Metabolic influences of fiber size in aerobic and anaerobic locomotor muscles of the blue crab, Callinectes sapidus
1 Department of Biological Sciences,University of North Carolina at
Wilmington, 601 South College Road, Wilmington, NC 28403-5915, USA
2 Department of Mathematics and Statistics, University of North Carolina at
Wilmington, 601 South College Road, Wilmington, NC 28403-5915, USA
* Author for correspondence (e-mail: kinseys{at}uncw.edu)
Accepted 3 August 2004
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Summary |
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Key words: crustacean, blue crab, Callinectes sapidus, muscle fiber, fiber size, fiber growth, anaerobic, aerobic, scaling, citrate synthase, lactate, diffusion, recovery, exercise, metabolism, TEM, mitochondria
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Introduction |
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An interesting feature of these white muscle fibers is that the
distribution of mitochondria varies as a function of fiber size. In juveniles,
mitochondria are uniformly distributed throughout the fibers and the
population is equally divided between subsarcolemmal and intermyofibrillar
fractions. However, in white fibers from adults, mitochondria are largely
subsarcolemmal (Boyle et al.,
2003). Thus, in large fibers, there is a cylinder of oxidative
potential around the periphery of the cell whereas the inner core of the fiber
has limited aerobic capacity. This developmental redistribution of
mitochondria dramatically increases intracellular diffusion distances between
mitochondria in large fibers, more so than would be expected from increases in
fiber diameter alone. Since contraction in these fibers is anaerobically
powered and relies on endogenous fuels, large cell size should not impact this
process. However, the small surface area to volume ratios (SA:V) and
intracellular diffusion limitations associated with large fiber size would be
expected to affect aerobic metabolism. This is consistent with observations
that post-contractile recovery in muscle from adult C. sapidus is a
very slow process (Milligan et al.,
1989
; Henry et al.,
1994
).
Burst contraction in crustacean muscles is similar to that in vertebrates,
where intracellular phosphagen and glycogen stores are depleted and lactic
acid accumulates (England and Baldwin,
1983; Booth and McMahon,
1985
; Head and Baldwin,
1986
; Milligan et al.,
1989
; Morris and Adamczewska,
2002
). In crustaceans, there is an initial reliance on the
hydrolysis of the phosphagen, arginine phosphate (AP), after which anaerobic
glycogenolysis is recruited. Glycogenolytically powered contractions are
slower than those powered by phosphagen hydrolysis
(England and Baldwin, 1983
;
Head and Baldwin, 1986
;
Baldwin et al., 1999
;
Boyle et al., 2003
), and
lactate only accumulates during an extended series of anaerobic contractions,
which are thought to be a normal part of the animal's behavior in the
environment (Booth and McMahon,
1992
).
In contrast to anaerobic contraction, the recovery from sustained exercise
in crustaceans is quite different from the vertebrate paradigm. An early phase
of recovery is a restoration of AP pools. This step of recovery is largely
powered by anaerobic glycogenolysis
(England and Baldwin, 1983;
Head and Baldwin, 1986
), which
contrasts with the exclusively aerobic resynthesis of the vertebrate
phosphagen, creatine phosphate
(Kushmerick, 1983
;
Meyer, 1988
;
Curtin et al., 1997
). Thus, in
crustaceans, most glycogen depletion and lactate accumulation occurs after
contraction (England and Baldwin,
1983
; Head and Baldwin,
1986
; Kamp, 1989
;
Henry et al., 1994
;
Morris and Adamczewska, 2002
;
Boyle et al., 2003
). The
reasons for this post-contractile lactate accumulation in crustacean muscle
are not known. However, it may be a mechanism for accelerating certain phases
of the recovery process to facilitate additional high-force contractions,
since exclusive reliance on aerobic metabolism would be expected to result in
an extremely slow recovery in very large fibers
(Boyle et al., 2003
).
Therefore, in crustaceans, anaerobic metabolism would be expected to
contribute to post-contractile recovery more in large fibers than in small
fibers.
Despite reliance upon anaerobic metabolism to power specific recovery
processes, complete recovery ultimately must depend on aerobic pathways. The
aerobic phase of recovery is dependent upon oxygen flux across the sarcolemma,
which is influenced by SA:V, and diffusive flux of ATP-equivalents from the
mitochondria at the fiber periphery to points of utilization in the fiber core
(Boyle et al., 2003). The
phosphoryl transfer from ATP to arginine, forming AP, is catalyzed by arginine
kinase (AK), which functions near equilibrium in crustacean muscle
(Ellington, 2001
;
Holt and Kinsey, 2002
). Since
the ATP-equivalent diffusive flux is carried almost exclusively by AP, aerobic
processes are dependent on the rate of AP diffusive flux
(Meyer et al., 1984
;
Ellington and Kinsey, 1998
;
Kinsey and Moerland, 2002
),
which is strongly hindered by structural barriers in crustacean muscle
(Kinsey et al., 1999
;
Kinsey and Moerland, 2002
;
Fig. 1). The scope of diffusion
limitation can be appreciated by examining the time required for intracellular
metabolite diffusion in muscle. In juvenile blue crabs, diffusion of AP across
a muscle fiber takes place in several seconds, whereas in adults the time
required for diffusion across a fiber can exceed 20 min
(Kinsey and Moerland, 2002
).
It is therefore expected that SA:V and diffusion limitations will increasingly
constrain the rate of aerobic post-exercise recovery as animals become
larger.
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In addition to white locomotor muscles, C. sapidus also have
smaller bundles of mitochondria-rich, dark fibers that power aerobic swimming
(Tse et al., 1983), and these
fibers must also maintain contractile function over a 3000-fold increase in
body mass during post-metamorphic development. There are two general
mechanisms by which an organism can increase its muscle mass during
post-embryonic growth (reviewed in
Rowlerson and Veggetti, 2001
).
New muscle fibers can develop through hyperplasia, as seen in cephalopods
(e.g. Pecl and Moltschaniwskyj,
1997
), or existing fibers can increase in diameter through
hypertrophy, as seen in fishes (e.g.
Weatherly and Gill, 1985
) and
crustaceans (Bittner and Traut,
1978
). Whereas the large developmental increase in body mass in
C. sapidus causes white locomotor muscle fibers to become giant
(Boyle et al., 2003
), the
aerobic function of the dark muscle fibers necessitates that they remain
small. C. sapidus appear to have resolved the conflicting demands for
hypertrophic development and aerobic contraction by subdividing the dark
fibers into smaller functional units (Tse
et al., 1983
). While the development of white locomotor fibers has
been previously described by Boyle et al.
(2003
), the ontogenetic
development of the aerobic fibers has not been addressed. For instance, it is
unclear whether a constant number of aerobic subdivisions is present in fibers
from all size classes, including the smallest fibers from juvenile animals
where diffusion would not be expected to limit aerobic processes, or whether
subdivisions form throughout development and function to maintain a constant,
small size of each aerobic functional unit.
The objectives of the present study were (1) to characterize the post-metamorphic developmental pattern of subdivision formation in the dark levator swimming muscle, which constitutes a giant fiber system that has undergone selection for aerobic function, and (2) to assess the reliance on anaerobic metabolism of white and dark fibers during post-contractile recovery. We investigated mitochondrial content, the scaling of aerobic enzyme activity, and subdivision formation in fibers of the dark levator muscle during post-metamorphic growth in C. sapidus. In addition, lactate levels following fatiguing exercise were monitored in white and dark levator muscle tissues as well as in hemolymph as a function of animal size. We hypothesized that (1) dark fibers would grow hypertrophically, but subdivisions would form throughout development to maintain a constant, small effective diameter, (2) post-contractile lactate accumulation would be greater and removal would be slower in white fibers from large crabs than from small crabs, and (3) post-contractile lactate accumulation would not occur in dark levator muscle due to the absence of SA:V and intracellular diffusive limitations in the small, subdivided fibers.
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Materials and methods |
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Transmission electron microscopy
To characterize the developmental pattern of the dark levator muscle, the
diameters of the fiber subdivisions and their corresponding mitochondrial
fractional areas were measured with transmission electron microscopy (TEM).
Twelve crabs ranging in body mass from <0.1 g to >190 g were divided
into four size classes referred to as very small, small, medium and large
(Table 1). Crabs were
sacrificed by removing the dorsal carapace. The dark levator muscles
(Cochran, 1935;
White and Spirito, 1973
;
Tse et al., 1983
) were exposed
by removal of the reproductive and digestive organs, gills and portions of the
endoskeleton and mechanically isolated from surrounding tissue. Resting length
of the muscle was measured from its origin on the median plate to the
insertion at the heavy tendon, while the fifth periopod (swimming leg) was
positioned as far anterodorsally as possible. A small bundle of dark levator
fibers was teased apart with a glass probe, tied off with 6-0 surgical silk
and then excised from the animal. Due to the heterogeneity of the fiber
composition in dark levator muscle, care was taken to isolate only the core of
the tissue, which is comprised of aerobic fibers
(Tse et al., 1983
). The fiber
bundle was placed at resting length in a primary fixative consisting of 1%
glutaraldehyde and 4% paraformaldehyde in 0.063 mol l-1
Sörenson's phosphate buffer, pH 7.38
(Egginton and Sidell, 1984
;
Preshnell and Schriebman,
1997
). The osmolarity of the fixative and all corresponding buffer
rinses was adjusted by the addition of 10% sucrose and a trace amount of
CaCl2 to prevent a change in cell volume. Tissues were held in
primary fixative for a minimum of 24 h at room temperature and then rinsed for
15 min in Sörenson's phosphate buffer. This process was followed by a
secondary fixation in 1% osmium tetraoxide in Sörenson's phosphate
buffer. Samples were then dehydrated with an ascending series (50%, 70%, 95%,
100%, 100%) of ethanol and embedded in Spurr's epoxy resin
(Spurr, 1969
; Electron
Microscopy Sciences, Hatfield, PA, USA). Samples were sectioned at 90 nm with
a diamond knife on a Reichert Ultracut E (Vienna, AT, USA) and collected using
a systematic random sampling method
(Howard and Reed, 1998
) to
ensure complete representation of the mitochondrial distribution throughout
the muscle. Five sections were collected from a random starting point, and
then a distance of 500 nm wasskipped before collecting another five sections.
This process was repeated until 5 µm of tissue was sectioned. Five sections
were mounted on each Formvar-coated (0.25% Formvar in ethylene dichloride)
high-transmission copper grid and were stained with 2% uranyl acetate in 50%
ethyl alcohol and with Reynolds' lead citrate
(Reynolds, 1963
). Sections
were examined with a Philips CM-12 TEM (Hillsboro, OR, USA) operated at 60 kV.
One section per grid was randomly chosen, and a montage of photographs was
generated from a 100 µm2 region of dark fibers at 3600x
using a 3
"x4" plate camera. Montage photographs were
obtained from five sections per animal to yield 60 total micrographs used for
mitochondrial fractional area and fiber subdivision diameter analysis; this
was to account for intra- vs inter-individual variation. Negatives
were developed and then digitized using a Microtek Scanmaker 4 (Carson, CA,
USA) negative scanner.
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Adobe Photoshop (version 7.0) was used to align individual images to form a montage. Subsequently, the margins of mitochondria were traced and filled to form polygons. Fibers were likewise traced and filled to form polygons. Areas and diameters of these polygons were then generated using Image Pro Plus (version 4.1.0.9; Media Cybernetics; San Diego, CA, USA). For each animal, fractional areas were calculated by dividing the mitochondrial area summed over all five micrographs for that animal by the total subdivision area summed over all five micrographs for each animal.
Citrate synthase activity
Citrate synthase (CS) activity measurements were used as a measure of
aerobic capacity and were examined as a function of animal size in dark
levator muscle. The scaling of white levator CS activity with body mass was
measured previously by Boyle et al.
(2003). Dark levator muscle
tissues were excised from 23 crabs ranging in body mass from <1 g to
>200 g (Table 1). Tissues
were homogenized with a PowerGen (Fisher, Hampton, NH, USA) homogenizer in
520 volumes of extraction buffer (50 mmol l-1 Tris, 1 mmol
l-1 EDTA, 2 mmol l-1 MgCl2, 2 mmol
l-1 dithiothreitol, pH 7.6) and sonicated with a sonic
dismembranator 60 (Fisher) on ice at 6 W using four bursts of 5 s each.
Samples were centrifuged at 16 000 g for 20 min, and the
supernatants were stored at 80°C until further analysis.
Citrate synthase activities were spectrophotometrically assayed at 25°C
using the methods of Walsh and Henry
(1990). The supernatant was
combined with 1 mmol l-1 5,5-dithio-bis (2-nitrobenzioc acid), 0.3
mmol l-1 acetylcoenzyme A and water in a 0.5 ml cuvette for a
baseline absorbance reading at 412 nm on an Ultrospec 4000 spectrophotometer
(Amersham Pharmacia Biotech, Buckinghamshire, UK) until the absorbance
stabilized. The reaction was initiated by the addition of 0.5 mmol
l-1 oxaloacetate, and the enzyme activity (µmol min-1
g-1) was calculated using the slope of the absorbance change
immediately following the addition of oxaloacetate.
In vivo fatigue
To determine the ontogenetic reliance on anaerobic metabolism in white and
dark levator fibers during post-contractile recovery, burst contractile
activity comparable with that during an escape response was elicited by
stimulating the thoracic ring ganglia
(Boyle et al., 2003). Only the
three largest size classes of crab (Table
1) were used for these experiments due to the difficulty of
obtaining sufficient quantities of tissue for metabolite extractions in the
very small animals. The size classes used were defined based on the
relationship between animal mass and white levator fiber size, where the mean
diameters (µm) of the fibers were 133.3±1.9, 226.0±4.2 and
462.4±22.0 for small, medium and large animals, respectively
(Boyle et al., 2003
).
Crabs were removed from aquaria and suspended in the air with a clamp such
that the swimming leg motion was unrestricted. Wire electrodes were placed in
two small holes drilled into the mesobranchial region of the dorsal carapace,
and a Grass Instruments SD9 physiological stimulator (Astro-Med, Inc., West
Warwick, RI, USA) was used to elicit contractile responses (80 Hz, 200 ms
duration at 10 V cm-1 between electrodes). A single 200 ms pulse
would induce a series of burst swimming contractions that typically lasted
from 2 to 20 s. Stimulations were repeated once every minute until the animal
was fatigued, determined by minimal response to stimulation. Following
exercise, crabs were immediately placed in aerated aquaria, provided with food
and allowed to recover for one of eight time periods (1, 10, 60, 90, 120, 240,
360, 480 min following exercise). Due to the expected longer recovery period
for large crabs (Henry et al.,
1994; Boyle et al.,
2003
), this group was allowed to recover for 720 minfollowing
exercise, and recovery at 90 min was not examined. At least five animals per
size class were not exercised. These animals were weighed and measured and
then allowed to rest overnight before the tissue collection. A minimum of four
crabs were used per time point, and the mean N value for all time
points was 6.8.
Immediately before sacrifice, hemolymph was collected with a syringe from the arthrodial membrane at the base of the swimming leg and frozen in liquid nitrogen. The crabs were sacrificed by removing the dorsal carapace. White and dark levator muscle tissues were quickly dissected as previously described, and samples were excised, freeze-clamped in liquid nitrogen and stored at 80°C until further analysis.
L-lactate assays
Frozen tissue samples (0.050.2 g) were homogenized in 929
volumes of chilled 7% perchloric acid, sonicated on ice at 6 W using four
bursts of 5 s each, then centrifuged at 4°C at 16 000 g
for 20 min. The supernatants were neutralized using 3 mol l-1
KCO3- in 50 mmol l-1 PIPES and centrifuged at
4°C at 16 000 g for 20 min. The resulting supernatants
were stored at 80°C.
The concentration of L-lactate in white levator, dark levator
and hemolymph was spectrophotometrically assayed following the procedures of
Lowry and Passonneau (1972) as
modified by Kinsey and Ellington
(1996
). A buffer containing
300 mmol l-1 hydrazine hydrate, 12 mmol l-1 EDTA and 4
mmol l-1 NAD+ at pH 9.0 was mixed with the supernatant
in a 0.5 ml cuvette and read at 340 nm on an Ultraspec 4000 (Pharmacia) to
obtain a baseline absorbance value. The reaction was initiated by the addition
of 18.5 units of L-lactate dehydrogenase, and the change in
absorbance was measured. The concentration in the sample was calculated
assuming that 1 g of white and dark levator muscle tissue has 0.75 ml of
intracellular water (Milligan et al.,
1989
).
Statistical analysis
Levene's test was used to test for heteroscedascity. One-way analysis of
variance (ANOVA) was used to test for the main effects of animal size class on
fiber subdivision diameter, mitochondrial fractional area, citrate synthase
activity and lactate concentration at rest, immediately following contraction
and 60 min post-contraction. Where significant size effects were detected,
Tukey's HSD test was used to make pairwise comparisons among the means from
animal size classes. Two-way ANOVA was used to analyze the post-contractile
lactate concentrations (10 min post-contraction and beyond) for the
interaction between animal size class and recovery time for each tissue type.
All statistical tests were analyzed with JMP software version 7.0.2 (SAS
Institute; Cary, NC, USA) or SAS software version 8.02. Results were
considered significant if P<0.05. The linear regression of CS
activity data and the area under the lactate recovery curve were calculated
using Sigma Plot software version 8.02 (SPSS Inc., Chicago, IL, USA). Standard
errors for the post-contractile lactate recovery curve integrals were
calculated from 200 bootstrap samples using six values per time point. Data
are represented as means ± S.E.M. throughout.
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Results |
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Neither the diameters of the dark levator muscle aerobic fiber subdivisions (F=0.25, d.f.=3, P=0.86, N=60) nor the mitochondrial fractional areas of the subdivisions (F=0.065, d.f.=3, P=0.98, N=60) changed during development (Figs 3, 4). The mean diameter of the subdivisions across all size classes was 35.6±2.7 µm, which is well within the range of cellular dimensions typical of aerobic fibers from other animals. Although the mean diameter of the dark levator fiber subdivisions did not change, the total number of subdivisions did increase throughout development. For example, the fibers from the very small size class have no subdivisions while subdivisions do exist in the fibers of the small, medium and large size classes (Fig. 3). The mitochondria were distributed almost exclusively around the periphery of these aerobic fiber subdivisions, and they represented between 20 and 30% of the total area of each subdivision (Fig. 4).
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Citrate synthase activity
CS activity in dark levator muscle scaled negatively with increasing body
mass (Fig. 5A). CS activity
levels were higher in dark levator than in white levator, as expected from the
difference in function of these muscles. A line was fitted to the data
according to the scaling relationship: CS
activity=axMb, where M is animal mass, a is
a constant and b is the scaling exponent
(Schmidt-Nielsen, 1984). When
grouped by animal size class, there was a significant effect of size on CS
activity (F=14.6, d.f.=2, P<0.001, N=23), and
mean CS activity in dark muscle from large animal size class was less than
half of the activity in muscle from the small size class.
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Lactate production and removal
The lactate concentration values were log10-transformed to
achieve homogeneous variance. One-way ANOVA was used to test for a significant
effect of animal size class on each of the tissue types for [lactate] at rest,
immediately following exercise and 60 min post-exercise. Resting lactate
concentrations were not significantly different among animal size classes for
white levator (F=2.0, d.f.=2, P=0.16, N=24;
Fig. 6A), dark levator
(F=3.31, d.f.=2, P=0.054, N=27;
Fig. 6B) or hemolymph
(F=1.02, d.f.=2, P=0.37, N=30;
Fig. 7C). All size classes of
crab responded similarly to stimulations, although smaller crabs tended to
have contractile bursts that were of a higher frequency but shorter duration
than large crabs. However, immediately following exercise there were no
differences among size classes in the amount of lactate produced during
exercise in white (F=1.02, d.f.=2, P=0.38, N=24) or
dark levator muscle (F=0.602, d.f.=2, P=0.56, N=17)
(Fig. 6), suggesting that
muscle from all size classes of animals was doing the same amount of
mass-specific anaerobic work during exercise. During recovery from exercise,
however, there were large differences in [lactate] among animal size classes.
This pattern was particularly apparent at 60 min post-exercise, where
[lactate] reached the highest levels observed in large white fibers, which led
to a significant effect of animal size class (F=14.3, d.f.=2,
P<0.001, N=25; Fig.
6A). There was also a significant effect of animal size class in
the dark levator at 60 min post-exercise (F=6.9, d.f.=2,
P=0.01, N=15; Fig.
6B), although the differences were not as great as were seen in
the white levator.
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A complete time course of post-exercise lactate production and removal is presented in Fig. 7. Two-way ANOVA was run separately for each tissue, and all three tissues had significant interactions between size and recovery time (white levator F=4.09, d.f.=10, P<0.001, N=196; dark levator F=2.07, d.f.=10, P<0.0356, N=164; hemolymph F=3.67, d.f.=10, P<0.0003, N=201). Lactate levels began to decline immediately following exercise in both muscle tissues from small and medium crabs, while lactate remained elevated in both muscle tissues from large crabs, and the subsequent time course of lactate removal in all tissues from large crabs required >480 min. Unexpectedly, dark levator muscle from large crabs appeared to require more time for lactate clearance than did small and medium crabs (Fig. 7B), although the differences between the size classes in lactate removal from dark levator muscle appeared to be considerably less than the differences between size classes in the white levator (Fig. 7A). The hemolymph time course mirrored that of the two muscle tissues, but peak lactate concentrations tended to lag slightly behind those for the muscles.
To analyze differences in the size dependence of lactate recovery in dark and white muscles, the area under the lactate recovery curve (Fig. 7A,B; 10 min post-contraction and beyond) was approximated with the trapezoidal rule of integration for each tissue and size class. In this analysis, the resting lactate concentrations (Fig. 6A,B) were subtracted from the post-exercise lactate concentration values in order to examine only the amount of lactate that was elevated above resting levels. There were no significant differences between the dark and white muscle in the small and medium animal size classes (Fig. 8). However, for muscle in the large animal size class, the [lactate]xtime integral was significantly higher in the white muscles than in the dark muscle (Fig. 8; P=0.0024, N=268).
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Discussion |
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Striated skeletal muscle is a post-mitotic and highly differentiated tissue
that has little capability of increasing by simple division of its component
fibers. In fishes, hyperplasia occurs until a certain point in development,
after which muscle mass is increased by hypertrophy of existing fibers
(reviewed in Weatherly and Gill,
1987), although there are exceptions to this generalization. For
example, fiber division occurs in adult specimens of the fishes Notothenia
coriiceps and Patagonotothen longipes, leading to a slight
increase (13%) in fiber number and muscle mass
(Johnston et al., 2003
). Fiber
splitting was also noted in the mullet, Mugil cephalus
(Carpené and Veggetti,
1981
), and in the European eel, Anguilla anguilla
(Willemse and Lieuwma-Noordanaus,
1984
). However, in these cases, fiber splitting led to the
generation of new fibers, rather than a subdivision of existing fibers. By
contrast, the principal means of increasing muscle mass during growth in
post-embryonic squid is hyperplasia (e.g.
Pecl and Moltschaniwskyj,
1997
; Preuss et al.,
1997
). Blue crab dark levator muscle is perhaps unique in that it
appears to grow hypertrophically, and, because of the large developmental
increase in body mass, the fibers that power swimming become exceptionally
large. However, the aerobic swimming function is maintained by fiber
subdivision and intra-fiber perfusion with hemolymph
(Fig. 2). The result of this
fiber subdivision is the developmental maintenance of a constant size of each
metabolic functional unit while there is a simultaneous increase in
the size of each contractile functional unit.
The capacity for aerobic swimming in portunid crabs presumably entailed the
evolution of aerobic, subdivided fibers from giant white fiber precursors,
similar to those described by Boyle et al.
(2003). This means that other
systems that support aerobic contraction must have co-evolved to function
within the framework of the subdivided aerobic fibers. For instance, the
circulatory system had to evolve intra-fiber perfusion, which is apparent in
Fig. 2, and the evolution of
this trait may have constituted a novel adaptation specifically designed to
promote aerobic swimming. Metabolic subdivision also may have posed new
challenges to coordinating contraction over an entire fiber. In this case,
however, crustaceans may have been particularly well suited to deal with this
potential evolutionary hurdle, since crustacean muscles are often
multi-terminally innervated and do not propagate action potentials in the
manner of vertebrates. Thus, if it were necessary to innervate each
subdivision to promote uniform contraction (an open question), crustacean
muscle may have been predisposed to evolve this trait. We can therefore
speculate that fiber subdivision is not widespread in the animal kingdom
because it is only necessary when aerobic fibers evolve from giant fibers, and
perhaps because other organisms are less able to accommodate the evolution of
the suite of systems required to support aerobic contraction in subdivided
fibers.
The present results also imply that there is a maximal size at which the
dark locomotor muscle fibers of blue crabs are able to contract aerobically
without becoming subdivided. Above this size, low SA:V and long intracellular
diffusive distances may begin to constrain the rate of oxygen and ATP fluxes
such that they are insufficient to keep pace with the high demands of
contraction. The lack of subdivisions in fibers from the smallest animals
indicates that these fibers are small enough to allow aerobic contraction.
This may indicate that there is no selective pressure to further reduce the
size of each metabolic functional unit in the smallest fibers. However,
spatial considerations may also limit the minimum size of subdivisions, since
exceedingly small fiber/subdivision size may not permit a sufficient volume of
the muscle to be devoted to myofilaments, leading to force production that is
inadequate for locomotion. Rome and Lindstedt
(1998) have characterized the
linkage between muscle design and function in the context of the percentage of
space allocated to myofibrils, the sarcoplasmic reticulum and mitochondria.
The developmental pattern in the dark levator muscle of blue crabs indicates
that, at least in some cases, fiber size and its influence on SA:V and
diffusive fluxes may also need to be considered to characterize fiber design
and function.
The CS activities and mitochondrial densities in aerobic muscle fibers were
roughly fivefold higher than that in white levator fibers
(Boyle et al., 2003) and are
consistent with observations that adult C. sapidus have locomotor
muscles that are highly vascularized
(McGaw and Reiber, 2002
) to
support their exceptional long-distance swimming capabilities
(Judy and Dudley, 1970
).
Although the negative allometric scaling of CS activity
(Fig. 5) was similar to the
findings in white levator muscle (Boyle et
al., 2003
), these data contrast with those for mitochondrial
fractional area in the dark aerobic fiber subdivisions, which did not scale
negatively with body mass (Fig.
4). This result was unexpected and it may indicate that the
mitochondria from the smaller animals are more densely packed with aerobic
enzymes. Alternatively, the discrepancy between the mitochondrial fractional
area and CS activity data could be due to differences in the method of data
collection. CS activity was referenced to the wet mass of the muscle, whereas
the TEM analyses were based on cross-sectional areas within a fiber
subdivision excluding extracellular and extra-subdivision spaces. There may be
systematic differences in the scaling of wet mass and subdivision
cross-sectional area that lead to the lack of agreement between these data
sets.
The second objective of this study was to examine some of the metabolic
implications of hypertrophic growth in crustacean muscle fibers with the
assumption that increasing fiber size in white muscle and the accompanying
mitochondrial shift towards the periphery of the fiber
(Boyle et al., 2003) lead to
excessive intracellular diffusion distances and low SA:V that constrain
aerobic metabolic processes (Fig.
1). A requirement for comparing the effects of fiber size on
recovery is that crabs from all size classes perform the same amount of
chemical work during exercise. During burst contraction, intracellular AP
stores are initially consumed, after which anaerobic glycogenolysis is
recruited to power sustained bouts of locomotion
(England and Baldwin, 1983
;
Booth and McMahon, 1985
;
Head and Baldwin, 1986
;
Milligan et al., 1989
;
Morris and Adamczewska, 2002
).
The resting concentration of AP (and glycogen) and the activity of AK do not
change with increasing body size in crustacean white muscle
(Baldwin et al., 1999
), so the
contribution of AP to ATP production during exercise is constant as well. It
follows that lactate production alone is an effective indicator of differences
among size classes in the amount of anaerobic work during exercise. The lack
of significant differences among size classes in muscle lactate production
during contraction (Fig. 6)
indicates that muscle tissues from animals of all size classes incurred equal
levels of mass-specific oxygen debt.
Boyle et al. (2003)
described some of the metabolic impacts of increased fiber size in white
levator muscle of blue crabs. They found that, following burst exercise,
glycogen recovery was much slower in larger animals, which they attributed to
the extreme diffusion distances in the large fibers. In addition, differences
in diffusive flux rates in fibers of different sizes may be amplified by the
time-dependent decrease in metabolite diffusion coefficients in muscle tissue,
which results from intracellular barriers that obstruct net molecular motion
across muscle fibers (Kinsey et al.,
1999
; Kinsey and Moerland,
2002
). Boyle et al.
(2003
) also found greater
glycogen depletion in white muscle fibers of larger animals, which they
suggested occurred because large fibers were relying more on anaerobic
glycogenolysis to speed up certain phases of recovery to more rapidly restore
contractile function. This conclusion was based on the relatively low ATP
yield per glucosyl unit during anaerobic glycogenolysis, which results in
greater glycogen depletion during recovery. The present study confirms the
hypothesis posed by Boyle et al.
(2003
) by providing direct
evidence that post-contractile anaerobic metabolism is size-dependent in white
muscle fibers from blue crabs. Since a very large fraction of the
post-contractile lactate production in white fibers diffuses into the blood,
the differences among size classes in the anaerobic contribution to recovery
are certain to be much more dramatic than is apparent from the muscle lactate
data alone (Figs 7,
8).
These arguments raise the question of which post-contractile recovery
processes are likely to be accelerated by invoking anaerobic metabolism, and
how this would benefit the animal. The most likely candidates are the recovery
of AP and the restoration of ionic gradients across the membrane. Although not
measured in the present study, there is evidence that AP recovery is
relatively rapid (Ellington,
1983; Head and Baldwin,
1986
; Thébault et al.,
1987
; Morris and Adamczewska,
2002
) and is associated with post-contractile glycogen depletion
and lactate accumulation in adult crustacean white muscle
(England and Baldwin, 1983
;
Head and Baldwin, 1986
;
Kamp, 1989
;
Henry et al., 1994
;
Morris and Adamczewska, 2002
;
Boyle et al., 2003
). Since AP
is the fuel initially used during burst activity
(England and Baldwin, 1983
;
Head and Baldwin, 1986
;
Kamp, 1989
;
Baldwin et al., 1999
), the
rapid replenishment of AP across the entire cell would facilitate additional
bouts of high-force anaerobic contractions. In contrast to crustaceans,
vertebrates rely exclusively on aerobic metabolism to power resynthesis of the
phosphagen creatine phosphate, and lactate does not accumulate following
contraction (Kushmerick, 1983
;
Meyer, 1988
;
Curtin et al., 1997
). The
vertebrate pattern of recovery therefore appears to closely resemble that in
muscle fibers from the small and medium animal size classes in this study
(Fig. 7A), which entails a
rapid, aerobic restoration of both phosphagen and lactate levels.
The relatively short intracellular diffusive distances in dark levator
muscle fibers (Tse et al.,
1983; Fig. 3) led
to the expectation that recovery following exercise would not lead to
size-dependent lactate production in this tissue, which is contrary to our
results (Figs 7,
8). However, these differences
were less dramatic than in white fibers, and post-contractile lactate
accumulation in the large animals was significantly lower in dark fibers than
in white, which is consistent with our hypotheses (Figs
7,
8). It is possible that dark
fibers also produce lactate following contraction in an effort to speed up
recovery, although it would be difficult to make the argument that this is due
to intracellular diffusion or SA:V constraints. In this view, the size
dependence would simply reflect the mass-specific decrease in aerobic capacity
that typically accompanies increases in body mass
(Fig. 5), and anaerobic
metabolism would be used to preserve a rapid post-contractile recovery rate as
animals grow larger. This interpretation implies that, at the very least, cell
size constraints are embodied in the differences in the size dependence of
lactate production between dark and white muscles seen in Figs
7,
8.
It is likely, however, that the differences between dark and white fibers
are actually much greater than is apparent in
Fig. 7. There are two reasons
to expect artificially large differences among size classes in dark fibers.
First, following exercise, the circulating hemolymph is highly concentrated
with lactate for an extended period of time. The bulk of the lactate that is
released to the hemolymph during, and particularly after, contraction is
expected to arise from the white fibers
(Boyle et al., 2003;
Fig. 7). The lactate-laden
hemolymph also perfuses the dark levator muscle, however, and there is likely
to be net diffusive flux of lactate from the hemolymph into the dark fibers.
Second, the fiber composition of the dark levator muscle is heterogeneous, and
the small, aerobic fibers located in the core are surrounded by giant white
fibers (Tse et al., 1983
). If
the white fibers are producing most of the lactate after contraction, the
close proximity of the dark fibers to the surrounding white fibers is likely
to also allow for net lactate diffusion into the dark fibers. Therefore, the
present whole-animal experiments may not be adequate to fully resolve
differences in the fiber size dependence of lactate dynamics between white and
dark muscle. An analysis of isolated fibers would be a useful approach to
address this issue.
Another argument for the presence of diffusive limitations in these large
white fibers can be made by examining the scaling of the activity of the
aerobic enzyme citrate synthase (Fig.
5). The body mass scaling exponent of CS activity for dark muscle
was more negative (b=0.19) than for the white muscle
(b=0.09), meaning there were greater differences among animal
size classes in dark muscle than in white
(Boyle et al., 2003). This
leads to the expectation that there would be greater differences in
post-exercise lactate dynamics among size classes in the dark muscle than in
the white muscle, which is contrary to both our predictions and
observations.
In summary, although aerobic fibers of the levator muscle in C.
sapidus grow hypertrophically, reaching very large sizes similar to
fibers in the white levator muscle, the fibers become increasingly subdivided
throughout development and maintain a mean subdivision diameter of 35
µm. This developmental pattern leads to a constant size for each metabolic
functional unit, while each contractile functional unit increases in size as
the animal grows. The time course of lactate removal from tissues following
exercise was consistent with the prediction that post-contractile metabolic
recovery is size dependent in white locomotor muscle fibers of C.
sapidus. Large white fibers appear to rely on anaerobic metabolism to
accelerate certain phases of the metabolic recovery process to offset
diffusive and/or SA:V limitations that may make aerobic metabolism
unacceptably slow. Although the aerobic locomotor fibers, which should not be
diffusion limited, also demonstrated a moderate size dependence of anaerobic
recovery, this result may be due to an inability to fully resolve differences
between muscle tissue types in a whole-animal experiment.
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References |
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