Aerobic capacities in the skeletal muscles of Weddell seals: key to longer dive durations?
1 Department of Internal Medicine, University of Texas Southwestern Medical
Center, Dallas, TX 75390, USA
2 Department of Marine Biology, Texas A&M University, Galveston, TX
77553, USA
3 Department of Biology, University of California Santa Cruz, Santa Cruz, CA
95604, USA
4 Department of Medicine, University of California San Diego, San Diego, CA
92093, USA
* Author for correspondence (e-mail: shane.kanatous{at}utsouthwestern.edu)
Accepted 20 August 2002
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Summary |
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Key words: Weddell seals, Leptonychotes weddelli, aerobic capacity, diving, skeletal muscle, mitochondria
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Introduction |
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Despite the marked adaptations for hypoxia resistance demonstrated by
harbor seals, Steller sea lions and Northern fur seals, these species are
relatively short-duration divers (average dive durations are approximately 2.5
min). In comparison, Weddell seals (Leptonychotes weddelli) represent
an elite diving species that may remain submerged for over 60 min. These seals
are highly adapted for an aquatic life in the shore-fast and pack-ice habitats
of Antarctica (Kooyman, 1981).
Deep foraging dives are usually 100-350 m deep (maximum recorded depth of 741
m) and between 15-20 min in duration (maximum recorded duration of 82 min)
(Davis et al., 1999
;
Castellini et al., 1992
;
Kooyman, 1981
;
Testa, 1994
;
Wartzok et al., 1992
). The
majority of free-ranging dives by Weddell seals is within their ADL and
suggests an exceptional ability to tolerate prolonged periods of apnea.
The purpose of the present study was to assess the aerobic capacity of the skeletal muscles of deep, long-duration divers. We hypothesized that skeletal muscle of Weddell seals would exhibit the same, or an even greater, increase in their capacity for aerobic, lipid-based metabolism than we had observed in the comparatively shallow, short-duration divers. However, we found that the skeletal muscles of Weddell seals do not have enhanced aerobic capacities compared with those of terrestrial mammals. This may result from energy-conserving modes of locomotion employed by Weddell seals that permit prolonged dive durations but at overall low levels of metabolism.
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Materials and methods |
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Electron microscopy and morphometry
Each glutaraldehyde-fixed muscle sample was cut into thin longitudinal
strips, stored in glutaraldehyde fixative and processed for light and electron
microscopy (EM), as previously described by Mathieu-Costello et al.
(1992). Four transverse
sections (1 µm) were collected from tissue blocks using an LKB Ultrotome
III (Peapack, NJ, USA) and stained with 0.1% aqueous toluidine blue solution
for light microscopy. Ultrathin sections (50-70 nm) were cut transversely to
the muscle fiber axis in each block. They were contrasted with uranyl acetate
and bismuth subnitrate, and electron micrographs for morphometry were taken on
70-mm film using a Zeiss 10 transmission electron microscope (Zeiss North
America, Thornwood, NY, USA).
Capillary density [QA(0); the number of capillaries per
fiber cross-sectional area] was obtained by morphometric analysis on
transverse sections using a 100-point eyepiece square-grid test system on 1
µm thick transverse sections examined at a magnification of 400x with
a light microscope. Capillary diameter
[(c)], average number of capillaries
around a fiber (NCAF) and mean fiber cross-sectional area
[
(f)] were measured using an image analyzer (Videometric 150,
American Innovision, San Diego, CA, USA) on the same transverse sections.
(c) was determined as the shorter
axis of close-to-circular profiles only (<20% difference between the
shorter and longer diameters), which assumed capillary cross-sectional
circularity. Capillary-to-fiber ratio [NN(c,f)] was
calculated as the product of QA(0) and
(f)
(Mathieu-Costello, 1993
).
The volume density of mitochondria, myofibrils and lipid droplets per volume of muscle fiber was estimated by point-counting on electron micrographs obtained by systematic sampling in ultrathin transverse sections from each of the four tissue blocks from each sample. Contact prints of the EM film were projected at a final magnification of 24 000x on a 144-point square-grid test system of a microfilm reader (Documator DL 2, Jenoptic, Jena, Germany).
Enzymatic activities and myoglobin concentration
Frozen muscle samples were thawed, weighed and homogenized at 0°C in
buffer containing 1 mmoll-1 EDTA, 2 mmoll-1
MgCl2 and 50 mmoll-1 imidazole, pH 7.6. The homogenates
were spun for 4-5 min at 10 000g, and the supernatant was
isolated. The assay conditions were as follows. Lactate dehydrogenase (LDH; EC
1.1.1.27): 50 mmoll-1 imidazole; 0.15 mmoll-1 NADH, pH
7.0 at 37°C; and 1 mmoll-1 pyruvate;
A340, millimolar extinction coefficient
340=6.22. ß-hydroxyacyl CoA dehydrogenase (HAD; EC
1.1.1.35): 50 mmoll-1 imidazole; 1 mmoll-1 EDTA; 0.1
mmoll-1 acetoacetyl CoA; and 0.15 mmoll-1 NADH, pH 7.0
at 37°C;
A340,
340=6.22.
Citrate synthase (CS; EC 4.1.3.7): 50 mmoll-1 imidazole; 0.25
mmoll-1 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB); 0.4
mmoll-1 acetyl CoA; and 0.5 mmoll-1 oxaloacetate, pH 7.5
at 37°C;
A412,
412=13.6.
Specific enzyme activities (µmol min-1 g-1 wet mass
of muscle) were calculated from the rate of change of the assay absorbance at
the maximal linear slope. The enzyme ratio CS:HAD was calculated to assess the
relative contribution of fatty acid metabolism to total aerobic
metabolism.
Myoglobin was assayed according to the method of Reynafarje
(1963) with the following
modifications. A portion (500 µl) of the homogenate was further diluted
with 1 ml of phosphate buffer (0.04 moll-1, pH 6.6). The resulting
mixture was centrifuged for 50 min at 28 000g at 4°C.
The supernatant was bubbled with carbon monoxide for 3 min. Spectrophotometric
absorbance was measured at 538 nm and 568 nm, and the concentration of
myoglobin in mg g-1 wet mass of muscle was calculated as:
(Abs538-Abs568)x5.865[1.5/0.5)x(mass of
sample)].
Fiber type
Cross sections of each muscle sample were cut into serial sections (7-9
µm) with a Shandon cryotome (Thermo Shandon, Pittsburgh, PA, USA)
maintained at -20°C. Sections were placed onto glass slides; four serial
sections per slide. Transverse orientation was verified using a standard light
microscope. Sections were stained using a metachromatic ATPase staining
protocol modified from Olgive and Feeback
(1990). Briefly, the procedure
was as follows: (1) ATPase preincubation for 8 min (pH of 4.5) at room
temperature, (2) three 2 min Tris rinses (pH 7.8), (3) incubation with ATP for
25 min (pH 9.4), (4) three calcium chloride rinses, (5) counterstaining in
0.1% toluidine blue for 1 min, (6) dehydration in ethanol and (7) clearing in
xylene. The proportion of slow oxidative (Type I), fast-twitch oxidative (Type
IIA) and fast-twitch glycolytic (Type IIB) fibers was determined by standard
point counting procedure and is presented as percentages relative to the total
number of fibers.
In addition to using dog hindlimb muscle controls, we further verified the
validity of the metachromatic stain results in seal muscle by comparison with
immunohistochemical staining for specific slow and fast myosin heavy chain
isoforms after the reactivity of the antibodies with seal proteins had been
verified by western analysis. Slides were fixed in ice-cold
alcoholformalinacetic acid fixative in a Coplin jar and washed
with phosphate-buffered saline (PBS). A proteinaceous blocking agent
(Powerblock Reagent, Innogenex, San Ramon, CA, USA) was applied to each
section to minimize non-specific antibody binding. A series of monoclonal
antibodies specific to myosin heavy chain isoforms Type I, Type IIa and Type
IIb were applied to one of the four sections on each of the slides and
incubated overnight in a humidity chamber at -4°C. Serial amplification of
the primary antibody was accomplished using an incubation of biotinylated
secondary antibody for 20 min, followed by a series of PBS washes followed by
a 20 min incubation with alkaline-phosphatase streptavidin conjugate
(Carson, 1990). After rinsing
in PBS, Fast Red substrate was applied, and color development was stopped by
washing in water. The slides were counterstained with Mayer's hematoxylin,
washed in water, and cover slipped with Dako glycergel (Carpinteria, CA, USA).
A sample of approximately 200-400 artifact-free and well-stained fibers was
counted from each section using a camera-mounted microscope attached to a PC
loaded with BIOQUANT software (Bioquant, Nashville, TN, USA) to determine the
percentage of Type I, Type IIa, and Type IIb fibers in each sample.
Statistical analysis
Statistical analysis was performed by analysis of variance (ANOVA) with
Tukey post-hoc tests (P<0.05, Sigmastat 2.0). Results are
presented as means ± S.E.M.
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Results |
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There were no significant differences in the volume densities of mitochondria (total volume density of mitochondria, 2.78±0.21% to 3.13±0.28%; volume density of subsarcolemmal mitochondria, 0.09±0.04% to 0.16±0.05%; volume density of interfibrillar mitochondria, 2.62±0.18% to 3.01±0.28%) or lipid droplets (0.06±0.05% to 0.16±0.07%) among the muscles of the Weddell seal (Table 1). However, the total volume density of mitochondria (5.9±0.5%), the volume density of subsarcolemmal mitochondria (0.6±0.1%) and the volume density of interfibrillar mitochondria (5.3±0.4%) in the hindlimb of the dog were significantly greater than in the muscles of the Weddell seal (Table 1). By contrast, there was no significant difference in the volume density of intracellular lipid droplets between the muscles of Weddell seals (0.06±0.05% to 0.16±0.07%) and dog hindlimb muscle (0.1±0.0%) (Table 1).
The M. longissimus dorsi, the principal swimming muscle of the seal, had a significantly greater fiber cross-sectional area and lower capillary density than the M. pectoralis and hindlimb muscle complex (Table 2). By contrast, there were no significant differences among the muscle groups of the Weddell seal in indices of capillary supply that were independent of fiber area (i.e. capillary-to-fiber ratio and average number of capillaries around a fiber) (Table 2). While the fiber cross-sectional areas of the Weddell seal muscles were significantly greater than those in the dog hindlimb, the indices of capillarity were significantly less (Table 2).
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Enzyme activities
As with the volume density of mitochondria, there were no significant
differences in the activities of the aerobic enzymes citrate synthase (CS) and
ß-hydroxyacyl CoA dehydrogenase (HAD) between swimming and non-swimming
muscles of the seals. However, CS activity and HAD activity in seal muscle
were significantly lower (47-50%) and significantly greater (2.3-4.1x),
respectively, than in dog hindlimb muscle
(Table 3). In the Weddell seal,
lactate dehydrogenase (LDH) activity, an index of anaerobic capacity, was
significantly lower (45%) in the hindlimb muscle complex than in the M.
pectoralis and was similar to that measured in the hindlimb of the dog. The
CS:HAD ratio, an index of the contribution of fatty acid metabolism to total
aerobic metabolism, ranged from 0.2 to 0.4 in the muscles of Weddell seals,
which indicated a complete reliance on fatty acids as the main fuel source for
aerobic metabolism. In other words, all of the carbon flux through the Krebs
cycle could be accounted for by carbon generated from fatty acid
catabolism.
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Myoglobin
The concentrations of myoglobin were not significantly different among
swimming muscles and non-swimming muscles of Weddell seals; however, they were
significantly greater (8-13-fold) than in the hindlimb of the dog.
Fiber type
There were significantly greater percentages of slow oxidative (Type I)
fibers in the swimming muscles (67±4.7% and 68.7±4.7% for the M.
longissimus dorsi and hindlimb, respectively) as compared with non-swimming
muscle (41.0±5.4% for the M. pectoralis) (Figs
3,
4). Conversely, there was a
significantly greater percentage of fast-twitch oxidative (Type IIA) fibers in
the non-swimming (58.8±5.4% for the M. pectoralis) as compared with the
swimming muscles (33.6±5.6% and 31.3±4.7% for the M. longissimus
dorsi and hindlimb, respectively). There was a complete absence of fast-twitch
glycolytic (Type IIB) fibers in the seal muscles. These results were
consistent using both the histochemical technique for myosin ATPase activity
and specific immunohistochemical staining for slow and fast myosin heavy chain
isoforms.
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Discussion |
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The mitochondrial volume densities and activities of aerobic enzymes in the
swimming muscles of Weddell seals were not significantly different from those
of the non-swimming muscles (Table
1). While the total volume density of mitochondria did not differ
from that of sedentary terrestrial mammals, the distribution of the
mitochondria throughout the muscles did. As shown previously for shallow
divers, the majority of mitochondria were interfibrillar, with less than 5% of
total mitochondrial volume density being subsarcolemmal. By contrast,
subsarcolemmal mitochondria usually account for 10% of the total
mitochondrial volume in sedentary terrestrial mammals and in excess of 30% in
athletic species (Kayar et al.,
1989
). Thus, while Weddell seal total volume density of
mitochondria did not differ from that of terrestrial mammals, there was a
difference in the distribution of mitochondria throughout the muscle. Another
similarity between the short- and long-duration divers was a reliance on fatty
acids as their principal fuel source for aerobic metabolism, as evidenced by
CS:HAD ratios, which were substantially less than 1
(Table 3).
Although the muscles of Weddell seals appear to be geared for lipid-based,
aerobic metabolism, they have a substantially reduced capillary supply
compared with that of terrestrial mammals. Kanatous et al.
(2001) found that the
capillary density in the swimming muscles of harbor seals was approximately
50% less than in hindlimb muscles of dogs with similar volume densities of
mitochondria. The same pattern is found in Weddell seals [body
mass=404±13.5 kg; QA(0)=259.8±26.7
mm2] compared with terrestrial mammals of comparable size, such as
the horse Equus equus [mean body mass=447±36 kg;
QA(0)=926±36 mm2] or the steer [mean
body mass=474±12 kg; QA(0)=727±6
mm2] (Kayar et al.,
1992
). This paradoxical result (a high reliance on aerobic
metabolism with diminished capillary supply) is explained by the high
concentration of myoglobin in Weddell seal muscles
(Table 3). The elevated
myoglobin concentrations appear to offset the diminished capillary supply by
increasing both the endogenous oxygen storage capacity and the apparent
intracellular diffusing capacity of oxygen in the muscle, allowing aerobic
metabolism to be maintained under the conditions of low oxygen partial
pressures and ischemia resulting from the dive response.
Interestingly, the fiber-type distribution of the Weddell seals, as
determined by a metachromatic ATPase-based stain and immunohistochemical
techniques, revealed a complete absence of fast-twitch glycolytic fibers (type
IIB) in both swimming and non-swimming muscles (Figs
3,
4). To our knowledge, this is
the first reported muscle-fiber-type analysis for Weddell seals. Previous
studies on short-duration divers found a generally mixed fiber type that
included all three major fiber classifications in the muscles
(Reed et al., 1994;
Kanatous et al., 1999
). The
fiber-type profile of the Weddell seals was consistent with our results for
LDH activity, an indicator of anaerobic capacity. The hindlimb was composed of
69% slow oxidative fibers and had the lowest activity of LDH, followed by
the M. longissimus dorsi and then the M. pectoralis, which had only 41% slow
oxidative fibers and the highest LDH activity
(Fig. 4,
Table 3). While the lack of
Type IIB fibers in muscles of Weddell seals was unexpected, it adds support to
the hypothesis that they preferentially maintain low levels of aerobic
metabolism during the majority of dives.
With advances in minimizing the size of electronics, an animal-borne video
system and data recorder was developed and deployed on different species of
marine mammals, including Weddell seals. This system permitted direct
observation of diving behavior and foraging strategy
(Davis et al., 1999;
Williams et al., 2000
),
allowing assessment of the locomotor strategies used by marine mammals
throughout their dives. Simultaneous measurements of metabolic rate and
flipper-stroke frequency provided new information on energy-conserving modes
of locomotion in diving Weddell seals. In previous studies, swim speed was
considered to be an important indicator of energetic cost. However, we
observed different energetic costs for gliding (no stroking) during descent,
intermittent stroking during horizontal travel and continuous stroking during
ascent, even though these activities occur at similar speeds (
2.0 m
s-1). Prolonged gliding (1-9 min in duration), which appears to be
associated with changes in buoyancy during descent, resulted in significant
energy savings. In addition to Weddell seals, flipper-stroke frequency and
swim speed have been measured in a northern elephant seal (Mirounga
angustirostris) and bottlenose dolphins (Tursiops truncatus)
(Williams et al., 2000
). These
two species also exhibited periods of prolonged gliding during descent.
Although swim speed has been used as an indicator of effort, our research has
shown stroke frequency to be a better indicator of energy expenditure. It
showed that marine mammals take advantage of a number of physical properties
of the aquatic environment and use behavioral strategies that reduce the
energetic costs of diving (e.g. up to 59.6% in Weddell seals) and that deep
divers employ these techniques more often or to a greater extent than do
shallow divers. These energetic savings allow marine mammals to increase their
dive durations beyond their predicted ADL (based on calculations of oxygen
consumption and body oxygen stores) and achieve remarkable depths despite
limited oxygen availability during diving
(Williams et al., 2000
).
In summary, the skeletal muscles of Weddell seals do not have enhanced aerobic capacities compared with those of terrestrial mammals but are adapted to maintain low levels of an aerobic lipid-based metabolism, especially under the hypoxic conditions associated with diving. This is in contrast to shorter-duration, active divers that exhibit aerobic capacities in their skeletal muscles that are similar to those of athletic terrestrial mammals. In other words, the combination of diving hypoxia and moderate levels of exercise appears to enhance aerobic capacity in their muscles to a degree seen in terrestrial animal athletes such as the dog and pony with much greater exercise capacity. The lower aerobic capacity of Weddell seal muscle reflects their energy-conserving modes of locomotion (via extended periods of gliding during descent) that enable longer, deeper dives. Based on these results, the skeletal muscles of marine mammals appear to adapt to the hypoxic conditions of diving in different manners.
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Acknowledgments |
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