0–50 mmol l−1KCl in 10 mmol l−1Tris
buffer, pH 8.5. Eluted myoglobin fractions were pooled, dialyzed against 10 mmol l−1NH
4HCO3, divided into samples,
lyophilized and stored at −80 °C.
Protein gel electrophoresis and western blotting
The purity of isolated Notothenia coriiceps myoglobin standard was established by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblot analysis as described previously (Sidell et al., 1997). Confirmation that the single immunopositive band produced by purified Notothenia coriiceps myoglobin on one-dimensional gels represented a single protein was obtained by two-dimensional electrophoresis performed according to the method of O’Farrell (1975) by Kendrick Labs, Inc., and by our laboratory, using a method modified from that of Hochstrasser et al. (1988) (Theresa Grove, personal communication, data not shown).
Identification of tissues expressing myoglobin protein and estimation of intracellular concentrations of myoglobin
The presence and intracellular concentrations of myoglobin in cardiac ventricular and pectoral fin adductor profundus muscle were determined by preparing a 10 % (w/v) homogenate (1 vol of original wet mass of tissue plus 9 vols of 20 mmol l−1 Hepes buffer, pH 7.8 at 4 °C). For each
preparation, 20–50 mg wet mass of frozen cardiac or pectoral tissue was placed in an ice-chilled glass tissue grinder with an appropriate volume of buffer and homogenized. The homogenate was centrifuged at 10 000 g for 10 min at 4 °C, and the resulting supernatant (heart ventricle or pectoral adductor) was collected. Protein concentrations of heart ventricle and pectoral muscle supernatants were determined by bicinchoninic acid (BCA) assay (Sigma), using a bovine serum albumin standard, and separated electrophoretically on duplicate SDS–PAGE gels (for electrophoresis and western blotting conditions, see Sidell et al., 1997).
Myoglobin concentrations in supernatant lanes of Coomassie-stained gels were determined densitometrically with a Sepra Scan 2001 flatbed scanner and software (Integrated Separation Systems). In addition to muscle supernatants, each gel contained a standard curve generated by loading known amounts of purified Notothenia coriiceps myoglobin. The concentration of purified Notothenia coriiceps myoglobin was determined spectrophotometrically.
Myoglobin concentrations in supernatant lanes were calculated from the linear relationship between micrograms of purified myoglobin loaded and integrated signal-area (r2=0.95–0.99 for all gels). All unknown values (myoglobin in
supernatants) consistently fell within the range of the standard curve.
RNA gel electrophoresis and northern blotting
Total RNA was isolated from finely ground, frozen heart ventricle and pectoral fin adductor profundus muscle
(50–300 mg) by acid guanidinium thiocyanate–phenol– chloroform extraction and reconstituted in sterile water (Chomczynski and Sacchi, 1987). The typical yield of total RNA was 1.5µg RNA mg−1tissue, with similar extraction
efficiencies among tissues. Extracted RNA was stored at −80 °C until use.
The concentration of extracted RNA was determined in triplicate by spectrophotometric analysis. The integrity of the RNA was verified by examination of 18S and 28S ribosomal bands stained with ethidium bromide, following separation of the RNA by electrophoresis on 1.2 % agarose formaldehyde gels (Sambrook et al., 1989). The RNA was then transferred to GeneScreen Plus nylon membrane (NEN) by capillary action and cross-linked to the membrane by ultraviolet irradiation at a dose of 0.12 J cm−2 (Kodak IBI Ultralinker
400). A 329 base pair (bp) myoglobin cDNA insert (corresponding to codons 5–114) isolated from Notothenia
coriiceps (kindly provided by Dr M. E. Vayda) was used to
construct a specific probe for myoglobin mRNA, as described previously (Sidell et al., 1997).
Slot blot quantification of myoglobin mRNA
Slot blot (Bio-Rad manifold) analysis was used to quantify the amount of myoglobin mRNA present in heart ventricle and pectoral adductor tissues. Prior to slot blotting, northern blots (for details, see Sidell et al., 1997) were performed on all samples to verify that total RNA was not degraded and to verify specific hybridization of myoglobin cDNA probe to an mRNA of the proper size (0.9 kb). Denatured Notothenia
coriiceps myoglobin cDNA insert served as an internal positive
control and was used to generate a standard curve within each blot. The starting concentration of cDNA was determined fluorometrically, then diluted serially from 100 to 1.5 pg. Yeast tRNA was used as a negative control on each blot.
Total RNA (5µg) (serial dilution 5.0–1.25µg) was loaded per sample. Myoglobin probe preparation, hybridization conditions and blot treatment were identical to those described above for the northern blot membrane. Washed blots were exposed overnight to preflashed Kodak Biomax MR X-ray film at −70 °C. Film was preflashed with a Vivitar 283 electronic flash unit masked to raise the fog level of the film to between 0.1 and 0.2 absorbance units above that of unexposed film. This procedure increased the sensitivity and linear range of the film. Myoglobin mRNA was quantified by densitometry (see above for scanner details) from the autoradiograph. To correct for background, the densitometric value obtained from the negative control (yeast tRNA) was subtracted from all densitometric values for unknowns. The standard curve from each blot was used to convert densitometric values of unknowns into picograms of myoglobin mRNA detected. These values were then expressed as picograms of myoglobin mRNA detected per microgram of total RNA loaded (pg Mb mRNAµg−1total RNA). Densitometric values of
unknowns consistently fell within the linear range of the standard curve (r2=0.96–0.99 for all blots).
Results
Tissue-specific expression of myoglobin protein
Considerable variation in expression of myoglobin was observed within the 13 (of the known 15) species of icefishes examined. Strong cross-reactivity with the polyclonal (not shown) and monoclonal anti-myoglobin antibodies (Fig. 2) was observed in heart ventricle of eight of the 13 icefish species examined (Chionodraco rastrospinosus, Chionodraco
hamatus, Chionodraco myersi, Pseudochaenichthys georgianus, Cryodraco antarcticus, Chaenodraco wilsoni, Chionobathyscus dewitti and Neopagetopsis ionah). Five
icefishes (Champsocephalus gunnari, Chaenocephalus aceratus, Dacodraco hunteri, Pagetopsis maculatus and Pagetopsis macropterus) lack detectable levels of myoglobin
protein in heart ventricle. Examination of pectoral adductor muscles from icefishes revealed that myoglobin protein is not expressed in this highly aerobic oxidative tissue by any species of this family (see also Sidell et al., 1997). Similar tissue-specific myoglobin protein expression was observed in the related red-blooded nototheniid species Gobionotothen
gibberifrons and Trematomus newnesi.
Intracellular concentration of myoglobin protein in heart ventricle
Intracellular concentrations of myoglobin in heart ventricle
were determined by SDS–PAGE. Gels were loaded with measured amounts of a myoglobin standard purified from
Notothenia coriiceps (Fig. 2). Densitometric measurement of
the curve generated by the standards was linear over the range of samples loaded. Myoglobin concentration estimates for channichthyid species ranged from 0.44±0.02 to 0.71±0.08 mg Mb g−1wet muscle mass (N=1–6) and those
for the two nototheniids were 0.85±0.11 and 1.12±0.07 mg Mb g−1wet muscle mass (N=6) (Table 1). Levels
of myoglobin protein in icefishes are comparable with those found in the red-blooded notothenioid fishes examined and are similar to values reported by Sidell et al. (1987) for another nototheniid, Notothenia rossii.
Tissue-specific expression of myoglobin mRNA
In the channichthyid and nototheniid species examined that do express myoglobin protein (see above), hybridization of a myoglobin-mRNA-specific probe identified a single band of 0.9 kb in northern blots of total RNA extracted from cardiac ventricle (see also Sidell et al., 1997). Four of the icefish species that lacked detectable levels of myoglobin protein (Chaenocephalus aceratus, Dacodraco hunteri, Pagetopsis
maculatus and Pagetopsis macropterus) also lacked detectable
[image:4.609.103.504.392.677.2]myoglobin mRNA. The tissue-specific pattern observed for protein expression was also exhibited for myoglobin message.
Fig. 1. Hearts from three species of notothenioid fishes. The channichthyid icefish Chaenocephalus aceratus has a pale yellow ventricle (far left) and lacks myoglobin protein expression. The channichthyid icefish Chionodraco rastrospinosus expresses myoglobin protein (0.64±0.07 mg Mb g−1wet mass) and displays a distinctly rose-colored ventricle (middle). In comparison, the related nototheniid species
Notothenia coriiceps has a characteristically red ventricle (far right) associated with the presence of myoglobin protein (concentration estimate
When detected, myoglobin message was present only in cardiac ventricle, while all oxidative skeletal muscle examined lacked detectable signal, indicating tissue-specific expression of the gene.
Concentration of myoglobin mRNA in heart ventricle
The concentration of myoglobin mRNA was determined by slot blot analyses using the same myoglobin-mRNA-specific probe utilized for northern blot analyses. Fig. 3 shows an example of the standard curve constructed by hybridization of a constant amount of 32P-labeled myoglobin cDNA probe with
known amounts of unlabeled myoglobin cDNA insert. Data obtained from identically treated samples of total RNA from heart ventricle and pectoral adductor profundus were plotted against the standard curve to quantify the amount of myoglobin mRNA present in these tissues. The concentrations of mRNA were expressed as picograms of myoglobin mRNA per microgram of total RNA (pg Mb mRNAµg−1total RNA).
Autoradiographs of blots revealed that values for unknowns consistently fell within the linear portion of the standard curve. Myoglobin mRNA was quantified from the same animals used for the determination of myoglobin protein concentration when sufficient tissue was available. For some species, tissue was limiting, and sample sizes for mRNA determinations are smaller than those for myoglobin protein.
Total myoglobin mRNA in heart ventricle for channichthyid species that express the protein ranged from 0.78±0.02 to 16.22±2.17 pg Mb mRNAµg−1total
RNA (N=1–6) (Table 1). The lowest myoglobin mRNA concentrations were found in Champsocephalus gunnari (0.33±0.09 pg Mb mRNAµg−1total RNA, N=6), a species of
icefish known to lack detectable myoglobin protein while still expressing mRNA (Sidell et al., 1997). Myoglobin mRNA concentrations for the two red-blooded nototheniid species fell between the high and low values determined for the Channichthyidae. We are unaware of any published values for myoglobin mRNA concentrations in teleosts. However, the values reported here for Antarctic fishes are approximately five times lower than those reported by Weller et al. (1986) for human cardiac myoglobin mRNA and fall
45.0
34.7
24.0
18.4
Mb 14.3
1 2 3 4 5 6 7 9 10 11 12
kDa
18.4
Mb 14.3
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
Integrated area
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
r2=0.98
Purified Notothenia coriiceps myoglobin (µg)
A
B
[image:5.609.48.358.83.579.2]C
within a range that can be characterized as a rare message (Alberts et al., 1994). In those species expressing the protein, steady-state concentrations of myoglobin protein are not paralleled by steady-state concentrations of myoglobin mRNA within and among icefish species, indicating that the synthesis and/or turnover of the protein is not directly determined by the size of the myoglobin mRNA pool.
Discussion
Expression of myoglobin protein
The present study extends our knowledge of the pattern of myoglobin protein and mRNA expression to 13 of the 15 known species of Antarctic channichthyid icefishes. Using the same definitive immunochemical approach, we have confirmed the results of Sidell et al. (1997) and further identified three additional species of icefish that produce myoglobin (Chionobathyscus dewitti, Neopagetopsis ionah and
Chionodraco myersi) and two additional species from which
the protein is absent (Dacodraco hunteri and Pagetopsis
maculatus) (see Table 1). Our results also document the very
high degree of tissue specificity in expression of myoglobin by notothenioid fishes. In a pattern that departs from the vertebrate norm, myoglobin protein is detectable only in heart ventricle and is absent from other aerobic muscles, including the primary oxidative skeletal muscle of these labriform swimmers, the pectoral adductor profundus.
We included lanes containing known amounts of myoglobin purified from Notothenia coriiceps into each of our SDS–PAGE analyses of muscle extracts. In addition to a simple test for the presence/absence of myoglobin protein, this approach permitted us to quantify the amount of myoglobin in each extract. Our results reveal that the
myoglobin concentration of heart ventricle is comparable in both red-blooded Antarctic nototheniid fishes and those hemoglobinless channichthyid icefishes that express the protein. Tissue myoglobin concentrations reported for these polar fishes (Table 1) are also similar to those of sedentary benthic fishes from temperate-zone latitudes, such as sea raven (Hemitripterus americanus) and long-horn sculpin (Myoxocephalus octodecimspinosus), 1.0 and 1.2 mg Mb g−1wet mass, respectively (Driedzic and Stewart,
1982). However, they are lower than those of more active temperate-zone species, such as striped bass Morone saxatilis (6.0 mg Mb g−1wet mass; Sidell et al., 1987). The myoglobin
[image:6.609.46.559.86.312.2]content of the heart muscle of fishes has been positively correlated with the ecological physiology of a variety of species ranging in lifestyle from benthic sedentary to active pelagic (Giovane et al., 1980). These observations make it tempting to suggest that the variation in myoglobin expression observed among Antarctic icefishes might be similarly attributable to life history differences. Indeed, a wide range of relative activity levels has been reported among icefishes on the basis of their feeding behaviors. Lifestyles range from sluggish demersal, such as Chaenocephalus aceratus, to semi-pelagic feeders, such as Pseudochaenichthys georgianus, to the pelagic Champsocephalus gunnari, which feeds exclusively on krill in the water column (Eastman, 1993). Myoglobinless species occupy both ends of this range of activities within the icefish family. Thus, no obvious causative relationship appears to exist between lifestyle and whether or not myoglobin is expressed in hearts of these animals. This lack of correlation with activity level is particularly curious in the light of experiments that strongly implicate a physiological role for the protein in these animals (Cashon et al., 1997; Acierno et al., 1997).
Table 1. Concentrations of myoglobin (Mb) and Mb mRNA in heart ventricles of Antarctic notothenioid fishes
Mb concentration Mb mRNA concentration
Taxon (mg Mb g−1wet mass) N (pg Mb mRNAµg−1total RNA) N
Channichthyidae
Chionodraco rastrospinosus 0.64±0.07 6 16.22±2.17 6
Chionodraco hamatus 0.62±0.04 6 0.78±0.02 4
Chionodraco myersi 0.71±0.08 4
Pseudochaenichthys georgianus 0.46±0.04 6 1.97±0.77 4
Cryodraco antarcticus 0.44±0.02 6 2.05 1
Chaenodraco wilsoni 0.65±0.08 6 5.31 1
Chionobathyscus dewitti 0.69±0.03 2
Neopagetopsis ionah 0.70 1
Champsocephalus gunnari ND 6 0.33±0.09 6
Chaenocephalus aceratus ND 6 ND 6
Dacodraco hunteri ND 4 ND 4
Pagetopsis macropterus ND 2 ND 2
Pagetopsis maculatus ND 1 ND 1
Nototheniidae
Gobionotothen gibberifrons 0.85±0.11 6 7.27±1.94 6
Trematomus newnesi 1.12±0.07 6 7.11±1.04 5
Expression of myoglobin mRNA
Using a myoglobin-specific cDNA probe, we were able to identify mRNA of the appropriate size in extracts from heart ventricle of several myoglobin-protein-expressing species of Antarctic fishes (see Table 1). Our northern blots did not reveal RNA encoding for myoglobin in hearts from four of the five species that do not express the protein. (Although not detectable by northern blot analyses, we do know that very low levels of transcript can be detected by polymerase chain reaction amplification of cDNA from Pagetopsis macropterus
hearts; Vayda et al., 1997.) Consistent detection of myoglobin mRNA in Champsocephalus gunnari, despite the absence of any detectable protein, has been reported previously (Sidell et al., 1997). A five-nucleotide duplication that is unique to this species causes a shift in reading frame of the message downstream from amino acid residue 91 and premature termination at residue 103 and is apparently responsible for the production of a defective translation product that is degraded immediately (Vayda et al., 1997). Finally, we have been unable to detect the presence of mRNA encoding for myoglobin in oxidative skeletal muscles from any of the channichthyid icefishes sampled and 12 other red-blooded notothenioid species, representing four different families (T. J. Moylan and B. D. Sidell, unpublished data). This observation strongly indicates that the event leading to loss of myoglobin expression in oxidative skeletal muscle occurred very early in the notothenioid lineage, presumably before the divergence of the extant families.
Having successfully quantified the levels of myoglobin in those icefish species that produce the protein, we hoped to gain some insight into factors that regulate its intracellular concentration. Perhaps one of the most straightforward possibilities is that the concentration of myoglobin in the tissue is determined directly by the pool size of mRNA encoding the protein. To test this possibility, we sought to quantify concentrations of myoglobin-specific mRNA in the same tissues. In these experiments, one typically also probes for a transcript encoding a constitutively expressed housekeeping gene to control for unequal RNA loading between samples. For this purpose, H. W. Detrich III (Northeastern University) generously provided a cDNA probe specific for β-tubulin prepared from Notothenia coriiceps, the same species from which the myoglobin probe was generated. Preliminary northern blot analyses with the tubulin probe indicated similar levels of β-tubulin mRNA in all tissues selected. However, more sensitive slot blot analyses revealed sufficient variability in tubulin mRNA expression to preclude its use as a control.
Because the extraction efficiencies for total RNA were similar among tissues, we expressed myoglobin mRNA levels as a fraction of total RNA loaded in each lane. Using this approach, total myoglobin mRNA in heart ventricles of channichthyid species ranged from 0.78±0.02 to 16.22± 2.17 pg Mb mRNAµg−1total RNA in species that produce the
protein. These are, to our knowledge, the first values reported for myoglobin mRNA content in fish tissues.
The published literature contains no unifying consensus on the relationship between concentrations of Mb protein and Mb mRNA in tissues. Weller et al. (1986) found myoglobin mRNA levels in human cardiac tissue of approximately 4µg Mb mRNAµg−1poly(A) RNA. If we assume that
poly(A) mRNA makes up 2–3 % of the total cytoplasmic RNA pool, this value corresponds to approximately 80 pg Mb mRNAµg−1total RNA, approximately five times
higher than that found in icefish. The concentration of myoglobin protein in human heart is approximately seven- to 10-fold higher than that in the hearts of Antarctic icefishes,
STD (−) 1 2 3 4 5
A
2.5
2.0
1.5
1.0
0.5
0
Integrated area
0 10 20 30 40 50
r2=0.98
Notothenia coriiceps cDNA STD (pg)
[image:7.609.58.288.76.428.2]B
Fig. 3. Determination of myoglobin (Mb) mRNA concentrations in heart ventricles of notothenioid fishes. Slot blot analysis performed on total RNA extracted from heart ventricles. Myoglobin mRNA was detected by hybridization with random-primed (Boehringer-Mannheim) 32P-labeled Notothenia coriiceps myoglobin probe. (A) Autoradiograph of slots used for the analysis. STD, Notothenia
coriiceps Mb cDNA insert loaded (50.0, 25.0, 12.5, 6.25, 3.10,
initially suggesting that a relationship might exist between myoglobin-specific mRNA pool size and the intracellular concentration of the protein. However, myoglobin protein levels in normal human muscle are generally higher in type I fibers than in type II fibers (Jansson and Sylven, 1983), but no obvious difference between the amount of myoglobin mRNA in the two fiber types can be detected by in situ hybridization (Mitsui et al., 1993). Weller et al. (1986) did find that levels of myoglobin protein and mRNA correlated in comparisons between skeletal muscle of seals and various muscle tissues from humans, but that mRNA levels in mouse skeletal muscle were higher than expected on the basis of these interspecific comparisons.
Our results indicate that a direct relationship between mRNA pool size and protein concentration does not, however, exist for myoglobin in tissues from Antarctic icefishes. In comparing across closely related channichthyid icefish species, we can conclude that the steady-state concentration of myoglobin protein does not parallel the pool size of myoglobin mRNA in the same tissue. As an illustration, a difference of approximately 20-fold in myoglobin mRNA level is found between the congeneric species Chionodraco rastrospinosus and Chionodraco
hamatus (Table 1). However, this marked difference in
mRNA pool size is not reflected in the concentration of myoglobin protein, which is not significantly different between hearts from the two species. These results suggest that myoglobin protein content in the hearts of Antarctic icefishes is not determined by the size of the mRNA pool encoding for the protein, but must be controlled at the points of translation or post-translationally (e.g. degradation rate constant for the protein).
Evolutionary context of myoglobin expression in icefishes
Mapping the pattern of myoglobin protein and myoglobin mRNA expression upon the phylogenetic tree of the icefish family permits us to address several features regarding the evolution of these traits. To accomplish this, we used a consensus phylogeny based upon a combination of morphological (Iwami, 1985) and more recent molecular biological mitochondrial (mt)DNA characters (Chen et al., 1998). Our first conclusion is that mutations leading to loss of cardiac myoglobin expression have occurred independently at least four times during the evolution of the family (Fig. 4). The five species that do not produce myoglobin protein (Champsocephalus gunnari, Chaenocephalus aceratus, Dacodraco hunteri, Pagetopsis macropterus and Pagetopsis maculatus) belong to four distinct clades, at least two of which
contain members that do express the protein. Second, the pattern of myoglobin mRNA expression suggests that the loss of myoglobin protein has occurred by at least three independent molecular mechanisms during the evolution of the icefish family. In Champsocephalus gunnari, myoglobin mRNA is present in modest amounts, but the corresponding protein cannot be detected. Although not detectable by northern blot analysis, very low concentrations of myoglobin mRNA are produced by Pagetopsis macropterus (Vayda et al., 1997). The mutations underlying the loss of myoglobin expression in these two species have now been identified and are different (Small et al., 1998). Chaenocephalus aceratus, in contrast, apparently does not transcribe the myoglobin gene at all (Small et al., 1998). All these processes are quite distinct from the mechanism responsible for the hemoglobinless state of this family, deletion of the gene encoding β-globin subunits from the genome (Cocca et al., 1995).
Genus
Number of species in genus
Species examined
Myoglobin
Protein mRNA
Champsocephalus 2 gunnari − +
Pagetopsis 2 macropterus* − − maculatus − −
Neopagetopsis 1 ionah + +
Pseudochaenichthys 1 georgianus + +
Dacodraco 1 hunteri − −
Channichthys 1
Cryodraco 1 antarcticus + +
Chionobathyscus 1 dewitti + +
Chaenocephalus 1 aceratus − −
Chionodraco 3 myersi + +
rastrospinosus + +
hamatus + +
[image:8.609.97.498.477.669.2]Chaenodraco 1 wilsoni + +
The seemingly random loss of myoglobin expression within the Channichthyidae at different times and by different mechanisms seems to be at odds with both biochemical (Cashon et al., 1997) and physiological (Acierno et al., 1997) information, indicating that the protein confers functional advantage to these animals, and molecular data (Vayda et al., 1997) that show very high levels of sequence conservation in the gene. Each of these latter lines of evidence suggests that selective pressure should mitigate towards retention of myoglobin expression. Perhaps the seemingly contradictory nature of these observations is really the product of our own tendency to want to see evolutionary events through the distorted lens of ‘black-and-white fallacy’. That is, both the trait is advantageous and its loss is lethal or, alternatively, it is at best neutral in functional terms and its loss can occur randomly. The pattern of myoglobin loss among Antarctic icefishes appears to illustrate that evolutionary patterns can be subtler than this binary view. Here, the truth appears to be ‘grey’ rather than ‘black or white’. In these animals, loss of the ability to express this physiologically functional protein appears to reduce the scope for cardiac performance, but is not lethal.
At the level of the individual organism, the non-lethality of the loss of myoglobin is relatively easy to explain on the basis of a combination of environmental and organismal characteristics. Because of both very cold temperature and very pronounced vertical mixing, the waters of the Southern Ocean are characterized by exceptionally high oxygen content. Controversies regarding metabolic cold-adaptation notwithstanding, absolute metabolic rates of Antarctic fishes are relatively low because of their cold body temperature. Thus, the conjunction of high oxygen availability with low absolute oxygen demand may explain why the loss of cardiac myoglobin is not a lethal mutation. However, these features do not address the more perplexing question that appears to fly in the face of modern population genetics theory: why is an apparently disadvantageous (on the basis of cardiac performance) trait not subject to negative selection against those species that lack myoglobin?
The crux of the question is whether the reduction in cardiac performance that accompanies loss of myoglobin expression is disadvantageous to icefish species. Implicit in this question is that the disadvantage would be one that occurs through competition. Can a competitive disadvantage exist in the absence of competition? We know that, some time between the mid-Tertiary and the present, there was a massive crash of species diversity in the Southern Ocean that left an ancestral stock of demersal notothenioids to colonize this expansive marine environment. Although the proximate cause of this event is not known with certainty, it is widely considered to be the reason for the ultimate dominance of notothenioid species in the fish fauna of Antarctica (Eastman, 1993). Perhaps the combined evolutionary features of relatively low niche competition in marine habitats depauperate of fish species and the uniquely cold and oxygen-rich waters of the Southern Ocean help explain why icefish species lacking functional myoglobin persist and thrive.
We gratefully acknowledge the following people for generously providing samples; Arthur DeVries (University of Illinois), Tetsuo Iwami (Tokyo Kasei Gakuin University, Japan) and Raffaele Acierno and Guido di Prisco (Italian National Antarctic Program). We are also indebted to the masters and crew of the R/V Polar Duke and the personnel at the US Antarctic Program’s Palmer Station who supported our work while in Antarctica. US National Science Foundation Grants OPP 92-20775 and 94-21657 to B.D.S. funded this work.
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