Vol. 279, Issue 2, R375-R388, August 2000
Thermal compensation of peripheral oxygen transport
in skeletal muscle of seasonally acclimatized trout
S.
Egginton,
S.
Cordiner, and
C.
Skilbeck
Department of Physiology, University of Birmingham, Birmingham B15
2TT, United Kingdom
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ABSTRACT |
Seasonal changes in
ultrastructure of locomotory muscle were quantified after
acclimatization to natural temperature and photoperiod. Only modest
changes were seen in the volume density (Vv) of
mitochondria in slow fibers ranging from 0.21 ± 0.01 (summer) to
0.24 ± 0.01 (winter), despite an increase in fiber size from 945 ± 19 to 1,594 ± 46 µm2, respectively,
resulting in a significantly greater total mitochondrial volume at low
temperatures. In contrast, intracellular lipid stores showed a
marked change with season, from a maximum Vv of lipid droplets of 0.16 ± 0.01 in winter, progressively declining
through spring and summer to a minimum of 0.07 ± 0.01 in
autumn. For both organelles, the surface density reflected
changes in Vv, indicating little modification of structure.
Seasonal effects may dominate those of environmental temperature on
mitochondrial separation, which in winter and spring fish at
4oC averaged 0.64 ± 0.06 and 1.20 ± 0.07 µm,
respectively. The extracellular transport of oxygen also varies with
season, the peak capillary density in autumn (2,851 ± 88 mm
2) resulting in a minimum tissue supply (domain) area
of 529 ± 9µm2 per capillary. As a consequence, the
predicted intracellular PO2 (~2.5 kPa) is
similar throughout the year.
capillary supply; intracellular lipid; mitochondrial separation; Oncorhynchus mykiss; oxygen tension
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INTRODUCTION |
TEMPERATURE IS
ONE OF THE MOST important physical factors determining biological
activity, especially in ectotherms, in which physiological rate
functions vary directly with changes in the thermal environment. Fishes
are an extremely diverse group that have successfully exploited thermal
niches from <0oC to >40oC. Although
individual species have to cope with a more restricted annual
temperature range, typically 10-20oC, this still poses
a significant challenge for maintenance of cellular function. In
particular, the rate of diffusion of substrates through tissue fluids
and activity of metabolic enzymes will be depressed at low
temperatures. Unless compensatory mechanisms are invoked, this would
result in a slowing of locomotory performance, growth and development,
reproduction, and other activities that determine ecological fitness.
Laboratory studies of temperature acclimation have revealed
physiological adaptations from the molecular to organismal levels of
organization, with an increased aerobic capacity in the cold often
compensating for the depressive effect of low environmental temperature
on the metabolic rate, particularly in eurythermal species
(23, 34). The most striking responses in
skeletal muscle of active teleosts are increased peripheral oxygen
supply as a consequence of an expanded capillary bed (22), enhanced substrate uptake resulting from an increased volume density (Vv) of mitochondria
[Vv(mit,f)] (15), and greater
potential ATP synthesis due to higher activity of enzymes from
oxidative pathways (especially 
-oxidation of lipids)
(18). In addition, temperate zone fishes often increase
their red muscle mass, which contributes to an expansion in sustainable
swimming speed in cold waters (35). At the upper end of a
species' thermal range, the scope for activity may also be compromised
due to high maintenance costs, although much less work has been carried
out on possible compensatory mechanisms under these conditions. It is
therefore usually assumed that either diffusive or catalytic
limitations must be overcome to maintain locomotory capacity
(isokinesis) in cold environments (see Ref. 38).
There are, however, a few studies that suggest our understanding of
this adaptive strategy is incomplete. For example, Dean (5) commented that slow fibers in cold-acclimated rainbow
trout appeared to have a similar mitochondrial content to those from warm-acclimated trout, and mitochondrial enzymes showed no positive compensation in cold-acclimated lake whitefish (1). This
may, in part, be due to the fact that, although tank-acclimation
experiments help to define possible mechanisms used to ameliorate
direct thermal effects, they are unable to replicate the integrative
response found under natural conditions. For example, seasonal
acclimatization must also involve responses to environmental factors
other than temperature, such as feeding, locomotory patterns of
activity, and changes in hormonal status due to altered photoperiod.
Consequently, when pond-dwelling chain pickerel were compared with fish
caught from the same location and acclimated in the laboratory to
similar temperatures, the latter consistently had higher enzyme
activities (28). In addition, whereas laboratory cold
acclimation of Crucian carp led to a striking proliferation of slow
muscle mitochondria (22), this was attenuated by seasonal
cold acclimatization (17). Laboratory acclimation studies
also tend to compare fish held at only two temperatures, usually around
the upper and lower critical temperatures, whereas the response to
environmental temperature is clearly nonlinear (see below).
We have therefore examined the response of rainbow trout to changes in
environmental temperature by sampling one cohort of fish, held in
outdoor raceways and subjected to natural photoperiod, at the
seasonally appropriate temperatures during spring (Sp), summer (Su),
autumn (Au), and winter (Wi; a range of 4-18oC).
Adaptations in the cardiovascular system and peripheral blood flow, as
well as swimming performance, were not linearly related to seasonal
temperature. Rather, the highest capacity for sustainable (aerobic)
exercise in acclimatized trout was found at the intermediate (11oC) habitat temperature (37).
Interestingly, whereas the capillary density (CD; and hence blood flow
capacity) varied directly with temperature in the myocardium, the
capillary-to-fiber ratio (C/F) in aerobic skeletal muscle showed the
inverse response. However, capillary length density was also maximal at
11oC as a result of significant fiber hypertrophy at the
lower temperature (12). Finally, enzyme activities
confirmed an increased oxidative capacity in the cold, although there
was only limited expansion of lipid metabolism (3),
perhaps in response to an active lowering of intracellular pH at low
temperatures to below that predicted by other ectotherm studies, which
may lead to metabolic depression (39).
To examine the structural limits placed on aerobic performance of
skeletal muscle, we therefore quantified the effects of seasonal
acclimatization on fine structure and its implication for peripheral
oxygen transport. We hypothesized that there would be a progressive
change in subcellular structure with season but that the integrative
basis of thermal acclimatization would necessitate more modest changes
than those described after laboratory temperature acclimation. Our
conclusions were tested by means of a mathematical model of
intracellular oxygen tension. Initial findings were presented in
abstract form (11).
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MATERIAL AND METHODS |
Experimental animals.
Rainbow trout [Oncorhynchus mykiss (Walbaum)] were
obtained from Leadmill trout farm (Hathersage, Derbyshire, UK), where
they were held in flow-through, open culture ponds exposed to natural photoperiod. Members of one cohort were serially sampled throughout the
year, when ambient water temperatures (±0.5oC) were
4oC in Sp (April), 18oC in Su (August),
11oC in Au (October), and 4oC in Wi (January).
Fish were fed on commercial trout pellets at a seasonally adjusted
ration to maintain optimal growth rate and quality required for
stocking purposes (3). Sequential sampling of fish at
random through the seasonal growth period could introduce a
scale-dependent bias in the analysis of any temperature effects, due to
the interaction with allometric changes in muscle structure (9). On the assumption that, for mature fish, intergroup
differences will be due mainly to size, rather than age, we chose
individuals of similar body mass and condition factor to minimize the
influence of extraneous variables on the acclimatization response
(Table 1).
Sample preparation.
From those fish meeting the minimum size requirement, six were selected
at random from each acclimatization group, separated into small holding
ponds, and briefly sedated in 1:50,000 tricaine methane sulfonate
(MS222; Sandoz) before stunning and spinal cord transection. Samples of
superficial slow ("red") muscle were dissected free from skin,
subdermal lipid, and underlying fast muscle along the horizontal septum
and directly below the dorsal fin. Deep fast ("white") muscle was
taken from a similar hypaxial position close to the vertebral column.
Muscle was pinned at resting length to strengthened cork strips and
fixed for 2-3 h at ~10oC in a buffered
glutaraldehyde/paraformaldehyde solution containing 0.05% sodium azide
(10). Samples were then trimmed into pieces with a cut
face of ~1 mm2, stored overnight in fresh fixative at
4oC then postfixed in buffered OsO4 for 1 h, dehydrated in ascending grades of alcohols, and vacuum embedded in
epoxy resin (Epon). Six blocks per fish were prepared for both muscle
types, and one was chosen at random for subsequent analysis.
Light microscopy.
Semithin (0.5 µm) sections were stained with toluidine blue to orient
the blocks for true transverse or longitudinal sections of muscle
fibers and to quantify the capillary supply at a magnification of ×500
using an unbiased sampling rule (8). Digitized images of
stained sections were used to determine the x,y
coordinates for muscle fiber boundaries and the associated capillary
coordinates; in-house software was used to calculate mean fiber area
[
(f)], C/F, and CD (vessels per unit
area of muscle fibers). As most capillary beds form a complex network
with numerous interconnections, so that both the effective capillary
length and surface area are greater than those estimated from simple
counts in tissue sections, we also calculated the capillary length
density [Jv(c,f)]. This parameter evaluates the
length of capillaries per unit volume of tissue derived from the CD and
an index of tortuosity (8), in this case assumed to have a
value of 1.05 (12), and can be multiplied by tissue volume
to determine the absolute volume of the capillary bed per fish (in
km/cm3). In addition, the radius of Krogh's tissue
cylinder for oxygen delivery was calculated as 1/
[
· Jv(c,f)].
The area of tissue supplied by individual capillaries (the domain of
influence) was calculated as a tessellation of nonoverlapping polygons,
representing the area of tissue closer to one capillary than any other.
Under conditions of maximal flow, assuming supply capacity to be
similar for all capillaries, the domain size will be inversely
proportional to the metabolic demand. Thus mean domain area will vary
inversely with capillary supply and may be thought of as a
space-filling alternative to the Krogh tissue cylinder method of
quantifying oxygen supply capacity of the microcirculation (13). The distribution of capillary domain area also
provides an index of the heterogeneity of capillary supply; as the
distribution is log-normal, this is best expressed as the standard
deviation of log-transformed area (LogSD) and a mean intercapillary
distance {ICD = [2(domain area/)]}. These
quantities are based on the spatial relationship between all
neighboring capillaries and probably better represent the heterogeneity
of intramuscular oxygen supply than indexes based on global estimates
of capillarization. Simple counts of capillaries may give an erroneous
picture of the potential oxygen transport to tissue due to
heterogeneities in both local supply and demand, so methods have been
developed to analyze the local capillary supply to individual fibers.
The number of capillaries around each fiber may be corrected for the
size of adjacent fibers [capillary-per-fiber density (CFD)]. However,
differences in size among muscle fibers is best accommodated by
calculating the overlap of capillary domains and fiber profiles giving
an index of local capillary supply [local C/F (LCFR)] in terms of
"capillary equivalents" of supply (13). This may then
be normalized for differences in individual fiber size to provide a
scale-independent index, the local CD [LCD (LCFR/fiber area)].
Electron microscopy.
Ultrathin (~80 nm) sections were double stained with methanoic uranyl
acetate (30%) and aqueous lead tartrate (2%), and electronmicrographs taken at an accelerating voltage of 60 kV, requiring minimum goniometer stage tilt to maximize image clarity. Micrographs were analyzed at a
final magnification of ×8,750-15,750 using a transparent overlay
of a stereological counting grid. A lattice spacing (d) of
1.3 cm (equivalent to 0.8-1.51 µm) was used for quantification of subcellular structure using standard point-count and line-intercept techniques for area and boundary length estimates, respectively (15). Stereology is the application of geometric
probability theory that permits extrapolation of structural information
from n to n + 1 dimensions (e.g., length to
surface, area to volume), provided random sampling criteria are
applied. Therefore, Vv and surface density (Sv)
in practice represent the area and boundary length of any structure as
a proportion of a reference cross-sectional area. Surface-to-volume
ratio (S/V) and profile cross-sectional area are calculated in a
similar manner. For measurement of mitochondrial cristae surface
densities (ratio of inner membrane surface area to mitochondrial
volume), electronmicrographs were viewed at a final magnification of
×36,000. The large fiber size precluded 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.
Capillary Vv and Sv can be calculated from the
product of Jv(c,f) and capillary cross-sectional area and
perimeter, respectively. The oxygen demand to be met by the capillary
supply may then be equated with the volume of mitochondria supplied by
each capillary, given as (mitochondrial Vv × domain area).
To estimate the radial distribution of mitochondria and intracellular
lipid depots from capillaries, the respective volume densities were
calculated by point counts in sampling zones delineated by concentric
annuli of equal area, centered over individual capillaries, and
extending beyond the one-half mean ICD (15). Intracellular diffusion distances within muscle fibers were estimated using linear
analysis techniques, measuring the distance between mitochondrial clusters along both axes (to remove directionality) of a randomly oriented test lattice. The average distance that a small molecule would
diffuse through the sarcoplasm before reaching a mitochondrial interface should then be one-half this distance. Mean free sarcoplasmic spacing was also calculated as 
a = [4
(1
Vv)/Sv] (14).
Mathematical modeling.
The combined effect that changes in fine structure have on
intracellular oxygen tension of slow muscle fibers was explored by
means of a mathematical model of diffusion (20). Briefly, the potential oxygen delivery (determined by the capillary supply) is
balanced by oxygen consumption (scaled according to mitochondrial volume), allowing for the diffusion distances involved (which vary with
fiber size), 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 fibers (subsarcolemmal and intermyofibrillar zones). For clarity, the results are presented as
wire-frame three-dimensional plots.
Statistical analysis.
Single-factor ANOVA was used for comparison of values, with
Fisher's protected least-significant differences to estimate
significance between groups. Sufficiency of sample size for
stereological parameters was tested by determining the stability of
population means on replicate analysis (8). Distribution
analysis of domain area and mitochondrial spacing used the
Kolmogorov-Smirnow procedure and comparisons were made by the
Mann-Whitney U test.
Chemicals.
All chemicals were of reagent grade or better quality (BDH, Poole,
Dorset). Glutaraldehyde and Araldite resin were of electron microscope
grade (Agar Scientific, Stanstead, Essex).
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RESULTS |
The microvasculature is highly responsive to seasonal temperature
changes.
Capillary domain analysis showed that supply area varied inversely with
CD, being closely related to fiber size (Table
2). Because this procedure required
analysis of material at a higher magnification than used previously to
describe the cold-induced angiogenesis, the values for C/F, CD, and
fiber size [(f)] are slightly
different from published values for this species (12), although the trend is the same. The initial increase in capillary supply on autumnal cooling was not accompanied by any significant increase in fiber size, and hence the supply area (capillary domain) was significantly reduced (Fig. 1).
Further angiogenesis in Wi fish is paralleled by fiber hypertrophy such
that the mean domain area returns toward that of Su-acclimatized fish.
The heterogeneity of capillary distribution (LogSD) is similar, with
only a modest increase in the transition between Su and Wi (Table 2),
whereas the influence of initial capillary growth is evident as a
broadening of the central distribution in Au (Fig. 1). The consequence
of such changes is that capillary separation varies according to season
rather than temperature: mean ICD is maximal at the lowest temperature
(Wi and Sp) and minimal in Au, with an intermediate value at the
highest temperature (Su). Intercapillary diffusion distance (estimated
as Krogh's radius) shows a similar trend (Table 2). The number of
adjacent capillaries corrected for differences in fiber size (CFD)
peaked in Au, as expected from the C/F and CD data. However, the LCFR
shows that in Wi slow fibers received nearly two capillary equivalents
of supply, compared with 1.4 in Su, although when normalized for fiber
size (LCD), the maximal local supply again appears to be in Au. That
both the global and local indexes of capillary supply show the same
trend is indicative of an unusually homogeneous muscle composition and
tightly regulated angiogenesis.
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Fig. 1.
Effect of seasonal acclimatization on capillary
supply of slow muscle in rainbow trout, expressed as the
distribution of capillary domain area. A: spring (Sp);
B: summer (Sp); C: autumn (Au); D:
winter (Wi). Angiogenesis stimulated by the initial cooling from Su led
to a reduced supply area in Au that returned to Su values (dashed line)
by Wi as a result of substantial fiber hypertrophy. Little further
change was observed on prolonged cold exposure into Sp; thereafter, the
supply area fell in line with a reduction in mean fiber size.
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Muscle fine structure displays limited seasonal remodelling.
Little change in gross morphology of the oxidative swimming muscle was
evident, with slow fibers retaining a prominent population of
mitochondria and lipid droplets throughout the year (Fig.
2). Extracellular lipid depots were also
evident, comprising ~15% of the interstitium at all temperatures.
Quantitative analysis shows a small transitional increase in
Vv(mit,f) on autumnal cooling in slow muscle fibers (Fig.
3). The accompanying changes in
mitochondrial surface area exposed to the sarcoplasm, expressed either
as an average per fiber volume (Sv) or per organelle
cluster (S/V), were also modest (Table
3). In addition, estimates of cristae Sv (µm2 inner membrane
surface/µm3 mitochondrial volume) were minimally affected
by thermal acclimatization, being 15.8 ± 1.09, 15.5 ± 0.8, and 17.4 ± 1.00/µm for and Sp, Su, and Au fish, respectively
(not significant). However, preservation of these structures is less
than perfect in published micrographs from fish muscle, even when
optimal fixation techniques were employed. This is presumably a
consequence of slow fixative diffusion at low temperatures exacerbated
by the long diffusion pathlengths in large fibers. We therefore advise
caution when comparing absolute values of cristae Sv among
studies.
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Fig. 2.
Electron micrographs of slow muscle fibers from trout
acclimatized to Sp (A), Su (B), Au
(C), and Wi (D). Note the similarity in
composition, especially in the relative area of fibers occupied by
mitochondria. Scale bar = 1.5 µm.
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Fig. 3.
Volume (Vv) and surface density
(Sv; µm1) of mitochondria (mit,f) and lipid
(lip,f) in slow (A) and fast muscle (B) fibers
from seasonally acclimatized trout. Contrast the relative constancy of
the mitochondrial population with the variability in intracellular
lipid depots in slow muscle. In fast muscle, the sparse distribution of
both organelles results in high data variance but is suggestive of a
progressive annual decline in mitochondrial content and changes in
lipid that are opposite those seen in slow muscle. * P < 0.05; ** P < 0.01 vs. Au.
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Importantly, the unchanged mitochondrial Vv does not imply
a static population of the ATP-generating machinery, because this represents a constant volume fraction of fibers whose size is doubled
on the transition from Au to Wi. Rather, these data indicate that
mitochondrial proliferation was matched to the rate of fiber hypertrophy or atrophy, producing organelles of similar structural composition throughout the year. However, as angiogenesis was significantly influenced by fiber size, the relationship between oxygen
supply and consumption was distinctly seasonal, with the structural
balance shifting from a proportionately greater CD in Au to a
relatively higher Vv(mit,f) in Wi (Fig.
4).
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Fig. 4.
The relationship between oxygen supply {capillary
length density [Jv(c,f)]} and oxygen consumption
[Vv(mit,f)] in trout slow fibers, showing the
seasonal variation that deviates significantly from the interspecific
regression obtained from published data (error bars fall within the
symbols). Note that during Wi, trout appear to be "undersupplied"
with capillaries, whereas they have "excess" capillaries in Su and
Au.
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Intracellular lipid content of slow fibers showed a complementary
decrease in Vv between Su and Au (Fig. 3), with the
mobilization of intracellular lipid stores at 11oC leading
to a significant decrease in surface density as the lipid droplets
become smaller (Fig. 2) and producing a greater S/V (Table 3). The
Vv of myofibrils [Vv(myf,f)] varies in
a complementary manner to those of mitochondria and lipid.
Consequently, the total myofibrillar volume-per-unit length (µm) of
slow fiber, given as [V(myf,f) = Vv(myf,f) × (f)], changes throughout the year, being 826, 570, 544, and 869 µm3 for Sp, Su, Au, and Wi
fish, respectively. This is paralleled by changes in V(mit,f)
such that the ratio of ATP production and consumption is broadly
matched, with V(myf,f) being 2.3- to 2.8-fold greater than
V(mit,f) throughout the year. There was no indication of
pathological changes in structure, e.g., the volume fraction of nucleus
per fiber [Vv(nuc,f)] was maintained among the
groups. Interestingly, the fibers in Sp fish had a significantly lower cytoplasmic Vv such that the intracellular compartments
were relatively condensed.
Fast muscle fibers had ~5-fold fewer mitochondria and 20-fold less
lipid than slow fibers throughout the year and were generally refractory to changes in environmental temperature. The exception was
mitochondrial Sv, which was significantly higher in Sp than in any other season, indicating a greater surface for metabolite exchange that parallels an increased Vv. However, the 60%
increase in both Vv and Sv was matched by only
a 30% increase in the S/V, indicating that mitochondrial biogenesis
results in clusters of organelles with a reduced specific surface area
for exchange. There were no other significant changes in gross fiber
composition (Fig. 3). In neither slow nor fast muscle fibers was there
any evidence for a differential response in fiber composition between the subsarcolemmal and intermyofibrillar regions (Table 3), implying a
direct effect of temperature on structure with potentially similar consequences for transmembrane and transcellular transport of metabolites.
Intracellular reorganization complements changes in organelle
volume.
A tight regulation of mitochondrial content is suggested by the close
parallel in radial distribution of mitochondria among acclimatization
groups, with an average of ~20% mitochondria across the diffusion
pathway from capillary to the center of muscle fibers and a regression
slope that was not significantly different from zero. This gave rise to
very similar slopes for the cumulative mitochondrial volume (Fig.
5A), indicating a similar
extraction capacity for oxygen. The Vv of lipid was quite
variable, ranging from zero to 35% for individual animals, although
the consistently higher lipid Vv at 4oC
resulted in a significantly greater slope for the cumulative radial
distribution in the order Wi > Su > Au (Fig.
5B). However, the modest effect of season on slow fiber
composition masks a significant reorganization of mitochondrial
distribution. The increased proportion of shorter distances, indicative
of either biogenesis and/or clustering (Figs. 2 and 6), is accompanied
by the progressively narrower mitochondrial separation (Li)
from Sp through to Wi (Table 4).
This conclusion holds irrespective of the measure of central tendency
adopted, as the form of distribution is broadly similar (Fig.
6), resulting in no significant
differences in estimates of skewness or kurtosis (Table 4). When the
mitochondrial surface bounding the free sarcoplasm is taken into
account, in calculating a, the trend is similar, but the
differences among groups appear less dramatic (Table 4).
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Fig. 5.
The cumulative volume of mitochondria (mito;
A) and intracellular lipid (B) along the radial
oxygen diffusion path from capillaries, analyzed by means of an annular
counting frame.  , Wi;  , Su;
 , Au. Note the similar gradient for mitochondria at
different temperatures, Vv(mit,f) = 154.9 + 20.98 × distance from capillary center
(r2 = 0.85). The gradient for lipid in the
cold-acclimatized fish, Vv(lip,f) = 207.3 + 28.40 × distance (r2 = 0.95), was significantly steeper than that for both Su (114.8 + 13.87 × distance; r2 = 0.90) and Au
(15.6 + 5.30 × distance; r2 = 0.11) fish.
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Fig. 6.
Frequency distribution of mitochondrial separation in
slow fibers from acclimatized trout. A: Sp; B:
Su; C: Au; D: Wi. The progressive appearance of a
cohort of short distances, from Su through Au to Wi, is consistent with
the biogenesis of new organelles. Intracellular reorganization returns
the distribution close to a unimodal distribution by Sp. However, mean
separation varies little with season.
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Intracellular oxygen tension is similar at all temperatures.
The volume of mitochondria supplied by each capillary scaled directly
with capillary spacing (i.e., with domain area) and was, on average,
similar for all acclimatization groups. Intracellular lipid volume
followed a similar scaling relationship but was significantly higher
over the whole range of domain area at the lowest temperature (Fig.
7). The integrative effect of these
changes in fiber structure and muscle capillary supply was examined by
means of a mathematical model of intracellular diffusion, with the
parameters used listed in Table 5.
Despite major differences in lipid content (30%), CD (50%), and fiber
size (50%) during the year, the resultant mean
PO2 varies less than 10% among acclimatization
groups, with minimum PO2 at the fiber center
varying only slightly more (Table 5). Thus complementary changes in the
structural determinants of peripheral oxygen transport are predicted to
result in a seasonally invariant profile of intracellular oxygen
tension (Fig. 8).
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Fig. 7.
Relative and total volume of mitochondria (A,
B) and lipid (C, D) per capillary
domain in trout slow muscle. , Wi; ,
Su; , Au. To maintain exchange capacity as the area
supplied by each capillary increases, the mitochondrial demand for
substrates (Vv) would have to decrease, such that the total
demand would remain constant, i.e., Vv(mit,f) = Vv × domain area. Su fish come closest to showing
this relationship, whereas Au and Wi fish instead maintain a constant
Vv, indicating active mitochondrial biogenesis at a rate
that keeps pace with fiber hypertrophy. The potential demand,
therefore, increases with supply area. The actual substrate flux will
be modulated by the effects of temperature on diffusion, with the
gradient of Vv(mit,f) varying in the order required to
oppose diffusive limitations. Intracellular Vv(lip,f) does
not vary with domain area [a(Dom)] in Wi, although the gradient of
Vv(lip,f) is no different from that seen in Su or Au.
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Fig. 8.
Calculated PO2 in slow muscle
fibers of seasonally acclimatized trout. A: Sp;
B: Su; C: Au; D: Wi. The integrated
response of changes in different structural compartments is predicted
to maintain a similar level of intracellular oxygenation throughout the
year.
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DISCUSSION |
The extensive literature on vertebrate thermal biology
suggests that adjustments in fiber size and mitochondrial densities are
widespread responses to alterations in environmental temperature (9). In fishes, the most extensive structural and
biochemical changes reported are found in some cyprinids, e.g., Crucian
carp show an impressive proliferation of mitochondria on cold
acclimation that appears to be paralleled by an extensive growth of
capillaries (22), suggesting an impedance match between
vascular oxygen supply and tissue oxygen demand. In addition, there is
an expansion of the slow muscle mass and changes in both metabolic and
contractile enzyme activities (34). However, changes in
structure do not necessarily have adaptive significance, and these
cyprinids may be unusual, possibly reflecting their relatively sluggish
activity pattern and hypoxia tolerance, whereas other more active
eurythermal species have an attenuated response to laboratory
temperature acclimation (34). The adaptive significance of
any change in physiology or morphology, therefore, has to be
interpreted with caution. In the striped bass, an active species
migrating up the Eastern seaboard of the United States at different
times of the year, there was no change in myofibrillar ATPase
(34), whereas angiogenesis served only to maintain, rather
than increase, CD in the face of cold-induced fiber hypertrophy
(15). We wished to establish whether strategies other than
those revealed by two-point comparisons imposed on captive fish might
be used to preserve muscle function during periods of fluctuating body
temperature and, therefore, examined the structural response of
skeletal muscle in trout undergoing seminatural seasonal
acclimatization. In the following, it is important to be clear that
morphometry can only characterize the potential capacity of a system,
e.g., at 
O2max, and can say little about
metabolic control of tissue at rest.
Fiber size determines the effective capillary supply.
Sustained swimming activity in teleost fishes is powered exclusively by
the band of slow muscle running superficially along the trunk
(34). To maintain the locomotory performance observed in
cold water (35), it is vital to overcome the impaired
contractile function imposed by low temperatures. An increase in
number, but not size, of fibers was observed in cold-acclimated
goldfish (26), whereas the 70% increase in slow muscle
mass of striped bass on cold acclimation (27) could not be
explained by the modest increase in mean fiber size observed in these
fish (15). These and other data suggest that fiber
hyperplasia underlies the increase in slow muscle mass on cold
acclimation. In contrast, the nearly twofold increase in fiber size of
trout sampled during the Au and Wi is more than adequate to explain the
increase in slow muscle mass from 4.3 to 5.2% body mass, respectively
(37), whereas the fiber S/V shows a complimentary seasonal
pattern to that of fiber area, suggesting that the prime response of
trout is one of fiber hypertrophy at low temperatures. This has
important implications for the efficiency of substrate exchange by the
capillary bed, as intracellular distances over which substrate
diffusion must occur increase at a time when the rates of diffusion are
reduced, thus potentially limiting the locomotory capacity in cold
waters. In striped bass slow muscle, C/F increased by 40% between
25oC- and 5oC-acclimated groups, although the
larger mean fiber size of cold-acclimated animals resulted in similar
values for CD and calculated dimensions of oxygenated tissue cylinders
around capillaries (15). Seasonal acclimatization of trout
induced a progressive expansion of the capillary bed from Su to Wi such
that C/F varied inversely with environmental temperature, but, whereas
fiber hypertrophy only occurred in the coldest group, CD was maximal at
the intermediate temperature of 11oC (12). We
have included an additional group of fish in the current study and
extended the analysis to explore further the relationship between these
parameters over an annual temperature cycle.
The striking cold-induced angiogenesis is attenuated on prolonged
exposure to low temperatures (Wi-Sp), implicating a transient hormonal
stimulus for capillary growth elicited by seasonal changes in
environmental temperature and/or photoperiod (9). By Sp, C/F closely resembled that of Su animals, although because this is
accompanied by a reduction in fiber area, the CD did not decrease to
the same extent, and, hence, the anatomical supply capacity of the
capillary bed was defended. Although the cylinder of tissue that may be
calculated from these data to be oxygenated by capillaries decreases a
little, the more realistic geometric analysis of supply by calculation
of domain area shows a near equivalent mean ICD. Importantly, the large
seasonal excursions in C/F and (f) do not substantially increase the heterogeneity of capillary spacing (LogSD), implying that angiogenesis is a tightly controlled local phenomenon that serves to maintain optimal extracellular delivery of
substrates to the working muscle. However, simple counts of capillaries, whether describing the global supply (C/F) or the number
of adjacent capillaries corrected for fiber area (CFD), cannot
adequately describe the combined influences of angiogenesis and
hypertrophy (13). An assessment of the local capillary
supply conditions is therefore required. These data indicate that on average, the potential substrate delivery for each muscle fiber (LCFR)
undergoes a regular annual cycle that tracks the seasonal growth
pattern, whereas the normalized supply (LCD) shows an impairment at
both seasonal extremes of temperature. This pattern of response is
consistent with earlier data on the scope for endurance swimming and
muscle blood flow, which revealed an integrated response to optimize
aerobic performance in the middle of the species' thermal range
(37).
Mitochondrial proliferation maintains, but does not increase,
organelle density.
Most ectotherms experience an acute reduction in oxygen consumption on
cold exposure followed by varying degrees of metabolic compensation on
prolonged acclimation. The ability to increase oxygen utilization
requires both catalytic and diffusive limits to be overcome by
adjustments in enzyme kinetics (36) and/or intracellular
structure (32), respectively. For all eurythermal fish
species so far examined, there is an inverse relationship between the
temperature of acclimation and the volume fraction of mitochondria in
slow fibers (9, 24). Mitochondrial
proliferation may help to preserve the rate of total cellular ATP
production by 1) increasing the intracellular concentration
of respiratory chain enzymes, which would offset reduced turnover in
the cold (23), and 2) reducing the mean
diffusion path length and increasing the exchange surface for
substrates within the cytoplasm (15, 40). In
contrast to this general pattern of response among
laboratory-acclimated fishes, seasonally acclimatized trout show a
striking similarity in Vv(mit,f), with the maximal
change of a little over 10% occurring during the transition between Su
and Au. This does not result from a static population of organelles,
but rather from active proliferation that parallels the extent of
muscle hypertrophy and atrophy such that the total mitochondrial
volume-per-unit fiber length [V(mit,f)] is similar between Su
and Au fish, increases 1.8-fold by Wi, and subsequently falls by 20%
in Sp. Interestingly, the maximal Vv(mit,f) in trout
(Wi, 376 µm3) is similar to that of striped bass
(5oC, 335 µm3), which may imply an upper
limit to the volume of metabolic machinery that can be accommodated in
a given cell. The Vv(mit,f) is a compromise between
conflicting functional demands. In fish slow muscle, this may approach the spatial limit possible in cells that also perform mechanical work and are exceeded only in modified, noncontractile tissue such as the heater organ of billfish (2,
41). A recent paper by Johnston et al. (24)
presents a compilation of mitochondrial Vvs in related
species that are higher than those of either bass or trout. Whether
this represents differences in thermal niche (stenothermal vs.
eurythermal), phylogeny (perciformes vs. salmonidae), or locomotion
(labriform vs. subcarangiform) is not clear. This constancy of
organelle density results in the same volume of mitochondria being
found at any given distance from a capillary in muscle from animals
over a 14oC range of environmental temperature. A
qualitatively similar radial distribution is usually only found in
muscle of isothermal animals, such as mammals (21), and in
icefish that live in the extremely cold but very stable waters of
Antarctica (16). This pattern of adaptation has important
implications for the role of mitochondria in overcoming the metabolic
limits of cold exposure, implying only modest compensation in substrate
availability within the cytoplasm or product delivery to the
myofibrillar network.
This picture of overall mitochondrial distribution is, however,
incomplete as mitochondrial proliferation in trout results in ribbons
of organelles quite different from the clustered arrays found after
cold acclimation in goldfish (40) and striped bass (15). As a result, the mean intermitochondrial diffusion
distance (Li) is progressively reduced from Su through Wi, thereby
aiding the exchange process before increasing again in Sp, at which
Vv(mit,f) approaches the seasonal minimum. The
influence of mitochondrial biogenesis on this process is evident from
the distribution of Li that shows a second peak in Wi fish,
corresponding to shorter diffusion distances caused by proliferation of
mitochondria by binary fission, with Au and Sp fish representing a
transition between this bimodal distribution and the more usual
log-normal distribution of Su animals. It is difficult to incorporate
mitochondrial ribbons into the PO2 model, and,
therefore, we probably underestimate the efficiency of transcellular
oxygen flux, strengthening the argument that oxygen is not a limiting
factor for these fibers at any temperature.
The size and shape of individual mitochondrial profiles, as well as
their cristae densities, are essentially unaltered by thermal
acclimation in either slow or fast muscle of striped bass (15) and trout (this study). Although our estimates of
cristae Sv are less than the 36-40/µm of St. Pierre
et al. (31), cristae packing in trout appears to be less
than that of the highly aerobic red muscle of tuna (63-70/µm)
and similar to other fishes (25-37/µm) and mammals
(20-40/µm; see Ref. 29). Although there would appear to be scope
for reducing the matrix space, the contribution of an increased (bass)
or maintained (trout) population of organelles to metabolic
compensation in the cold is primarily a volumetric response to
temperature, i.e., a quantitative rather than qualitative difference
among acclimated/acclimatized groups of animals. As cristae density
will correlate with specific oxidative capacity if respiratory chain
enzymes were maximally packed, the increased oxidative capacity of
trout red muscle estimated from activities of mitochondrial enzymes in
crude tissue homogenates (3) may be expected to parallel
differences in mitochondrial densities. That they don't suggests
either a change in specific mitochondria activity, which in the absence
of a significant change in cristae surface density would imply an
altered respiratory chain density, or changes in the fatty acid (FA)
composition of phospholipids within the mitochondrial membranes, which
are known to have a significant impact on enzyme activities. Indeed,
the latter response may be very important, as activity of
membrane-bound enzymes, such as carnitine palmitoleoyltransferase
(CPT), show a more complete compensation than do those of soluble
enzymes (32). In addition, one cannot exclude the
possibility of changes in enzyme isoforms with thermal
acclimatization that may also alter metabolic capacity in the
absence of noticeable ultrastructural changes. Nevertheless, the
present data are consistent with the finding of no significant effect
of thermal adaptation in maximal rates of oxygen consumption for
isolated mitochondria among species living at different temperatures (25), although Guderley and Johnston (19)
demonstrated an enhanced oxidative capacity after cold acclimation of
sculpin. Further, St. Pierre et al. (31) suggested that
acclimatization induces compensation in mitochondrial respiration after
seasonal acclimatization of trout as a result of shifts in enzyme
levels and cristae packing. Differences in holding conditions (range of
temperature and photoperiod) may underlie the apparent contrast with
the present study in the thermal plasticity of mitochondrial structure
or respiratory capacity. Whereas we showed little change in cristae
Sv, the minor differences reported by St. Pierre et al.
(31) appear to be inadequate to significantly affect the calculated PO2 distribution (Fig. 8). However,
because enzyme activities change considerably more than ultrastructural
indexes, one would not expect a simple one-to-one relationship between respiratory capacity and mitochondrial content (40),
although they are broadly correlated over a wide range of capacity
(41), and therefore modest differences in cristae
Sv may translate into greater differences at the enzymatic level.
Complementary changes in other compartments may aid metabolic
compensation.
Unlike the response of striped bass, where a similar volume of
myofibrils per slow fiber in both cold- and warm-acclimated fish was
supplied by a greater volume of mitochondria, in trout, the specific
myofibrillar volume changes with season. As a consequence, the ratio of
potential ATP demand by the contractile machinery to ATP-generating
capacity varies only between a factor of 2.3 (Wi) and 2.8 (Su). These
values are similar to those from warm-acclimated striped bass, whereas
in cold-acclimated fish, it is close to unity (calculated from data in
Ref. 15). This appears to run counter to the adaptationist arguments
requiring an increase in respiratory chain enzyme content to overcome
catalytic limitations in the cold. An alternate strategy would be to
ameliorate the reduction in diffusion coefficient of substrates at low
temperatures by structural reorganization of intracellular
compartments, particularly the mitochondria, to minimize the distance
over which metabolites have to travel by diffusion (40).
For aqueous substrates, the compensation is nearly perfect in goldfish
(33) but less good in striped bass (15) and
quite modest in trout (this study). However, other compartments may
play a crucial role.
The mitochondrial content and CD of fish slow muscle approaches that of
the mammalian myocardium in its aerobic potential. Highly oxidative
tissues are known to maintain low intracellular concentrations of
modulators that promote glycolysis and glycogenolysis and undergo
smaller changes in levels of intracellular activators during shifts in
work intensity (7), and this is thought to be associated
with a shift in metabolic fuel preference from carbohydrate substrates
toward FAs at low temperatures (4). Although the level of
intracellular lipid may not be taken as direct evidence for a shift in
fuel preference, as dietary acquisition probably outstrips catabolic
activity, it is consistent with such enzymatic data. A significant
decrease in intracellular lipid would, however, seem clearly to
indicate mobilization and, presumably, catabolism of fat. In trout,
this is also consistent with a significant amount of extracellular
lipid deposition, which would appear to be balanced by rates of
mobilization for most of the year (~1 ml lipid/100 g body mass in Wi,
Sp, and Su), with an accumulation in Au (1.3 ml lipid/100 g body
mass). Importantly, the intracellular balance between
lipogenesis and lipolysis in the cold tends to favor the former, as
cold acclimation is associated with a 13-fold greater volume of
intracellular lipid droplets in slow fibers of striped bass acclimated
to 5oC than to 25oC, reaching a volume
fraction of 8% (15). The presence of significant amounts
of intracellular lipid in striped bass slow fibers served to preserve
adequate transcellular oxygen flux at low temperatures, thus obviating
the need for a significant degree of angiogenesis (20).
Although trout accumulate twice this amount when acclimatized to
4oC (Table 3), this starts from a much higher volume
fraction at 18oC and corresponds to only a 4.6-fold
increase in total intracellular lipid when changes in fiber size are
allowed for, compared with a 17-fold difference in bass. However, it is
this change in Vv of lipid droplets
[Vv(lip,f)] that largely accounts for the decrease in Vv(myf,f) in Wi fish. The inverse relationship
between Vv and S/V of intracellular lipid suggests that the
cyclical change in depot volume is achieved by expansion and
contraction of individual droplets rather than the cyclical production
of new depots. The radial distribution of lipid in trout shows that all
along the intramuscular oxygen transport pathway, there is a greater
volume of lipid fuel available at 4oC than at higher
temperatures, which, together with a reduced diffusion distance, will
favor greater FA oxidation. The 2.4-fold increase in
Vv(lip,f) is paralleled by a 1.5-fold increase in activity of CPT (2), an enzyme that is widely recognized
as an indicator of the capacity for -oxidation of FAs and a
potentially important contributor to regulation of carbon flux through
this pathway (30). At the tissue level, FA oxidation may
be determined by FA availability, determined by both lipase activity
(which is increased on cold acclimation) and FA uptake (which is
largely dependent on the concentration gradient between compartments). Whether or not this accumulation accurately reflects the shift toward
lipid metabolism, such a quantity would serve to further ameliorate a
decrease in transcellular oxygen flux at low temperatures (6, 20).
The above arguments only hold for red muscle, as the structural
capacity for ATP production by mitochondria in white muscle is in
excess of actual tissue demand, and although this tissue is heavily
reliant on lipid oxidation, it has a lower CPT activity per volume
mitochondria than red muscle (29). In addition, the relatively sparse capillary supply adds further weight to the argument
that mitochondria in white muscle are not limited by oxygen delivery in
the same way as are those in red muscle, due to the intermittent demand
for ballistic force production fuelled by anaerobic glycolysis.
In conclusion, we have shown that subtle changes underlie the
integrative response to cold acclimatization, requiring comparatively modest responses within any one system to achieve substantial differences in gross physiological activity. Seasonal acclimatization of rainbow trout produces relatively small changes in mitochondrial Vv, in contrast to the large increases reported for other
species after tank acclimation to low temperatures. This apparent lack of metabolic compensation at low environmental temperatures is exacerbated by an increased fiber girth, thus increasing the
intramuscular diffusion distances. The local capillary supply is
sensitive to changes in fiber size, being greatest at 11oC,
and is therefore likely to be limiting for aerobic muscle performance at the seasonal extremes in temperature. This is countered by structural reorganization, which leads to decreased intracellular diffusion distances and increased lipid content in the cold. The cumulative effect of these changes leads to a potentially high and
unchanged mean fiber oxygen tension throughout the year, a response
appropriate for an active species inhabiting fast-flowing waters.
Perspectives
Use of laboratory acclimation studies fail to address the possible
synergistic effect of tank stress and altered temperature on
physiological responses in addition to attenuating any integrated response to parallel changes in environmental quality with
season. In addition, there is an implicit assumption in comparative
biochemistry and physiology research that any observed differences must
have adaptive significance. In attempting to encompass these issues, it
is clear that only limited structural remodelling is required for
thermal compensation of peripheral oxygen transport in fish muscle.
| |
ACKNOWLEDGEMENTS |
We are grateful to H. Guderley and C. D. Moyes for a number of
helpful comments.
| |
FOOTNOTES |
This work was supported by the Natural Environment Research Council.
Address for reprint requests and other correspondence:
S. Egginton, Dept. of Physiology, Univ. of Birmingham, PO Box 363, Birmingham B15 2TT, UK (E-mail: s.egginton{at}bham.ac.uk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 1 April 1999; accepted in final form 11 February 2000.
| |
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ABSTRACT
INTRODUCTION
