|
|
||||||||
Department of Physiology, University of Birmingham, Birmingham B15 2TT, United Kingdom
| |
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
| |
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).
| |
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)].
(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).
| |
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.
|
|
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.
|
|
|
|
(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).
|
|
|
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).
|
|
|
| |
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.
| |
REFERENCES |
|---|
|
|
|---|
1.
Blier, P,
and
Guderley H.
Metabolic responses to cold acclimation in the swimming musculature of lake whitefish, Coregonus clupeaformis.
J Exp Zool
246:
244-252,
1988.
2.
Block, BA.
Structure of the brain and eye heater tissue in marlins, sailfish and spearfishes.
J Morphol
190:
169-189,
1986[Web of Science][Medline].
3.
Cordiner, S,
and
Egginton S.
Effects of seasonal temperature acclimatization on muscle metabolism in rainbow trout, Oncorhynchus mykiss.
Fish Physiol Biochem
16:
333-343,
1997.
4.
Crockett, L,
and
Sidell BD.
Some pathways of energy metabolism are cold adapted in Antarctic fishes.
Physiol Zool
63:
472-488,
1990.
5.
Dean, JM.
The metabolism of tissues of thermally acclimated trout (Salmo gairdneri).
Comp Biochem Physiol A Physiol
29:
185-196,
1969.
6.
Desauliniers, N,
Moerland TS,
and
Sidell BD.
High lipid content enhances the rate of oxygen diffusion through fish skeletal muscle.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R42-R47,
1996
7.
Dudley, GA,
Tullson PC,
and
Terjung RL.
Influence of mitochondrial content on the sensitivity of respiratory control.
J Biol Chem
262:
9109-9114,
1987
8.
Egginton, S.
Morphometric analysis of tissue capillary supply.
In: Vertebrate Gas Exchange from Environment to Cell, edited by Boutilier RG.. Berlin, Germany: Springer Verlag, 1990, p. 73-141.
9.
Egginton, S.
Anatomical adaptations for peripheral oxygen transport at high and low temperatures.
S Afr J Zool
33:
119-128,
1998.
10.
Egginton, S,
and
Cordiner S.
Effect of fixation protocols on muscle preservation and in situ diffusion distances.
J Fish Biol
47:
59-69,
1994.
11.
Egginton S and Cordiner S. Changes in muscle fibre fine structure
induced by seasonal acclimatisation of rainbow trout (Abstract).
Society for Experimental Biology, Lancaster, 1996. (http://link.springer.de/link/service/journals/00898/meeting/lanc96/a7.htm).
12.
Egginton, S,
and
Cordiner S.
Cold-induced angiogenesis in seasonally acclimatized rainbow trout (Oncorhynchus mykiss).
J Exp Biol
200:
2263-2268,
1997[Abstract].
13.
Egginton, S,
and
Ross HF.
Planar analysis of tissue capillary supply.
In: Oxygen transport in biologoical systems, edited by Egginton S,
and Ross HF.. Society for Experimental Biology Seminar Series 51. Cambridge, UK: Cambridge Univ. Press, 1992, p. 165-195.
14.
Egginton, S,
Ross HF,
and
Sidell BD.
Morphometric analysis of intracellular diffusion distances.
Acta Stereologica
6:
449-454,
1988.
15.
Egginton, S,
and
Sidell BD.
Thermal acclimation induces adaptive changes in subcellular structure of fish skeletal muscle.
Am J Physiol Regulatory Integrative Comp Physiol
256:
R1-R9,
1989
16.
Fitch, NA,
Johnston IA,
and
Wood RE.
Skeletal muscle capillary supply in a fish that lacks respiratory pigments.
Respir Physiol
57:
201-211,
1984[Web of Science][Medline].
17.
Gorlich, A,
Kozlowska M,
Romek M,
and
Kilarski WM.
Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle.
J Fish Biol
49:
1280-1290,
1996.
18.
Guderley, H.
Functional significance of metabolic responses to thermal acclimation in fish muscle.
Am J Physiol Regulatory Integrative Comp Physiol
259:
R245-R252,
1990
19.
Guderley, H,
and
Johnston IA.
Plasticity of muscle fibre mitochondria with thermal acclimation.
J Exp Biol
199:
1311-1317,
1996[Abstract].
20.
Hoofd, L,
and
Egginton S.
The possible role of intracellular lipid in oxygen delivery to fish skeletal muscle.
Respir Physiol
107:
191-202,
1997[Web of Science][Medline].
21.
Hoppeler, H,
Mathieu O,
Weibel ER,
Krauer R,
Lindstedt SL,
and
Taylor CR.
Design of the Mammalian respiratory system. VIII. Capillaries and mitochondria in muscles.
Respir Physiol
44:
129-150,
1981[Web of Science][Medline].
22.
Johnston, IA.
Capillarization, oxygen diffusion distances and mitochondrial content of carp muscles following acclimation to summer and winter temperatures.
Cell Tissue Res
222:
325-337,
1982[Web of Science][Medline].
23.
Johnston, IA.
Cold adaptation in marine organisms.
Trans Roy Soc (Lond) Series B
326:
655-667,
1990.
24.
Johnston, IA,
Calvo H,
Guderley H,
Fernandez D,
and
Palmer L.
Latitudinal variation in the abundance and oxidative capacities of muscle mitochondria in perciform fishes.
J Exp Biol
201:
1-12,
1998
25.
Johnston, IA,
Guderley H,
Franklin CE,
Crockford T,
and
Kamunde C.
Are mitochondria subject to evolutionary temperature adaptation?
J Exp Biol
195:
293-306,
1994[Abstract].
26.
Johnston, IA,
and
Lucking M.
Temperature-induced variation in the distribution of different types of muscle fibre in the goldfish (Carassius auratus, L.).
J Comp Physiol [A]
124:
111-116,
1978.
27.
Jones, PL,
and
Sidell BD.
Metabolic responses of striped bass (Morone saxatilis) to temperature acclimation. II. Alterations in metabolic carbon sources and distribution of fiber types in locomotory muscle.
J Exp Zool
219:
163-171,
1982.
28.
Kleckner, NW,
and
Sidell BD.
Comparison of maximal activities of enzymes from tissues of thermally acclimated and naturally acclimatized chain pickerel (Esox niger).
Physiol Zool
58:
18-28,
1985.
29.
Moyes, CD,
Schulte PM,
and
Hochachka PW.
Recovery metabolism of trout white muscle: role of mitochondria.
Am J Physiol Regulatory Integrative Comp Physiol
262:
R295-R304,
1992
30.
Rodnick, KJ,
and
Sidell BD.
Cold-acclimation increases carnitine palmitoyltransferase-I activity in oxidative muscle of striped bass.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R405-R412,
1994
31.
St. Pierre, J,
Charest PM,
and
Guderley H.
Relative contribution of quantitative and qualitative changes in mitochondria to metabolic compensation during seasonal acclimatisation of rainbow trout Oncorhynchus mykiss.
J Exp Biol
201:
2961-2970,
1998[Abstract].
32.
Sidell, BD.
Cellular acclimatisation by quantitative alterations in enzymes and organelles.
In: Cellular Acclimatisation to Environmental Change, edited by Cossins AR,
and Sheterline P.. London: Cambridge Univ. Press, 1983, p. 103-120.
33.
Sidell, BD,
and
Hazel JR.
Temperature affects the diffusion of small molecules through cytosol of fish muscle.
J Exp Biol
129:
191-203,
1987
34.
Sidell, BD,
and
Moerland TS.
Effect of temperature on muscular function and locomotory performance in teleost fishes.
Adv Comp Environ Physiol
5:
115-156,
1989.
35.
Sisson, JE,
and
Sidell BD.
Effect of thermal acclimation on muscle fiber recruitment of swimming striped bass (Morone saxatilis).
Physiol Zool
60:
310-320,
1987.
36.
Somero, GN.
Proteins and temperature.
Annu Rev Physiol
57:
43-68,
1995[Web of Science][Medline].
37.
Taylor, SE,
Egginton S,
and
Taylor EW.
Seasonal temperature acclimatisation of rainbow trout: cardiovascular and morphometric influences on maximal sustainable exercise level.
J Exp Biol
199:
835-845,
1996[Abstract].
38.
Taylor, EW,
Egginton S,
Taylor EW,
and
Butler PJ.
Factors which may limit swimming performance at different temperatures.
In: Global Warming
Implications For Freshwater and Marine Fish, edited by Wood CM,
and McDonald DG.. Society for Experimental Biology Seminar Series 61. Cambridge, UK: Cambridge Univ. Press, 1997, p. 105-133.
39.
Taylor, SE,
Egginton S,
Taylor EW,
Franklin CE,
and
Johnston IA.
Estimation of intracellular pH in muscle of fishes from different thermal environments.
J Therm Biol
24:
199-208,
1999.
40.
Tyler, S,
and
Sidell BD.
Changes in mitochondrial distribution and diffusion distances in muscle of goldfish upon acclimation to warm and cold temperatures.
J Exp Zool
232:
1-9,
1984.
41.
Weibel, ER.
Design and performance of muscular systems: an overview.
J Exp Biol
115:
405-412,
1985
This article has been cited by other articles:
![]() |
S. Young and S. Egginton Allometry of skeletal muscle fine structure allows maintenance of aerobic capacity during ontogenetic growth J. Exp. Biol., November 1, 2009; 212(21): 3564 - 3575. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. B. Lortie and T. W. Moon The rainbow trout skeletal muscle beta -adrenergic system: characterization and signaling Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2003; 284(3): R689 - R697. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. O. Portner Physiological basis of temperature-dependent biogeography: trade-offs in muscle design and performance in polar ectotherms J. Exp. Biol., August 1, 2002; 205(15): 2217 - 2230. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Guderley and J. St-Pierre Going with the flow or life in the fast lane: contrasting mitochondrial responses to thermal change J. Exp. Biol., August 1, 2002; 205(15): 2237 - 2249. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |