Muscle fine structure may maintain the function of oxidative fibres in haemoglobinless Antarctic fishes
1 School of Marine Sciences, University of Maine, Orono, ME 04469,
USA
2 Department of Physiology, University of Birmingham, B15 2TT, UK
* Present address: Department of Molecular, Cellular and Developmental Biology,
University of Colorado at Boulder, Boulder, CO 80309, USA
Author for correspondence (e-mail:
s.egginton{at}bham.ac.uk)
Accepted 21 October 2002
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Summary |
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Key words: muscle, haemoglobin, Antarctic icefish, capillary supply, metabolic enzyme, lipid, mitochondria
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Introduction |
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There are two types of labriform swimming: drag-based and lift-based.
Pectoral fins move perpendicular to the flow during the power stroke of
drag-based swimming, while in lift-based swimming, fins move up and down like
bird wings (Lindsey, 1978).
Notothenioids employ drag-based labriform locomotion, controlled by six
muscles of the pectoral fin (Johnston,
1989
). The power stroke is produced by the pectoral adductor
profundus, which constitutes approximately 3% of body mass. This muscle is
composed of 80% oxidative fibres, which contain large densities of
mitochondria and aerobically poised enzymes, and a rich capillary network
(Walesby and Johnston, 1980
;
Johnston and Harrison, 1985
;
Dunn et al., 1989
). Despite
its high aerobic capacity and role in powering sustained swimming, pectoral
muscle does not express the oxygen-binding protein myoglobin (Mb), which in
other vertebrates both stores and facilitates the diffusion of oxygen within
aerobic muscle (Wittenberg and Wittenberg,
1989
). 25 species from five families of notothenioids have been
examined and all lack Mb in the oxidative skeletal muscle of the pectoral
girdle (Moylan and Sidell,
2000
; and T. J. Moylen and B. D. Sidell, unpublished
observation).
One family of Antarctic fishes, the Channichthyidae, lack both Mb in
pectoral muscles and the circulating oxygen-binding protein haemoglobin (Hb).
Lack of Hb gives channicthyids a translucent, ice-like appearance that
inspired their common name of icefish. Despite having an oxygen carrying
capacity nearly an order of magnitude lower than that of red-blooded
notothenioids (Egginton,
1994), icefish have similar oxygen consumption rates and activity
patterns to their red-blooded relatives of the family Nototheniidae
(Ruud, 1954
;
Holeton, 1970
).
We sought to understand how the aerobic pectoral locomotory muscles maintain function despite the lack of facilitated transport by Hb in icefish. We chose species for this study that are evolutionarily closely related, and all lack Mb expression; they only differ in their expression of Hb. We examined differences in ultrastructure and capillarity of the pectoral adductor muscle between two benthic/epibenthic nototheniids (Notothenia coriiceps and Gobionotothen gibberifrons) and two benthic/epibenthic channichthyids (Chaenocephalus aceratus and Chionodraco rastrospinosus). We also estimated key enzyme activities within these four species to determine if loss of oxygen-binding proteins has reduced oxidative metabolic capacity.
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Materials and methods |
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Tissue preparation for microscopy
The oxidative pectoral muscle, m. adductor profundus, was fixed while
pinned at resting length to strengthened cork strips (N. coriiceps
and C. aceratus), or while attached to the underlying cartilage
(C. rastrospinosus). Tissue was fixed in an ice-cold buffered
glutaraldehyde/paraformaldehyde solution containing 0.05% sodium azide
(Egginton and Cordiner, 1994)
or a solution of 3% glutaraldehyde, 0.1 mol l-1 sodium cacodylate,
0.11 mol l-1 sucrose and 2 mmol l-1 CaCl2, pH
7.4, of similar osmolality. Samples were then trimmed into blocks with a cut
face of approx. 1 mm2 and post-fixed in buffered 1% OsO4
for 1-2 h. Blocks were cut from the outer edge of the tissue, where the
fixative had penetrated well, and care was taken to avoid areas that appeared
damaged. Samples were subsequently dehydrated and embedded in epoxy resin
(Araldite) under vacuum. 2-4 blocks were prepared from each individual (5-6
individuals per species), and one was chosen at random for subsequent
analysis. In addition to the present measurements of capillary size and
cristae surface density, indices of capillarity and tissue ultrastructure for
G. gibberifrons were taken from Londraville and Sidell
(1990a
,b
).
Light microscopy
Semi-thin (0.5-1.5 µm) sections were stained with 1% Toluidine Blue to
orient the blocks for true transverse (TS) or longitudinal (LS) sections of
muscle fibres. Parameters of capillary supply were calculated from samples
viewed at a magnification of x500 (N. coriiceps, C. aceratus)
or x400 (C. rastrospinosus) using an unbiased sampling rule
(the `forbidden line' procedure; Egginton,
1990). Mean fibre area,
f, capillary to
fibre ratio, C:F, and numerical density of capillaries,
NA(c,f), were calculated from digitized images using
in-house software or SigmaScan (Jandel). Mean fibre area was calculated from
measurements of a minimum of 20 fibres per section; C:F and
NA(c,f) were measured using a square-lattice test pattern
with an interval spacing equal to 25-37.5 µm (sample area of 0.051-0.079
mm2). The test pattern was place on a digitising tablet and viewed,
along with the sections through a light microscope fitted with a drawing tube.
Between two and four non-overlapping regions were sampled from each section.
Capillary tortuosity, c(K,0), i.e. the deviation from an anisotropic
orientation of capillaries with respect to the long axis of muscle fibres, was
estimated from the ratio of unbiased capillary counts in LS and TS, assuming a
spherical-normal distribution (Egginton,
1990
). We also calculated the capillary length density,
Jv(c,f), which is a measure of the length of capillaries per unit
volume of tissue and accounts for the degree of tortuosity in the capillary
bed, estimated as c(K,0) x NA(c,f). The
maximum diffusion distance, also known as Krogh's radius (Kr), is
related to capillary length density [Jv(c,f)] as:
Kr=1/[
Jv(c,f)]1/2
(Weibel, 1984
). Capillary
volume and surface densities were calculated from the product of
Jv(c,f) and capillary cross sectional area and perimeter,
respectively.
Electron microscopy
Ultrathin (approx. 80 nm) sections were double-stained with 2% uranyl
acetate followed by 0.5% lead citrate, or methanoic uranyl acetate (30%) and
aqueous lead tartrate (2%). Electron micrographs were taken at an accelerating
voltage of 60 kV using the systematic area-weighted subsampling method
(Cruz-Orive and Weibel, 1981).
10-12 micrographs per block were analysed at a final magnification of
x8500-15 750. Surface area of the capillary,
(c), and
the boundary length of the capillary,
(c), were measured by projection onto
a digitising tablet. 5-20 capillaries were measured from each individual. The
volume density (volume of cellular components per unit volume of fibre) of
mitochondria, Vv(mit,f), and intracellular lipid droplets,
Vv(lip,f), were quantified using point-counting and a square lattice
test grid with a spacing (d) equal to 0.8-1.65 µm on the projected
image (Weibel, 1979
).
Cristae surface density (surface area of inner-mitochondrial membranes per
volume of mitochondria), Sv(cs,mit), was estimated from 10-12
micrographs taken at x36 000-39 000, as described previously
(Egginton and Sidell, 1989;
O'Brien and Sidell, 2000
). The
field of view was chosen using the systematic-area-weighted-quadrats
subsampling method (Cruz-Orive and Weibel,
1981
), and the mitochondria most clearly viewed in cross-section
were photographed. Sv(cs,mit) was calculated using the line-intercept
method with a square lattice test pattern overlaid on top of projected
micrographs at a final magnification of x72 000 (d=0.10 µm;
N. coriiceps and C. aceratus) or x91,000
(d=0.11 µm; C. rastrospinosus). In addition,
Sv(cs,mit) from G. gibberifrons was analyzed from material
previously collected by Londraville and Sidell
(1990a
), using the same
sampling method, with sections restained for best clarity and photographic
prints of mitochondria (x81 000; d=0.12 µm). There was no
significant difference between measurements of Sv(cs,mit) made on
projected micrographs and printed micrographs of mitochondria from C.
rastrospinosus (O'Brien and Sidell,
2000
). Mean free sarcoplasmic spacing was also calculated as
a=[4(1-Vv)/Sv]
(Egginton et al., 1988
).
Mitochondrial cristae surface densities per gram of tissue were calculated
using a value of 1.055 g cm3 for the density of muscle
(Webb, 1990
).
Enzyme analysis
Measurements of maximal activities of phosphofructokinase (PFK), carnitine
palmitoyltransferase I (CPT-I) and cytochrome oxidase (CO) require fresh
tissue and were performed shortly after tissue dissections. Tissues used for
measuring the maximal activities of citrate synthase (CS),
ß-hydroxyacyl-CoA-dehydrogenase (HOAD), pyruvate kinase (PK) and lactate
dehydrogenase (LDH) were quickly frozen in liquid nitrogen, and stored at
-70°C until assays were completed. Maximal activity of hexokinase (HK)
measured on both fresh and frozen tissues after control experiments determined
that this enzyme was stable to freezing. Measurements were made using a
Perkin-Elmer Lambda 6 spectrophotometer equipped with a six-carriage cuvette
carrier chilled with a circulating and refrigerated water bath. Assays were
performed in triplicate at 1±0.5°C.
Tissue was homogenized in a 10% w/v ice-cold buffer (40 mmol l-1 Hepes, 1 mmol l-1 EDTA, 2 mmol l-1 MgCl2, pH 7.8 at 1°C), except for tissues used in CO and CPT-I assays (see below). Dithiothreitol (DTT) was added to a final concentration of 2 mmol l-1 in buffer for PFK, LDH and HK assays. Tissue was first minced on a chilled stage, then homogenized with two 10-15 s bursts of a Tekmar Tissuemizer, keeping the homogenate on ice between bursts. Homogenisation was completed by hand using a Tenbroeck ground glass homogeniser.
Background enzyme activities were measured in the absence of initiating substrate and subtracted from total activity in the presence of substrate. Maximal activities were determined by measuring the rate of oxidation or reduction of pyridine nucleotides at 340 nm for 5 min, except when noted differently. Details of assay conditions are described below.
Phosphofructokinase (PFK, EC 2.7.1.11)
The assay used was modified from that described by Opie and Newsholme
(1967) and Read et al.
(1977
). The final reaction
mixture contained 7 mmol l-1 MgCl2, 200 mmol
l-1 KCl, 1 mmol l-1 KCN, 2 mmol l-1 AMP, 0.15
mmol l-1 NADH, 2 mmol l-1 ATP, 4 mmol l-1
fructose 6-phosphate (F6P), 2 U ml-1 aldolase, 10 U ml-1
triosephosphate isomerase, 2 U ml-1 glycerol-3-phosphate
dehydrogenase, 75 mmol l-1 triethanolamine, pH 8.4 at 1°C.
Reactions were initiated by addition of a mixture of ATP and F6P.
Lactate dehydrogenase (LDH, EC 1.1.1.27)
Procedure for this assay was that described by Hansen and Sidell
(1983). The final reaction
mixture contained 5 mmol l-1 pyruvate, 0.15 mmol l-1
NADH, 1 mmol l-1 KCN, 50 mmol l-1 imidazole, pH 7.7 at
1°C. Reactions were initiated by addition of pyruvate.
Pyruvate kinase (PK, EC 2.7.1.40)
This assay was conducted as described by Hansen and Sidell
(1983). The final reaction
mixture contained 150 mmol l-1 KCl, 1 mmol l-1 KCN, 10
mmol l-1 MgSO4, 0.15 mmol l-1 NADH, 5 mmol
l-1 ADP, 2.5 mmol l-1 phosphoenol pyruvate (PEP), 10 U
ml-1 LDH, 50 mmol l-1 imidazole, pH 7.1 at 1°C.
Reactions were initiated by addition of PEP.
3-hydroxyacyl CoA dehydrogenase (HOAD, EC 1.1.1.35)
The methods for this assay were first described by Beenakkers et al.
(1967) and subsequently
modified by Hansen and Sidell
(1983
). The final reaction
mixture contained 1 mmol l-1 EDTA, 1 mmol l-1 KCN, 0.15
mmol l-1 NADH, 0.1 mmol l-1 acetoacetyl CoA, 50 mmol
l-1 imidazole, pH 7.7 at 1°C. Reactions were initiated by
addition of acetoacetyl CoA.
Citrate synthase (CS, EC 4.1.3.7)
For this assay, we used the protocol described by Srere et al.
(1963), with minor changes.
The final reaction mixture contained 0.25 mmol l-1
5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB), 0.4 mmol l-1
acetyl CoA, 0.5 mmol l-1 oxaloacetate, 75 mmol l-1
Tris-HCl, pH 8.2 at 1°C. Reaction was initiated by addition of
oxaloacetate. Progress of the reaction was monitored by following production
of the reduced anion of DTNB at 412 nm.
Hexokinase (HK, EC 2.7.1.1)
We used the methods described by Zammit and Newsholme
(1976) for this assay. The
final reaction mixture contained 7.5 mmol l-1 MgCl2, 0.8
mmol l-1 EDTA, 1.5 mmol l-1 KCl, 0.4 mmol l-1
NADP, 2.5 mmol l-1 ATP, 10.0 mmol l-1 creatine
PO4, 1.0 mmol l-1
-D-glucose, 0.9 U
ml-1 creatine phosphokinase, 0.7 U ml-1
glucose-6-phosphate dehydrogenase, 75 mmol l-1 Tris-HCl, pH 7.6 at
1°C. Reactions were initiated by the addition of glucose.
Cytochrome oxidase (CO, EC 1.9.3.1)
The method of Wharton and Tzagoloff
(1967) was used to measure
activity. Tissue was homogenized in 50 mmol l-1
K2HPO4/KH2PO4, 0.05% Triton X-100,
pH 7.5. The assay medium consisted of 10 mmol l-1
K2HPO4/KH2PO4, 0.65% (w/v) reduced
(Fe2+) cytochrome c and 0.93 mmol l-1
K3Fe(CN)6. Reaction was initiated by the addition of
enzyme. Maximal activities were measured by following the oxidation of reduced
cytochrome c at 550 nm.
Carnitine palmitoyltransferase-I (CPT-I, EC 2.3.1.21)
Maximal activities of CPT-I were measured in isolated mitochondria
(Rodnick and Sidell, 1994).
Tissue was homogenised in 10% w/v of ice-cold 40 mmol l-1 Hepes, 10
mmol l-1 EDTA, 5 mmol l-1 MgCl2, 150 mmol
l-1 KCl, 35 mmol l-1 sucrose and 0.5% BSA, pH 7.27 at
1°C, using a Duall ground glass homogeniser. A sample of crude homogenate
was reserved for measuring total CPT activity. Homogenate was centrifuged at
270 g for 10 min. Supernatant was collected and recentrifuged
at 270 g. Supernatant was collected and centrifuged at 15,000
g for 20 min. The mitochondrial pellet was gently resuspended
in homogenisation buffer (-BSA) and centrifuged at 15,000 g
for 20 min. The pellet was gently resuspended in homogenization buffer lacking
BSA to a final concentration of approximately 5 µg protein
µl-1. A sample of the mitochondrial suspension was frozen at
-70°C for later protein determination using the bicinchoninic acid method
(Smith et al., 1985
).
The final assay medium consisted of 1.0 mmol l-1 EGTA, 220 mmol l-1 sucrose, 40 mmol l-1 KCl, 0.13% BSA, 0.1 mmol l-1 DTNB, 40 µmol l-1 palmitoleoyl-CoA, 1 mmol l-1 carnitine, 20 mmol l-1 Hepes, pH 8.0 at 1°C. Activity was simultaneously measured in six cuvettes. Malonyl-CoA, a known inhibitor of CPT-I, was added to three of the six cuvettes to a final concentration of 10 µmol l-1. Reactions were initiated by the addition of carnitine. Maximum activity was measured by following the production of the reduced anion of DTNB at 412 nm. Maximal activities of CPT-I were estimated as the fraction of total activity inhibited in the presence of malonyl-CoA.
Statistical analysis
Data were analyzed using factoral ANOVA, with a post-hoc Fisher's
least-significant-difference test used to assess comparisons among the four
species.
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Results |
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PFK catalyzes one of the non-equilibrium reactions in glycolysis and
measurement of its maximal activity is a good indicator of anaerobic metabolic
potential (Crabtree and Newsholme,
1972a). Maximal activities of LDH and PK are also frequently used
as indices of anaerobic metabolic capacity. Although the activities of each
enzyme differ between red and white-blooded fishes, when all three enzymes are
considered together, there is no clear pattern of differences in anaerobic
potential between fishes that express Hb and those lacking the protein.
Activity of HK, an enzyme generally indicative of capacity for aerobic
oxidation of glucose, was relatively low and not significantly different among
all species examined, and thus does not correlate with the presence or absence
of circulating haemoglobin.
Maximal activity of CPT-I is a good index of potential for ß-oxidation
of fatty acids (Crabtree and Newsholme,
1972b), and activities were generally higher in red-blooded
species compared to icefish. Although there was no significant difference
between N. coriiceps and C. rastrospinosus
(P=0.10), there were differences among both nototheniids and both
channichthyids. In particular, activity of CPT-I was greater in C.
rastrospinosus compared to C. aceratus (P=0.004).
Despite the substantially greater densities of intracellular lipid droplets in
red-blooded nototheniids compared to hemoglobinless channichthyids (cf.
Table 3), no compelling
parallels were observed in activities of either of the enzymatic markers of
ß-oxidation capacity, HOAD and CPT-I
(Table 1).
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Capillary and fibre dimensions
The mean cross-sectional area of oxidative fibres,
f, were larger in icefish compared to red-blooded
nototheniids (Fig. 1), and
fibres from C. rastrospinosus were significantly larger than those
from C. aceratus (Table
2). The capillary-to-fibre ratio (C:F) of all fishes is around one
for the nototheniids, which is characteristic of sluggish fishes, but is
higher in the channichthyids, and greatest in pectoral muscle from C.
rastrospinosus. The combination of
f and C:F
results in a variable capillary density, NA(c,f), among
species, being lowest in C. rastrospinosus as a result of its
exceptionally large fibre size (Table
2).
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C. aceratus had capillaries with large surface areas due to the
particularly large bore, which would minimize the pressure against which the
heart must work to pump a large volume of blood through the circulatory
system. It is surprising, therefore, that individual capillaries are smaller
in the other icefish, C. rastrospinosus, compared to both C.
aceratus and the red-blooded nototheniids
(Table 2). Few capillaries were
viewed in which the major axis measured less than 15% longer than the minor
axis in nototheniids, a criterion frequently used for selecting capillaries
for measuring cross sectional area, a(c)
(Mathieu-Costello et al.,
1992), suggesting a low degree of anisotropy with respect to the
fibre long axis. The capillary bed was indeed more tortuous in icefish
[tortuosity index, c(K,0)=1.44 compared to red-blooded species,
c(K,0)=1.11], but the wide range in both fibre size and capillary
supply resulted in measurements of capillary length density, Jv(c,f),
that showed no clear pattern among species
(Table 2). Values for capillary
volume and surface densities (data not shown) were dominated by the effect of
Jv(c,f), and varied in a similar manner among species. Consequently,
Krogh's radius Kr was also not consistently different between the
nototheniids and channichthyids: N. coriiceps Kr=17.4±1.02
µm; G. gibberifrons Kr=21.4±0.51 µm; C. aceratus
Kr=19.0±0.79 µm; C. rastrospinosus Kr=29.4±1.07
µm (P<0.05 among, but not between nototheniids and
channichthyids).
Fibre ultrastructure
Mitochondria occupy a larger proportion of cell volume in oxidative muscle
from the haemoglobinless channichthyids than in red-blooded nototheniids
(Fig. 2), but with a 14%
difference between the icefish C. aceratus and C.
rastrospinosus (Table 3).
The surface area of inner mitochondrial membranes per unit volume mitochondria
(cristae surface density) was similar between C. aceratus and C.
rastrospinosus, and values for both icefish were lower than in N.
coriiceps and G. gibberifrons
(Table 3). Little variation was
seen in the volume density of intracellular lipid droplets within the two
groups, but Vv(lip,f) was lower in icefish than in the red-blooded
species.
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The potential consequence of differences in mitochondrial volume density, Vv(mit,f), and cristae surface density, Sv(cs,mit), was estimated by calculating mitochondrial cristae surface density per gram of tissue Sv(cs,m). Results show that complimentary differences in Vv(mit,f) and Sv(cs,mit) result in values of Sv(cs,m) that are nearly equivalent among all species (Table 3). Although Sv(cs,m) is usually correlated with maximal activity of cytochrome oxidase (CO), this is not the case in Antarctic fishes. The specific CO activity per g tissue is greater in G. gibberifrons compared to the two icefish (Table 1), but the specific cristae surface area is equal to or less than that of icefish (Fig. 3, Table 3). These data indicate that CO and other components of the electron transport chain may be more densely packed within the mitochondrial cristae of G. gibberifrons compared to channichthyids. The greater packing of electron transport proteins may not be reflected in the ratio of cristae surface area to capillary surface area (Fig. 4) as this parameter is heavily influenced by the differences in capillary density, NA(c,f).
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Discussion |
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Mitochondrial ultrastructure
Cristae surface densities, i.e. surface area per unit volume of
mitochondrion, are nearly equivalent (40 m2 cm-3) among
different mammals and muscle types
(Hoppeler et al., 1981b;
Schwerzmann et al., 1989
). As
a result, mitochondrial volume densities can be correlated reliably with
oxygen consumption rates in mammalian muscle
(Hoppeler and Lindstedt,
1985
). In contrast, cristae surface densities vary among Antarctic
fish species and are lower in icefishes compared to red-blooded fishes in both
skeletal and cardiac muscle (Archer and
Johnston, 1991
; Johnston et
al., 1998
; O'Brien and Sidell,
2000
). Thus, higher VV(mit,f) in icefish
muscle may not require correspondingly high densities of capillaries because
each mitochondrion has a lower capacity for oxidative metabolism than those
from red-blooded species. As a consequence, both cristae density and
mitochondrial density must be considered in order to characterize capillary
supply requirements of Antarctic fish muscles.
We can normalise for differences in mitochondrial ultrastructure among
species by examining the capillary surface area supplying a given unit of
mitochondrial inner membrane. The ratio between capillary surface density and
cristae surface density is approximately 1:200 in several types of oxidative
muscles measured in 13 mammalian species
(Hoppeler and Billeter, 1991).
This value is much higher than that in the Antarctic fish so far examined:
N. coriiceps (1:527), G. gibberifrons (1:602), C.
aceratus (1:434) and C. rastrospinosus (1:1182) in the present
study, and previously (C. aceratus, 1:799;
Archer and Johnston, 1991
).
Values calculated for a temperate zone teleost Morone saxatilis
(1:432; Egginton and Sidell,
1989
), are similar to those for the most sluggish species, but
lower than for the more active notothenioids. These observations seem
counterintuitive. One would expect an equivalent or perhaps even greater
capillary surface area per surface area of cristae in icefishes, because their
blood has a lower oxygen carrying capacity than that of red-blooded
fishes.
Cristae surface densities alone, however, may not be a good measure of
oxidative capacity in Antarctic fishes. When two sub-Antarctic species were
compared, Paranotothenia magellanica had higher mitochondrial cristae
surface densities (45.1±4.7 µm-1) than Eleginops
maclovinus (39.2±3.9 µm-1), yet isolated
mitochondria from both species had similar rates of oxygen consumption
(Johnston et al., 1998). These
differences may result from differences in lipid composition of the
inner-mitochondrial membranes, catalytic activities of electron transport
proteins, or densities of electron transport chain elements.
Maximal activities of cytochrome oxidase (CO) per gram of muscle were
greater in red-blooded nototheniids than in icefish, yet the specific surface
area of cristae was nearly equivalent. Together, these data indicate that CO
and other elements of the electron transport chain may be less densely packed
within the inner-mitochondrial membranes of icefish than in red-blooded
species, and that lower cristae surface densities per mitochondrion in icefish
were compensated by higher volume densities of mitochondria. Similar trends
have been observed in cardiac muscle of Antarctic species
(O'Brien and Sidell, 2000).
Overall, the low density of electron transport proteins within cristae of
icefish pectoral muscle suggests that a correspondingly low capillary surface
area per unit inner-mitochondrial membrane may match rates of oxygen
utilization. This may be a feature of cold-adapted species, e.g. striped bass
acclimated to low temperature (5°C) had a lower ratio of capillary:cristae
surface densities (1:810) than did individuals acclimated to 25°C (1:432;
Egginton and Sidell,
1989
).
Fibre ultrastructure may maintain O2 flux
A high density of lipid-rich subcellular structures may help compensate for
low capillarity in icefish muscle and may contribute to maintaining oxygen
diffusion to mitochondria. Oxygen is more than four times more soluble in
non-polar solvents than in water (Battino
et al., 1968). The higher solubility of oxygen in lipid than
aqueous cytoplasm has led several investigators to hypothesize that
intracellular lipid may be an important storage site for oxygen, and a
low-resistance pathway for oxygen diffusion
(Longmuir, 1980
;
Ellsworth and Pittman, 1984
;
Sidell, 1998
). For example,
the extent of intracellular lipid was inversely correlated with capillary
supply in cyprinids (Sänger,
1999
), and oxygen flux may be maintained in striped bass at cold
temperature by virtue of the greater than 13-fold increase in volume of
intracellular lipid droplets that occurs during cold acclimation
(Egginton and Sidell, 1989
;
Hoofd and Egginton, 1997
).
Evidence is available to suggest that similar apparent mismatches in supply
(capillary density) and demand (mitochondria) in Antarctic teleosts also may
be overcome by increases in intracellular lipid density
(Londraville and Sidell,
1990a
). Finally, empirical measurements of oxygen movement through
tissues of differing lipid content support this hypothesis, because the
diffusion constant for oxygen increases in cold-acclimated striped bass in
parallel with increased lipid density
(Desaulniers et al.,
1996
).
Very low densities of intracellular lipid droplets were observed in fibres
of C. aceratus and C. rastrospinosus. However, both of these
species have extremely high densities of mitochondria, which increase the
amount of lipid-rich intracellular membrane. The hydrocarbon core of
biological membranes may function similarly to lipid droplets as an avenue for
oxygen diffusion (Longmuir,
1980; Koyama et al.,
1990
). Three-dimensional analyses of mitochondria have shown that,
in muscles containing high densities of mitochondria (>20%), some
mitochondria form a continuous reticular structure, with mitochondria
connected both longitudinally along the fibre axis and transversely, in
regions adjacent to the I-band (Bakeeva et
al., 1978
; Kayar et al.,
1988
). In transverse sections of icefish pectoral adductor muscle,
myofibrils appear as rosettes surrounded by mitochondria. This membranous
network that spans the muscle fibre may be an important mechanism for
delivering oxygen to the centre of fibres in the absence of Hb and Mb.
The unusually high densities of mitochondria in icefish oxidative muscle
also reduce the diffusion distance that metabolites must travel between the
mitochondria and myofibrils, and between mitochondrial clusters. Consequently,
the mean diffusion distance between the cytoplasm and mitochondria
a was 1.23 µm in C. aceratus and 2.89 µm in
N. coriiceps, the latter being similar to that found in the
relatively sluggish goldfish (
a=2.9 µm;
Tyler and Sidell, 1984
).
Similarly, the distance between mitochondrial clusters within icefish fibres
is approximately one-half that of red-blooded species (data not shown).
Physiological significance of fibre size
The size of muscle fibres generally increases as body temperature decreases
in fishes, both within individuals in response to cold temperature acclimation
and among species inhabiting different latitudes
(Egginton and Johnston, 1984;
Egginton and Sidell, 1989
;
Rodnick and Sidell, 1997
;
Johnston et al., 1998
).
Surprisingly, fibre size is also greater in species lacking haemoglobin
(Fitch et al., 1984
;
Archer and Johnston, 1991
;
Johnston et al., 1998
; this
study) compared to red-blooded species. Oxygen delivery to mitochondria within
the muscles of Antarctic fishes thus must overcome a two-pronged challenge.
Not only is the distance that oxygen must travel to reach the centre of the
fibre large, but also the cold body temperature of these animals results in a
lower diffusion constant for the gas through a cytoplasm of high viscosity.
The situation is further exacerbated by the lack of intracellular
oxygen-binding myoglobin to facilitate oxygen diffusion in the skeletal
muscles of these species (Moylan and
Sidell, 2000
). Constraints on intracellular delivery of oxygen
seem particularly acute in the channichthyid icefish, which also lack
oxygen-binding haemoglobin in their circulation. To avoid failure to meet
aerobic demands of the tissue in these animals, one could reasonably expect to
find specific mechanisms to enhance oxygen delivery to the tissue, and/or
mechanisms that result in reduced oxygen demand. High mitochondrial densities
within oxidative muscle of icefishes may serve such a function.
Despite large fibre radii in icefish, high densities of mitochondria are
found at the centre of fibres in these species. In fact, mitochondrial
densities at the centre of oxidative fibres of C. aceratus are
equivalent to those nearest the capillary, unlike that observed in G.
gibberifrons, where mitochondrial densities decline toward the centre of
the fibre (Archer and Johnston,
1991). These results suggest oxygen diffusion is not limiting in
icefish muscles, and thus, oxygen flux to the fibre centre may be maintained
via the hydrocarbon cores of the reticular network of mitochondrial
membranes in fish muscles (Egginton et
al., 2000
; O'Brien and Sidell,
2000
).
Even in light of the above arguments, the effects of both cold-temperature
and oxygen-binding protein expression on fibre size seem anomalous. However,
one positive outcome from increasing fibre size may be to conserve energy.
Approximately 20-40% of an organism's energy budget is used to maintain ionic
gradients (Jobling, 1994).
Minimising ion leakage is one mechanism animals use to reduce metabolic costs
and endure environmental stress. Cell membranes are less permeable to ions in
cold-tolerant animals (hibernators) compared to those that are cold-sensitive
(Hochachka, 1986
), and turtles
reduce membrane permeability to help them survive long periods of hypoxia
(Hochachka et al., 1996
).
Large fibre size in icefish decreases the surface-to-volume ratio of the cell,
and the relative surface area available for the diffusion of ions. Increasing
fibre size thus may decrease the energetic demands of icefish, and may be
possible only because the mitochondrial reticulum helps to maintain oxygen
diffusion within these large fibres.
Two characteristics of their vascular systems may provide additional
insurance that oxygen supply is maintained to the centre of large muscle
fibres in icefish. First, icefish have a well-developed hypobranchial
circulatory system, which delivers freshly oxygenated blood directly from the
gills to the pectoral muscles (Egginton
and Rankin, 1998). Flow capacity of the hypobranchial system in
icefish is approximately 30% of total cardiac output. In contrast, in
red-blooded nototheniods, the hypobranchial system is formed from fewer and
smaller efferent branchial arteries compared to icefishes, and is a less
important component of the pectoral vasculature system
(Egginton and Rankin, 1998
).
Second, the capillary bed of icefish oxidative muscle is highly tortuous,
forming extensive loops along the muscle fibres, which maximises the surface
area for gas exchange (Egginton and
Rankin, 1998
).
Oxidative capacity
Our measurements of enzyme activities indicate that the lower aerobic
metabolic capacity of pectoral adductor muscle in icefish correlates with
lower capillarity of the musculature. Maximal activities per gram tissue of
citrate synthase and cytochrome oxidase, two enzymes indicative of aerobic
metabolic capacity, are lower in muscles of icefish compared to red-blooded
species. CPT-I catalyzes an important reaction in the metabolism of fatty
acids, and is a good indicator of potential for fatty-acid oxidation. Maximal
activity of CPT-I is greater in N. coriiceps and G.
gibberifrons compared to the two icefish. Despite an apparently lower
capacity for aerobic metabolism, icefish do not compensate with a greater
reliance on anaerobic metabolic pathways to fuel swimming. In general,
Antarctic fish species have a lower capacity for anaerobic metabolism than
temperate zone species, possibly because of the greater thermal sensitivity of
these enzymes compared to aerobically poised enzymes
(Crockett and Sidell,
1990).
In conclusion, low capillary densities relative to mitochondrial densities in pectoral adductor of icefish reflect differences in ultrastructure and in metabolic requirements compared to those of red-blooded Antarctic fishes. Differences in mitochondrial architecture between icefish and red-blooded species indicate that oxidative capacity per mitochondrion may be lower in icefish. Thus, equivalent volumes of mitochondria in channichthyids and red-blooded nototheniids may not require equivalent lengths of capillary to maintain oxidative phosphorylation (Fig. 4). Although large mitochondrial density does not increase the oxidative metabolic capacity of icefish muscle, lipid-rich membranes may maintain oxygen flux because oxygen is more soluble in their hydrocarbon core than in cytoplasm. The reticular structure of the mitochondrial population may further provide an important avenue for oxygen diffusion in icefish that have two obstacles to overcome in maintaining oxygen flux: lack of oxygen-binding proteins and large-sized fibres. Despite ultrastructural characteristics that may enhance oxygen diffusion, aerobic metabolic capacities per gram muscle are lower in icefish than in red-blooded species, thus restricting them to low energy niches and making them potentially more sensitive to changes in their thermal environment.
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