1277
The Journal of Experimental Biology 203, 1277–1286 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JEB2492
CONCENTRATIONS OF MYOGLOBIN AND MYOGLOBIN mRNA IN HEART
VENTRICLES FROM ANTARCTIC FISHES
THOMAS J. MOYLAN AND BRUCE D. SIDELL*
School of Marine Sciences, University of Maine, 5741 Libby Hall, Orono, ME 04469-5741, USA
*Author for correspondence (e-mail: [email protected])
Accepted 9 February; published on WWW 23 March 2000
Summary
We used a combined immunochemical and molecular
was used to quantify mRNA in five Mb-expressing icefishes.
approach to ascertain the presence and concentrations
Mb mRNA was found in low but detectable amounts in
Champsocephalus gunnari, one of the species lacking
of both the intracellular oxygen-binding hemoprotein
detectable Mb. Mb mRNA concentrations in heart
myoglobin (Mb) and its messenger RNA (mRNA) in 13 of
ventricle from Mb-expressing species ranged from
15 known species of Antarctic channichthyid icefishes.
0.78±0.02 to 16.22±2.17 pg Mb mRNA µg−1 total RNA). Mb
Mb protein is present in the hearts of eight species
protein and Mb mRNA are absent from the oxidative
of icefishes: Chionodraco rastrospinosus, Chionodraco
skeletal muscle of all icefishes. Steady-state concentrations
hamatus, Chionodraco myersi, Chaenodraco wilsoni,
of Mb protein do not parallel steady-state concentrations
Pseudochaenichthys georgianus, Cryodraco antarcticus,
of Mb mRNA within and among icefishes, indicating that
Chionobathyscus dewitti and Neopagetopsis ionah. Five
the concentration of Mb protein is not determined by the
icefish species lack detectable Mb protein: Chaenocephalus
size of its mRNA pool.
aceratus, Pagetopsis macropterus, Pagetopsis maculatus,
Champsocephalus gunnari and Dacodraco hunteri.
Mb
concentrations
range
from
0.44±0.02
to
Key words: myoglobin, mRNA, Antarctic fish, Channichthyidae,
0.71±0.08 mg Mb g−1 wet mass in heart ventricle of species
Nototheniidae, heart, cardiac muscle, mRNA.
expressing the protein. A Mb-mRNA-specific cDNA probe
Introduction
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
1278 T. J. MOYLAN AND B. D. SIDELL
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
pooled on the basis of relative mobility compared with
hemoglobin and high absorbance at 415 nm, but low
absorbance at 280 nm. For further purification, pooled
myoglobin-containing fractions were applied to a Whatman
Myoglobin and myoglobin mRNA in fish heart 1279
DE-52 anion-exchange column (5 ml bed volume) and eluted
with a 60 ml gradient of 0–50 mmol l−1 KCl in 10 mmol l−1 Tris
buffer, pH 8.5. Eluted myoglobin fractions were pooled,
dialyzed against 10 mmol l−1 NH4HCO3, 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 onedimensional 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 1.5 µg RNA mg−1 tissue, 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 (5 µg) (serial dilution 5.0–1.25 µg) 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 mRNA µg−1 total RNA). Densitometric values of
unknowns consistently fell within the linear range of the
standard curve (r2=0.96–0.99 for all blots).
Values are presented as means ± S.E.M.
1280 T. J. MOYLAN AND B. D. SIDELL
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 tissuespecific 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−1 wet muscle mass (N=1–6) and those
for the two nototheniids were 0.85±0.11 and
1.12±0.07 mg Mb g−1 wet 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
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−1 wet 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
3.04 mg Mb g−1 wet mass; T. J. Moylan and B. D. Sidell, unpublished data).
Myoglobin and myoglobin mRNA in fish heart 1281
A
1
2
3
4
5
6
7
9
10
11
12
kDa
45.0
34.7
24.0
18.4
Mb
14.3
B
18.4
Mb
14.3
0.20
C
0.18
0.16
Integrated area
0.14
0.12
r2=0.98
0.10
0.08
0.06
0.04
0.02
0
0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Purified Notothenia coriiceps myoglobin (µg)
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
Fig. 2. Determination of myoglobin concentrations in
heart ventricles of notothenioid fishes. (A) 15 %
tricine gel of supernatants from heart ventricle (35 µg
each) of the channichthyid icefish Cryodraco
antarcticus. Lanes 3, 5, 7, 10, heart ventricle
supernatant from four individuals; lane 1, heart
ventricle supernatant from Chaenocephalus aceratus
(negative control). Lanes 2, 4, 6, 9, 11, standard
curve of 0.4, 0.8, 1.2, 1.6, 2.0 µg of purified
Notothenia coriiceps myoglobin, respectively.
Numbers alongside lane 12 correspond to sizes (in
kDa) of molecular mass standards in that lane.
Quantification of myoglobin protein (Table 1) was
accomplished by densitometric scan of the
myoglobin-associated
band
(Mb,
arrow).
(B) Immunoblot of a duplicate gel to A (above) with
monoclonal mouse anti-human myoglobin antibody.
The results indicate the expression of myoglobin
protein in heart ventricles of Cryodraco antarcticus
and the absence of detectable myoglobin protein in
Chaenocephalus aceratus. Only the molecular mass
range containing myoglobin (arrow) is shown.
(C) Plot of the standard curve from A (above). The
integrated area of scanned bands from standards is
plotted against micrograms of protein loaded. The
area of myoglobin bands from unknowns consistently
fell within this standard curve.
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 mRNA µg−1 total 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 mRNA µg−1 total
RNA (N=1–6) (Table 1). The lowest myoglobin mRNA
concentrations were found in Champsocephalus gunnari
(0.33±0.09 pg Mb mRNA µg−1 total 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
1282 T. J. MOYLAN AND B. D. SIDELL
Table 1. Concentrations of myoglobin (Mb) and Mb mRNA in heart ventricles of Antarctic notothenioid fishes
Mb concentration
(mg Mb g−1 wet mass)
N
Channichthyidae
Chionodraco rastrospinosus
Chionodraco hamatus
Chionodraco myersi
Pseudochaenichthys georgianus
Cryodraco antarcticus
Chaenodraco wilsoni
Chionobathyscus dewitti
Neopagetopsis ionah
Champsocephalus gunnari
Chaenocephalus aceratus
Dacodraco hunteri
Pagetopsis macropterus
Pagetopsis maculatus
0.64±0.07
0.62±0.04
0.71±0.08
0.46±0.04
0.44±0.02
0.65±0.08
0.69±0.03
0.70
ND
ND
ND
ND
ND
Nototheniidae
Gobionotothen gibberifrons
Trematomus newnesi
0.85±0.11
1.12±0.07
Taxon
Mb mRNA concentration
(pg Mb mRNA µg−1 total RNA)
N
6
6
4
6
6
6
2
1
6
6
4
2
1
16.22±2.17
0.78±0.02
6
4
1.97±0.77
2.05
5.31
4
1
1
0.33±0.09
ND
ND
ND
ND
6
6
4
2
1
6
6
7.27±1.94
7.11±1.04
6
5
Concentration data are presented as mean ± S.E.M.; ND, not detected by western or northern blotting.
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−1 wet 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−1 wet mass; Sidell et al., 1987). The myoglobin
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 semipelagic 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).
Myoglobin and myoglobin mRNA in fish heart 1283
A
STD
1
(−)
2
3
4
5
2.5
B
Integrated area
2.0
1.5
r2=0.98
1.0
0.5
0
0
10
20
30
40
50
Notothenia coriiceps cDNA STD (pg)
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 (BoehringerMannheim) 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,
1.55 pg), (−), yeast tRNA negative control; lanes 1, 2, 3, 4 and 5 are
representative loadings of sample total RNA from unknowns (5.0,
2.5 and 1.25 µg). (B) Plot of the standard curve used in determination
of myoglobin mRNA concentrations. Densitometric values
associated with the integrated area of scanned slots (STD) are shown
as a function of picograms of Notothenia coriiceps Mb cDNA insert
loaded. Absorbance values of unknowns consistently fell within the
linear range of the standard curve.
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 mRNA µg−1 total 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
4 µg Mb mRNA µg−1 poly(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 mRNA µg−1 total 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,
1284 T. J. MOYLAN AND B. D. SIDELL
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).
Genus
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).
Number of
species
in genus
Champsocephalus
Pagetopsis
2
2
Neopagetopsis
Pseudochaenichthys
Dacodraco
Channichthys
Cryodraco
Chionobathyscus
Chaenocephalus
Chionodraco
1
1
1
1
1
1
1
3
Chaenodraco
1
Species
examined
Myoglobin
Protein mRNA
gunnari
macropterus*
maculatus
ionah
georgianus
hunteri
−
−
−
+
+
−
+
−
−
+
+
−
antarcticus
dewitti
aceratus
myersi
rastrospinosus
hamatus
wilsoni
+
+
−
+
+
+
+
+
+
−
+
+
+
+
Fig. 4. Phylogenetic topology of myoglobin and myoglobin mRNA expression in heart ventricle among channichthyid species. The
phylogenetic relationships within the Channichthyidae are based on a cladistic analysis of morphological and molecular characters (redrawn
from Iwami, 1985; Chen et al., 1998). Data for Chionobathyscus dewitti, Neopagetopsis ionah, Chionodraco myersi, Dacodraco hunteri and
Pagetopsis maculatus are from the present paper. Data for all other species are from Sidell et al. (1997). *Although not detectable by northern
blot analyses, we do know that polymerase chain reaction amplification of cDNA from Pagetopsis macropterus hearts shows that very low
levels of transcript are produced in this species (Vayda et al., 1997).
Myoglobin and myoglobin mRNA in fish heart 1285
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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