Peripheral oxygen transport in skeletal muscle of Antarctic and sub-Antarctic notothenioid fish
1 Department of Physiology, University of Birmingham, Birmingham B15 2TT, UK,
2 Department of Physiology, University of Nijmegen, 6525 Nijmegen, The Netherlands,
3 CONICET, CADIC, Ushuaia, Argentina and
4 School of Biology, Gatty Marine Laboratory, University of St Andrews, Fife KY16 8LB, Scotland
*e-mail: s.egginton{at}bham.ac.uk
Accepted 7 January 2002
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Summary |
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Key words: diffusion, icefish, mathematical model, oxygen tension, stereology, notothenioid fish.
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Introduction |
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Notothenioids show some remarkable adaptations to life in sub-zero temperatures, including the production of plasma glycopeptide antifreezes (for a review, see Eastman, 1993). Members of the family Channichthyidae are particularly striking in that they lack functionally significant quantities of the respiratory pigments haemoglobin and skeletal muscle myoglobin, although some species have retained myoglobin expression in the myocardium (Moylan and Sidell, 2000
). Oxygen-carrying capacity is thus impaired, with arterial oxygen content only approximately 10 % that in the red-blooded Notothenidae (Holeton, 1972
; Egginton, 1994
). For oxygen supply within the tissues, the oxygen partial pressure is the important variable so, as long as oxygen supply is maintained, capillary PO2 will not be impaired and muscle function should not therefore be compromised. However, the extent to which other cardiovascular and structural adaptations compensate to minimise impaired peripheral oxygen transport is unclear.
The Southern Ocean offers a uniquely stable thermal environment within which cold adaptation of fishes may be expected to have occurred, obviating the need to retain the functional plasticity required in more variable ecosystems (Somero, 1995). Specifically, the annual range of the inshore marine environment is only approximately 1.5 to +1.5°C. In deep water, which occurs closer than around other continents, temperature hardly varies from around the freezing point of sea water, 1.86°C (Eastman, 1993
). The notothenioids were presumably part of the ancestral fauna associated with habitats of the continental shelf during its southerly tectonic movement prior to cooling of the Southern Ocean. However, physical isolation caused by the opening of the Drake Passage and establishment of the circumpolar current has provided the potential for unique features resulting from endemic speciation, which molecular evidence suggests is a relatively recent event (Clarke and Johnston, 1996
). What might at first appear to be a cold adaptation might instead be a specialisation or ancestral characteristic of the notothenioids.
Many Antarctic fishes use a labriform type of sustained swimming, making use of well-developed pectoral fins and associated muscles (Archer and Johnston, 1987). Resting metabolic rates are low compared with temperate species, and the factorial scopes for aerobic activity are modest (Wells, 1987
; Forster et al., 1987
; Johnston et al., 1991
). Johnston et al. (1998
) found that the temperature-dependence of state 3 respiration of isolated mitochondria in perciform species fitted a single quadratic relationship irrespective of habitat temperature. This indicated that the rate of oxygen consumption per unit mitochondrion volume was relatively fixed and that increasing the volume of mitochondrial clusters was the primary mechanism for enhancing the muscle aerobic capacity in cold-water fish. Indeed, ultrastructural studies have found high densities of mitochondria in the slow muscle of Antarctic fish [35.6 % in juvenile Notothenia neglecta (Johnston and Camm, 1987
), 50.1 % in adult Chaenocephalus aceratus (Johnston, 1987
) and 45 % in Psilodraco breviceps (Archer and Johnston, 1991
)] approaching or even exceeding those for myocardium of active endotherms (finch 34 %, mouse 37 %) (Bossen et al., 1978
). Cold acclimation also results in an increase in mitochondrial volume density in the muscle of many temperate fish species (Johnston and Maitland, 1980
; Egginton and Sidell, 1989
). Thus, differences in muscle mitochondrial content in Antarctic notothenioids may be related to their phyletic derivation or simply be an extension of the response observed during winter in temperate fishes (cold acclimatisation) that has become fixed in the genome (cold adaptation).
One striking feature of muscle structure in Antarctic notothenioids is the presence of very large diameter fibres; the fibres can exceed 50 µm in diameter in aerobic muscles and 500 µm in fast muscles (Kilarski et al., 1982; Battram and Johnston, 1991
; Fernandez et al., 2000
). Similar large-diameter muscle fibres are also found in notothenioid species from the Beagle Channel living at much higher temperatures (Johnston et al., 1998
; Fernandez et al., 2000
). The potential problems with oxygen transport in cold-water notothenioids are therefore accentuated by long diffusion distances, which are well beyond those normally experienced in cold-acclimated fishes (Egginton, 1998
). To investigate this further, we have compiled data on the fine structure of slow muscle fibres from the locomotory (pectoral) muscle of perciform species inhabiting a wide range of environmental temperatures from the Southern Ocean to the Mediterranean Sea and used a modelling approach to examine the fundamental structural features necessary for adequate oxygenation of skeletal muscle in different thermal environments.
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Materials and methods |
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Sample preparation
Four to six fish were selected at random from each group and briefly sedated in bicarbonate buffered 1:50 000 (m/v) tricaine methane sulphonate (MS222; Sandoz) prior to stunning and spinal cord transection. The pectoral muscles deliver the main propulsive force during swimming, with slow (red) fibres located mainly adjacent to the pectoral girdle. Samples of the slow adductor muscle were dissected free from skin, subdermal lipid and overlying fast muscle. Muscle was pinned at resting length to strengthened cork strips and fixed for 23 h at approximately 10°C in a buffered glutaraldehyde solution (Egginton and Sidell, 1989; Archer and Johnston, 1991
). Samples were then trimmed into pieces with a cut face of approximately 1 mm2, stored overnight in fresh fixative at 4°C then post-fixed in buffered 1 % OsO4 (m/v) for 1 h, dehydrated in ascending grades of alcohols and vacuum-embedded in epoxy resin (Araldite/Epon). Six blocks per fish were prepared, and one was chosen at random for subsequent analysis.
Light microscopy
Semi-thin (0.5 µm) sections were stained with Toluidine Blue to orientate the blocks for true transverse or longitudinal sections of muscle fibres (around 50 per field, two fields per section) and to quantify the capillary supply at a magnification of x500 as capillary-to-fibre ratio (C:F), using an unbiased sampling rule (Egginton, 1990) and rounded to the nearest half-capillary for ease of computation. Mean fibre area was estimated as the inverse of fibre density per field or as the mean of digitised areas (previous experience has shown these approaches to produce equivalent values).
Electron microscopy
Ultrathin (approximately 80 nm) sections (one per block, chosen at random) were double-stained with methanoic uranyl acetate (30 % m/v) and aqueous lead tartrate (2 %), and electron micrographs were taken at an accelerating voltage of 60 kV from one or two grids per block (one field per grid, 2030 fibres per field). Micrographs were analysed at a final magnification of x8750 to x15 750 using the transparent overlay of a stereological counting grid. A lattice spacing (d) of 1.3 cm (equivalent to 0.81.51 µm) was used for quantification of subcellular structure using standard point-counting and line-intercept techniques for area and boundary length estimates, respectively (Egginton and Sidell, 1989). Providing that random sampling criteria are applied, volume density (Vv) in practice represents the area of any structure as a proportion of a reference cross-sectional area. The large fibre diameter precluded the use of the whole cross section as reference phase, so muscle was subsampled by the method of systematic area-weighted quadrats, whereby different regions are sampled in proportion to their volume fraction, giving an unbiased estimate of population means (Cruz-Orive and Weibel, 1981
). Data were collected separately for subsarcolemmal and intermyofibrillar zones, from which mean fibre volumes could also be calculated.
Mathematical modelling
The combined effect that differences in fine structure have on the intracellular oxygen tension of the aerobic pectoral muscle fibres was explored by means of a mathematical model of intracellular diffusion (Hoofd and Egginton, 1997). This model is based on the structural parameters affecting both oxygen delivery and consumption and, hence, provides an estimate of the potential for intracellular oxygenation assuming maximal blood flow and mitochondrial respiration. Briefly, the potential oxygen delivery (determined by the capillary supply) is balanced by oxygen consumption (scaled according to mitochondrial volume), allowing for the diffusion distance involved (which varies with fibre radius) and oxygen permeability (given by the ratio of intracellular lipid to aqueous sarcoplasm), corrected for the kinetic effects of temperature (Q10), and partitioned among the distinct structural regions found in fish muscle fibres (subsarcolemmal and intermyofibrillar zones).
The input variables used, and adjusted in an iterative manner to explore their relative importance, were: temperature (°C), oxygen permeability, pO2 (mol m1 kPa1 s1), rate of oxygen consumption, O2 (mol O2 m3 tissue s1), intermyofibrillar zone radius/fibre radius, mitochondrial volume density, Vv(mit,f), lipid volume density, Vv(lip,f), the number of capillaries around a fibre and the angular location of capillaries around a fibre.
The model of necessity invokes a number of assumptions: (i) that muscle fibres have a circular cross section and a radius equal to the mean radius for that tissue, derived from empirical measurements; (ii) that capillaries have a circular cross section, which in red-blooded species is 3.1 µm in diameter (based on measurements from Notothenia coriiceps slow muscle) and in icefish is 4.5 µm (based on measurements from Chaenocephalus aceratus slow muscle); (iii) that mitochondria consume oxygen at a rate of 4.0 ml O2 ml1 min1 at 37°C and operate with a Q10 of 2.0; (iv) that capillaries are spaced at equidistant angles around the muscle fibre; (v) that mitochondria are not distributed homogeneously (the respective volume densities in the subsarcolemmal and intermyofibrillar zones are quantified separately); (vi) that, to accommodate data from different studies, lipid is distributed homogeneously throughout the fibre (see below); (vii) that Kroghs diffusion constant relative to water (Kr) is 5 for lipid (KL) and 0.4 for cytoplasm (KC) (a cytoplasmic value of KC=0.5 is assumed for icefish because of their lower protein concentration); (viii) that permeability is the weighted average of Kroghs diffusion constants for the lipid and cytoplasmic fraction, pO2={Vv(lip,f)KL+[1Vv(lip,f)]KC}=(DO2xSO2), where DO2 and SO2 are oxygen diffusivity and solubility, respectively; (ix) that assumed myoglobin is zero or negligible in all species since myoglobin is not expressed in notothenioids and would, in any case, be ineffective at low temperatures; (x) that mitochondrial cristae density and respiratory chain enzyme activity are similar among species, with the lower cristae density of icefish incorporated as a variant; and (xi) that blood flow is maximal and capillary PO2 is 6 kPa.
The model allows the influence of the following variables to be examined: (i) Kroghs diffusion constant; (ii) fibre size; (iii) capillary size (iv) homogeneity of mitochondrial and lipid distribution and (v) interspecific differences when values are normalised for a common temperature.
Statistical analyses
The data were based on mean fibre composition, so statistical comparisons accounting for intraspecific variance were not possible, but we used a Students t-test to indicate where significant interspecific differences were found on the basis of the mean ± S.D. PO2.
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Results |
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Discussion |
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Temperature
Bearing in mind these limitations, model iterations are helpful to illustrate the physiological limits likely to be imposed by current phenotype, and also to gauge the effect of any change in environmental temperature as a result of climatic change or altered distribution patterns. For example, should Chaenocephalus aceratus be faced with the annual range of temperatures experienced by Champsocephalus esox, we calculate a mean fibre PO2 of 0.89±1.51 kPa at 4°C and 0.45±1.31 kPa at 10°C (means ± S.D.), with a significant proportion of mitochondria along the fibre radius experiencing anoxia. Clearly, these animals are well-adapted to the extreme stenothermal environment of the Southern Ocean, but structurally would appear to be ill-equipped to cope with any perturbation in temperature, indicating a loss of plasticity. The environmental temperature in the Beagle Channel ranges from 4°C in winter to a maximum of 1011°C in summer. This may be extended by several degrees in both directions in shallow bay waters, and the intertidal nesting species Patagonotothen tessellata and Harpagifer bispinnis survive extreme diurnal temperature ranges during summer spawning and in persistent cold water while guarding their nests during nocturnal low tides in July and August.
There is evidence for a possible adaptation in peripheral oxygen transport among the Channichthyes, with Champsocephalus esox calculated to exhibit a mean intracellular PO2 of 1.43±1.51 kPa (Kr=0.4) or 2.36±1.20 kPa (Kr=0.5) (means ± S.D.) at 0°C, far higher than that predicted for Chaenocephalus aceratus at 0°C. Aerobic muscle performance is likely also to be aided by the presence of ventricular myoglobin in some channicthyid species (Acierno et al., 1997). In Champsocephalus esox, the ventricular muscle has an orange-pink appearance (J. Calvo, personal observation) that may indicate the presence of myoglobin in this species as well, which would help preserve cardiac output under conditions of metabolic stress, although it may simply reflect cytochrome colour. One captive specimen of Champsocephalus esox survived 6 h of exposure to 14°C while maintaining the ability to swim (J. Calvo, personal observation) at this temperature, where intracellular PO2 is calculated to average 0.47 kPa, with regions of anoxia. In contrast to the channichthyids, the red-blooded Antarctic notothenioids, including the sluggish Notothenia coriiceps, are predicted to show a marked resilience to elevated temperature and, with care, may be gradually acclimated in the laboratory to temperatures experienced by their sub-Antarctic cousins (S. Egginton, unpublished data). For example, Lepidonotothen nudifrons would be predicted to decrease mean intracellular PO2 by 75 % when raised from 0 to 20°C, whereas Trematomus newnesi would only have a modest 8 % reduction because of its smaller fibre size (Table 2).
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Increasing fibre girth may be a secondary effect of the differences in habitat temperature during development as a result of the protracted larval stage of the Antarctic species, which can be extended over most of the winter, spring and summer months (Kock, 1992). The majority of Antarctic and sub-Antarctic notothenioids show an unusual pattern of muscle growth characterised by limited fibre recruitment, resulting in slow and fast fibre diameters in excess of 60 and 550 µm, respectively (Fernandez et al., 2000
). However, it is likely that the presence of large-diameter fibres has a phylogenetic explanation and simply reflects a limitation on fibre recruitment in these species rather than some specific adaptation to life in cold waters (I. A. Johnston, unpublished results). There is taxonomic variation in fibre number, with Eleginops maclovinus only having one-fifth of the number of fibres of a salmon of the same body size but 10 times more fibres than other species in the family Notothenidae that can reach the same body size (I. A. Johnston, unpublished observations). It may be that low temperature is permissive for large-diameter fibres, i.e. having large fibres may not be a disadvantage and it may help to reduce resting metabolic rate. This may explain the apparent paradox of large fibres under conditions of low diffusivity, suggesting that selection pressure during notothenioid radiation was not primarily directed at maintaining a high intracellular oxygen tension. Nevertheless, this wide range of fibre size is likely to have a direct effect on mean fibre PO2. At the extreme of large fibres (icefish), calculated PO2 was zero at the fibre centre (Fig. 1). This may seem odd because mitochondria are distributed right to the centre of the fibres in this species (Archer and Johnston, 1991
); however, this simply means that mitochondria can respire at sub-maximal
O2 and that a proportion would be non-functional at
O2max, thus limiting aerobic power output.
Mitochondrial content
In most vertebrates, the mitochondrial structure and mass-specific rate of oxygen consumption appear to be relatively invariant, such that mitochondrial volume varies directly with O2 (Hoppeler et al., 1987
). The model uses a standard value for mitochondrial respiration of 4.0 ml O2 ml1 min1, based on studies of whole-muscle
O2 at the
O2max for individual species (Hoppeler et al., 1987
), scaled for the effect of temperature (Q10=2). Although a number of studies have suggested there is little effect of acclimation or environmental temperature on mitochondrial structure (Egginton and Sidell, 1989
; Johnston et al., 1994
), and hence mass-specific
O2, recent data suggest that some notothenioids may have an unusually low respiratory capacity (Johnston et al., 1998
). In this case, the high value for Vv(mit,f) may be an inadequate index of fibre oxidative capacity, although whether this is a result of lower cristae density or is compounded by an altered respiratory chain density is unclear.
However, using the 30 % lower cristae surface density reported by Archer and Johnston (1991) to rescale the
O2 in the model makes little qualitative difference to our conclusions because it would result in a twofold higher calculated mean PO2 in m. pectoralis, e.g. increasing it to 2.62±1.06 kPa (mean ± S.D.) for the channichthyid at 0°C. Even if cristae density were 60 % lower than that of nototheniids assumed in these calculations, the mean PO2 of Chaenocephalus aceratus would still only be the same as that of Notothenia coriiceps (3.10±0.91 kPa) (mean ± S.D.) and markedly less than that of the red-blooded notothenioids, which have small diameter fibres. However, it would take a rather modest reduction in
O2 of only approximately 10 % to raise the minimum oxygen tension towards that adequate for unimpeded mitochondrial respiration (0.3 kPa), a likely consequence of a relatively low specific activity of citrate synthase in these animals (S. Egginton, S. Skilbeck and S. Cordiner, in preparation).
Diffusion constant
We have compiled data on the fine structure of slow muscle fibres from the locomotory muscle of perciform species inhabiting a wide range of environmental temperatures and examined the consequences for intracellular oxygen delivery. While the major determinants of tissue oxygen tension are the capillary supply and intracellular diffusion distance, other factors such as the composition of the cytosol may play a modifying role in the diffusivity of oxygen (Hoofd and Egginton, 1997). For example, the value for Kroghs diffusion constant relative to water normally used is based on a nominal cell protein concentration of 30 mg ml1, whereas we have found significantly lower values for trunk muscle protein concentration in channichthyids (N. M. Whiteley and S. Egginton, unpublished data). It is calculated that, in pectoral muscle fibres of Chaenocephalus aceratus, this difference may significantly increase Kr and hence lead to a potentially higher mean PO2 than that predicted using the standard input variables. Although the value of Kr=0.5 used in the present study may be conservative, for the minimum PO2 to be increased above zero would require a more watery muscle than is actually found.
Fibre composition
Changes in fibre composition that occur during cold acclimation which may affect the intracellular flux of oxygen include increased lipid and mitochondrial content (Egginton and Sidell, 1989). Because oxygen solubility is higher in lipid than in aqueous solution, and the diffusion coefficient for oxygen was unaltered by acclimation, the calculated diffusion constant is greater in cold- than in warm-acclimated fish (Desaulniers et al., 1996
; Hoofd and Egginton, 1997
). However, in the notothenioid species we examined, the lipid content was relatively low, suggesting that other structural parameters may play a limiting role. The influence of mitochondrial content has been discussed previously. In addition, as the model uses assumptions designed to examine the capacity for oxygen transport to tissue, any deviations from optimal conditions will clearly reduce the apparent efficiency of the process. For example, the model assumes a homogeneous cytoplasmic composition in each domain, whereas in reality both capillary supply and mitochondrial distribution are often quite heterogeneous in vivo, leading to some impairment of supply on the basis of simple diffusion. This is particularly the case for the distribution of intracellular membranes, and the permeability to oxygen is therefore likely to be particularly heterogeneous.
Membranes are unlikely to be oxygen barriers, but may restrict the intracellular distribution of myoglobin and lipid. However, any reticulum of lipid-rich structures such as the sarcoplasmic reticulum or ribbons of mitochondria may act as low-resistance conduits for intracellular flux of oxygen (Longmuir, 1980). Although the model is unable to incorporate such structural heterogeneity, we can estimate the likely influence that different cellular organisation may have. For example, values for intracellular PO2 in Notothenia coriiceps based on normalised data (Table 3) but at 0°C were 3.29±0.79 and 2.30 kPa (mean ± S.D. and minimum), which would be reduced to 3.21±0.80 and 2.42 kPa, respectively, if the mitochondria were homogeneously distributed between the subsarcolemmal and intermyofibrillar zones across the fibre, to 3.20±0.80 and 2.20 kPa for homogeneous lipid distribution and to 3.12±0.81 and 2.33 kPa if both components were homogeneously distributed between the subsarcolemmal and intermyofibrillar zones. The other species were predicted to be affected to a similar degree if these organelles were not distributed in a heterogeneous manner.
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Concluding remarks
Antarctic notothenioids have large diameter fibres and a high mitochondrial content, which tend to decrease mean PO2. At the maximal fibre O2, capillary supply is unlikely to maintain a high PO2 for all species: hypoxaemia is likely to develop in the pectoral muscle fibres of the icefish Chaenocephalus aceratus, which lacks respiratory pigments, while red-blooded nototheniids potentially have little problem in maintaining a high PO2. Sub-Antarctic notothenioids have a similar muscle fine structure to those caught within the Antarctic Convergence, resulting in a relatively high mean PO2 in red-blooded species. Even within the large diameter fibres of the icefish Champsocephalus esox, PO2 exceeds 1 kPa at winter temperatures (4°C), although oxidative metabolism is predicted to be impaired at the summer maximum (10°C). At the other thermal extreme, related Mediterranean perciform species have a negligible drop in intracellular PO2 across a fibre, and these fibres are of similar size to those of Trematomus newnesi from the Antarctic. We tentatively identify a hierarchy of importance for the various influences on muscle fibre oxygenation as follows: temperature, capillary number, fibre size, Vv(mit,f), diffusion constant, fibre composition and capillary radius. No one factor can explain the observed differences among species, e.g. cold-adapted fish still have a low PO2 at 10°C when normalised for fibre radius, as a result of high mitochondrial content and low capillary supply (Table 3).
These data suggest that, within a single phylogenetic group, integrative structural adaptations potentially enable a similar degree of tissue oxygenation over a 20°C range of environmental temperature in the nototheniids that is overwhelmed by the lack of respiratory pigments in the channichthyids. Importantly, PO2 does not correlate with either activity pattern or ecological niche since high values are found at both thermal extremes of the perciform habitat.
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Acknowledgments |
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References |
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