|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 13, 2009
Journal of Experimental Biology 212, 673-683 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023481
Skeletal muscle fiber types in the ghost crab, Ocypode quadrata: implications for running performance
1 Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260,
USA
2 Department of Exercise and Nutrition Sciences, University at Buffalo, Buffalo,
NY 14260, USA
Author for correspondence (e-mail:
smedler{at}buffalo.edu)
Accepted 4 December 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: ghost crab, muscle fiber type, skeletal muscle
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
; Hafemann and Hubbard,
1969) and at slower speeds can maintain activity for more than an
hour without rest (Full,
1987). Sustained slow locomotion relies on aerobic metabolism
whereas short sprints depend on glycolytic muscle activity and result in
lactic acid build-up (Full,
1987). The factorial scope of exercising ghost crabs
(approximately 8–12 times the resting metabolic rate) is more typical to
that of active ectothermic vertebrates and mammals than to an invertebrate
(Full, 1987;
Full and Herreid, 1983).
Biomechanical studies reveal that in spite of some obvious differences between
crabs and terrestrial vertebrates, ghost crabs exhibit many of the same
features of locomotion as other running animals. These include similar stride
frequencies during running and a trot-to-gallop transition at critical speeds
(Blickhan and Full, 1987;
Full and Weinstein, 1992).
Crustacean muscles exhibit an array of diverse physiological properties and it
is therefore relevant to ask how skeletal muscle organization might relate to
running performance. Furthermore, it is currently unknown how the kinetic
properties of skeletal muscles that power ghost crab locomotion might compare
with the more extensively studied contractile parameters of terrestrial
vertebrate muscles.
In spite of the fact that running capability is derived directly from the
specific contractile and metabolic capacities of skeletal muscles in the ghost
crab's legs and thorax, very little is known about the cellular and molecular
organization of their muscles. Decapod crustaceans exhibit a wide variety of
muscle fiber types specialized for a variety of specific functions
(Atwood, 1976;
Silverman et al., 1987).
Several different fiber types have been identified based on differences in
fiber ultrastructure, histochemistry, contractile properties and myofibrillar
isoform composition. Studies classifying discrete fiber types based on ATPase
histochemistry have identified four fiber types in decapod crustaceans
(Gruhn and Rathmayer, 2002;
Gunzel et al., 1993;
Rathmayer and Maier, 1987).
Using myofibrillar isoform assemblages to classify specific fiber types, at
least three different fiber types can be recognized: slow phasic
(S1), slow tonic (S2) and fast
(Medler et al., 2004;
Medler and Mykles, 2003;
Mykles, 1985;
Mykles, 1988). Each of these
types possesses a different myosin heavy chain, as well as different isoforms
of tropomyosin, troponin T (TnT), troponin I (TnI) and others. More recently,
two types of fast fibers have been identified in a species of crayfish that
exhibit differences in sarcomere width, TnI isoform composition and
Ca2+ activation properties
(Koenders et al., 2004). There
is currently no common basis for integrating the different crustacean muscle
classification schemes into discrete fiber types but it is clear that a number
of distinct fiber types exist and that these types are functionally diverse.
In fact, classifying crustacean muscles into discrete categories may not
reflect the continuum of fiber types actually present in crustacean muscles
(Medler et al., 2004).
In the current study, we investigated the organization of the extensor and flexor carpopodite muscles that comprise the muscle mass within the meropodite (Fig. 1). These antagonistic muscles alternately extend and flex the distal segments of the crab legs during running and represent some of the primary muscles involved in locomotion. Our goal was to define physiologically relevant parameters that are known to affect muscle function in other crustacean muscles. These included identification of alternate isoforms of myofibrillar proteins, as well as fiber type – specific aerobic potential identified through histochemical methods. In conjunction with these studies, we made measurements of running speed and stride frequency in crabs that ranged in size from 200 mg to 75 g. We also used kinematic analyses of digital video recordings from running crabs to estimate muscle shortening velocities in vivo. Based on these analyses, we report that the active shortening properties of the muscles are about that expected for a similar sized mammal but substantially slower than a comparably sized lizard. In addition, we identify specializations in muscle fiber type that may help explain how the muscles are used for different running speeds. Finally, we report a significant level of complexity in the myofibrillar isoform assemblages present in different muscle fiber types. These include three alternate isoforms of myosin heavy chain (MHC), TnT and TnI expressed in different combinations in single fibers. We discuss the possible significance of these different fiber types within the broader context of integrated locomotory function.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Determination of muscle shortening velocity in vivo
We recorded crabs with a digital video camera (60 Hz) while they were
running on a treadmill. A small spot of red paint was applied to the distal
end of the meropodite and to the distal end of the propopodite of the second
walking leg. Treadmill speed was increased gradually until animals reached a
trot-to-gallop transition. Stride frequency and angular velocities of the
second walking leg were determined afterward either by visual observation of
individual frames (stride frequency) or by using motion analysis software for
angular velocities [MotusTM v. 8.4 motion analysis software (Vicon-Peak,
London, UK)].
We determined the amounts of muscle strain corresponding to angular changes in the meropodite–carpopodite joint by manually rotating the leg through its range of motion by pulling on the central tendon (apodeme) with a pair of forceps. The amount of apodeme movement (mm) was measured under a stereomicroscope as the leg joint was flexed or extended. The simple geometry of the flexor and extensor muscles allowed us to directly convert apodeme movement into absolute muscle shortening. Muscle fiber lengths were measured with digital calipers and absolute muscle shortening was converted to relative muscle shortening (% of fiber length). We assumed that the fiber lengths measured in dissected legs approximated those of resting fiber lengths in vivo.
We estimated muscle shortening velocity in two ways. First, we measured
angular velocities from digital video recordings of running crabs and then
used these to estimate corresponding muscle shortening velocities as described
above. Second, for muscles shortening with a sinusoidal strain trajectory, the
frequency of contraction is approximated as one half of the ratio of muscle
shortening velocity to the total strain amplitude
(Medler and Hulme, 2009).
Using the strain amplitudes determined from our kinematic analyses, we were
then able to estimate muscle shortening velocities as a function of
contractile frequency. To estimate maximal shortening velocity
(Vmax), we assumed that the muscles operating in
vivo were shortening at approximately 30% of Vmax
(Josephson, 1993;
Rome and Lindstedt, 1997).
Muscle tissue preparation
Crab legs were quickly frozen by rapid immersion in liquid N2
and stored at –80°C for later analysis. For protein analyses, we
removed the exoskeleton and cuticle from the anterior and posterior regions of
the meropodite and frozen legs were freeze-dried under vacuum. The dried legs
with muscles intact were stored at –20°C in sealed plastic tubes
containing desiccant. This procedure preserves the anatomical orientation of
the fibers and allows for the removal and analysis of individual muscle
fibers. For the isolation of RNA, whole pieces of frozen muscle were cut with
a razor blade and the tissues were processed immediately.
Measurement of muscle fiber lengths and fiber pinnation angles
To determine fiber lengths, we removed individual dried muscle fibers from
intact muscles from different regions along the length of the muscle (proximal
mid distal). The point of insertion was measured as the distance
from the proximal end of the meropodite. Fiber lengths were measured to the
nearest millimeter with electronic calipers (VWR Bridgeport, NJ, USA). Fiber
lengths were compared statistically using an unpaired t-test, pooling
values from the proximal and distal regions and comparing their lengths with
those from the mid-region.
Muscle fiber pinnation angles were measured from frozen muscles, where the
carpopodite–propopodite joint was in a neutral position (
90 deg. as
in Fig. 1A). Multiple angles
were measured for each extensor and flexor muscle and these were averaged for
each individual. Three crabs of different sizes (9.7, 20.6 and 55 g) were used
for these measurements and there was no indication that insertion angles
varied with animal size.
Determination of sarcomere length
We dissected muscles in a Ringer's solution free of Ca2+ and
containing EDTA to promote relaxation. Small bundles of fibers were removed
from different anatomical regions of the muscles and placed in a small
reservoir of the Ringer's solution on a microscope slide and observed at a
final magnification of x200 with either DIC optics or with the condenser
diaphragm closed down to increase contrast. Images of the fibers were captured
digitally and then printed. Mean sarcomere length was calculated by measuring
the distances across several sequential sarcomeres (10) along a fiber. We
calibrated distances with a stage micrometer placed in the same position as
the muscle fibers. In addition, sarcomere lengths were measured from
histochemically stained muscle sections.
Analysis of myofibrillar proteins by SDS PAGE and western blotting
Individual fibers were removed from different anatomical regions of
freeze-dried muscles and placed directly in 30–50 µl of SDS sample
buffer [62.5 mmol l–1 Tris-HCl (pH 6.8), 12% glycerol, 1.25%
SDS and 1.25% β-mercaptoethanol] in 1.5 ml microcentrifuge tubes. We then
homogenized fibers in the sample buffer using a plastic hand-held pestle that
fitted directly into the tube. Aliquots of the fiber samples (5 µl) were
loaded on PAGE gels.
For resolution of MHC isoforms, samples were separated on 16 cm gels for 24
h under constant voltage (200 V) at 8°C following the general protocol of
Blough and colleagues (Blough et al.,
1996). Resolving gels consisted of 8% polyacrylamide (200:1
acrylamide:N,N'methylenebisacrylamide) and 12% glycerol. Stacking gels
were composed of 4% polyacrylamide (20:1
acrylamide:N,N'methylenebisacrylamide) and contained no glycerol. After
completion of electrophoresis, gels were fixed overnight in 50% methanol and
silver stained according to the methods of Wray and colleagues
(Wray et al., 1981). To
determine the ratio of multiple MHC isoforms co-expressed in single fibers, we
measured the relative amount of each protein for 40 fibers taken from four
crabs of different sizes (6.2, 8.75, 18.7 and 50 g) using densitometry of
scanned gels (NIH Image 1.63).
Isoforms of TnT, TnI and P75 were identified through SDS-PAGE and western blotting procedures. Samples were separated on gels containing 8% acrylamide (37.5:1 acrylamide: N,N'methylenebisacrylamide) under constant voltage (200 V) for 45 min, using a Bio-Rad Mini-Protean 3 gel apparatus (Hercules, CA, USA). Separated proteins were then transferred to PVDF membranes under constant voltage (100 V) for 1 h at 4°C.
Blots were probed for TnT, TnI or P75 using polyclonal antibodies raised in
rabbits against these proteins isolated from lobster muscles
(Medler and Mykles, 2003;
Medler et al., 2004;
Medler et al., 2007). After
transfer of proteins, membranes were washed in deionized water and then
allowed to air dry and stored for up to several days before probing with
antibodies. Dried membranes were wet in 100% methanol for 30 s and then washed
in deionized water for 5 min. They were then blocked in a solution of 2%
non-fat dry milk in a Tris-buffered saline solution (20 mmol
l–1 Tris, 500 mmol l–1 NaCl, pH 7.5)
containing 0.05% Tween detergent (TTBS) for 1 h. Blots were incubated in the
same milk solution containing anti-TnT, anti-TnI or anti-P75 antibody
(1:10,000 or 1:20,000 dilution) for 1 h. Blots were then washed three times (5
min per wash) in TTBS. They were next incubated in anti-rabbit biotin IgG
(Vector Labs, Burlingame, CA, USA; 1:5000 dilution) for 1 h and again washed
three times in TTBS. Blots were then incubated in an avidin–biotin
complex conjugated to horseradish peroxidase (ABC solution; Vector Labs) for
30 min. Finally, antibody complexes labeling isoforms of TnT, TnI or P75 were
visualized by chemiluminescent detection
(Covi et al., 1999).
Isolation of cDNAs encoding MHC isoforms
The 3' end of three MHC isoforms [encoding a portion of the rod
region of the MHC molecules and the 3' untranslated region
(3'-UTR)] were cloned using a 3'-RACE procedure. RNA was isolated
from the muscles of crab legs with TRIzol reagent (Invitrogen Inc., Carlsbad,
CA, USA) and used for first strand cDNA synthesis. The first strand synthesis
reaction was carried out using Super Script II RNase-Reverse Transcriptase
(Invitrogen Inc.) and contained 100 ng of total RNA, 2.5 µmol
l–1 dNTP, 1x first strand buffer, 5 mmol
l–1 dithiothreitol (DTT), 2.5 units of RNase inhibitor and 1
µl of 10 µmol l–1 adapter primer (Invitrogen adaptor
primer, Cat. No. 10542 – 017):
5'-GGCCACGCGTCGACTAGTAC(T)17-3'. Next, a PCR was performed using
the cDNA from the first strand synthesis as a template. The forward primer was
designed to anneal to a conserved region of the MHC, as deduced by analysis of
three lobster MHC isoforms: 5'-GAAGGCTAAGAAGGCCATGGTTGA-3'. The
reverse primer was designed to anneal to a sequence within the adapter primer:
5'-GGCCACGCGTCGACTAGTAC-3'. The PCR reaction consisted of 95°C
(5 min) [60°C (30 s), 72°C (30 s), 95°C (30 s)] repeated for 35
cycles and 72°C (10 min). Takara Ex-Taq-HS was used for all PCR reactions
(Takara Bio USA, Madison, WI, USA). PCR products were identified on 1% agarose
gels stained with ethidium bromide. These bands were gel purified and cloned
into the pCR 2.1 TOPO plasmid using the TOPO TA cloning kit (Invitrogen Inc.).
Transformed bacteria were grown overnight on pre-warmed LB plates containing
ampicillin (100 µgml–1) coated with X-gal. We identified
colonies with PCR inserts by their white color in blue/white screening and the
presence of inserts was confirmed by PCR using vector primers (M13 reverse and
T7). Several positive clones of each isoform were identified and sequenced by
the DNA sequencing lab at Roswell Park Cancer Center (Buffalo, NY, USA).
Histochemical identification of metabolic fiber types
The NADH–diaphorase reaction was used to identify aerobic fibers in
the crab leg muscles. A solution containing NBT (0.8 mg ml–1)
and NADH (0.64 mg ml–1) in 50 mmol l–1
sodium phosphate (pH7.3) was made fresh. Frozen muscles were embedded in OCT
compound (Sakura Finetek, Torrance, CA, USA) and sections (10–20µm)
were cut on a Cryostat (Leica Microsystems, Wetzlar, Germany). Sections were
mounted on microscope slides and stored at –20°C until ready for
staining. Prior to staining, sections were thawed and allowed to air dry for
15–50 min. Sections were then rinsed quickly by immersing in deionized
water to remove any residual OCT compound. Tissues were then covered with the
NBT/NADH solution and incubated in the dark at room temperature for 30 min.
After staining, sections were rinsed in several washes of deionized water and
then dehydrated through a graded ethanol series. Sections were then covered
with mounting medium and covered with a coverslip. We identified fibers with
positive staining regions by their distinct purple color.
Statistical analyses
Linear regression analyses were performed on running velocity and frequency
as a function of body mass, with all values being log-transformed prior to
analysis. An unpaired t-test was used to compare the lengths of
muscle fibers from different muscle regions. In all cases, significance was
accepted at 
<0.05. For descriptive statistics, values are
means±standard deviation unless otherwise noted. All statistical
analyses were performed with Statview 5.0.1 (SAS Institute, Cary, NC,
USA).
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Two distinct fiber types comprise both the extensor and flexor muscles. The proximal and distal fibers of the extensor and flexor are visibly light brown to pink in coloration whereas those occupying the mid-regions of the muscles are distinctly white. These differences are consistent with the oxidative capacity of the muscles, as indicated by NADH–diaphorase histochemistry. The pigmented fibers stain intensely from this reaction, indicating high numbers of mitochondria within the fibers (Fig. 2). These fibers also appear to possess internal subdivisions similar to those reported for other aerobic crustacean muscles (membrane in-folding) (Fig. 2B). The fibers from the mid-regions of the muscles were much larger in diameter and showed weak staining with the NADH–diaphorase reaction, which is limited to the fiber periphery (Fig. 2C). In addition, the proximal and distal fibers are significantly shorter than those of the mid-region (P<0.0001) (Fig. 3). Sarcomere lengths within fibers were variable and depended on the anatomical location of the fibers. The large diameter fibers of the muscle mid-region possessed mean sarcomere lengths of 3.5±0.4 µm (range: 2.65–4.0 µm) whereas the proximal and distal fibers were more variable. Within the proximal and distal muscle regions, some of the fibers possessed short sarcomeres like the mid-region fibers but most had longer sarcomeres, with a mean length of 6.2±2.3 µm (range: 4–10 µm).
|
|
Running performance
Running speed was significantly correlated with body size in ghost crabs
ranging in size from 200 mg to 63 g, with the fastest animals attaining speeds
of just over 100 cm s–1
(Fig. 4A). Running speed was a
function of mass0.31, which means that change in speed as a
function of size became less among the larger crabs
(Fig. 4A, inset). In the
current study, speed became essentially size independent in crabs of 30 g and
larger (86.7±11.9 cm s–1; N=14). By contrast,
stride frequency declined as a function of body size in crabs, with the
smallest crabs (10 g) measured reaching frequencies of just over 8 Hz
whereas larger crabs (60–70 g) ran at approximately 5 Hz
(Fig. 4B). Overall, frequency
was found to be a function of body mass–0.13. Comparison with
stride frequencies of a sister species of ghost grab, Ocypode
ceratopthalma, revealed a similar pattern of declining frequencies in
larger animals, although the slope of the relationship was considerably
steeper (mass exponent=–0.49) (Fig.
4C) [data for O. ceratopthalma derived from Burrows and
Hoyle (Burrows and Hoyle,
1973)].
|
Muscle shortening velocity and strain in vivo
Shortening velocities of the extensor and flexor carpopodite muscles were
determined from angular velocity during running and from muscle geometry
(Fig. 5). Alternatively, we
used stride frequency to estimate shortening velocity, assuming that the
shortening cycle was sinusoidal. Using the former method, we found shortening
velocities ranged from 0.8–3.1 muscle lengths per second (l
s–1) in crabs ranging in size from 10–55 g. For
estimates directly from stride frequencies, shortening velocities ranged from
1.1–3.2 l s–1 in crabs ranging from
4.3–62.7 g. Shortening velocity declined significantly as a function of
body size using either method of estimation (data not shown). To provide a
single point of comparison, we estimated the shortening velocity of both
muscles for a mouse-sized crab (25 g;
Table 1). We estimated maximum
shortening velocity (Vmax) by assuming that the muscles
were operating near a shortening velocity that would provide maximum power
output (30% of Vmax). Vmax for
the extensor carpopodite was estimated to be approximately 4.6–4.7
l s–1, depending on which method was used to
estimate shortening velocity in running crabs. For the flexor carpopodite, our
estimate of Vmax was 5.6–7 l
s–1. The mean muscle strain during rapid running was
11.6±5.8% of muscle length for the extensors and 17.6±8.8% for
the flexors.
|
|
Myofibrillar isoforms
We identified several isoforms of myofibrillar proteins that differed among
fiber types in ghost crab muscles. These included multiple isoforms of MHC,
TnT and TnI (Fig. 6). There
were at least three isoforms of MHC, three of TnT and two of TnI.
|
|
; Medler et al.,
2004). We believe that these three sequences encode the MHC
proteins identified in the muscles but further work is needed to establish
which nucleotide sequence encodes each of the three proteins.
|
|
We identified three isoforms of TnT, which mirror the expression of MHC isoforms (Fig. 6B; Fig. 9). TnT3 was expressed in the mid-region fibers whereas TnT2 was expressed in the more proximal and distal fibers of both large and small crabs. In largest crabs (>50 g), the TnT1 isoform was co-expressed with TnT2. Based on our analyses, we believe that the fibers expressing TnT1 are likely to be the same fibers that express the MHC2 isoform.
|
|
;
Medler and Mykles, 2003;
Mykles, 1985). We did not
detect any body size related patterns in the expression of this protein.
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Tse et al., 1983). Overall,
the fiber type distribution is essentially the same as that described for the
flexor carpopodite of the crab, Carcinus maenas
(Parsons, 1982;
Parsons and Mosse, 1982). In
C. maenas, the more aerobic fibers are slower in their intrinsic
contractile properties whereas those of the mid-region are fast
(Parsons, 1982). The extensor
muscles of crayfish, Procambarus clarkii and lobsters, Homarus
americanus, also exhibit a similar pattern of fiber type distribution,
where slow tonic (S2) fibers are concentrated at the proximal and
distal regions of the muscles (Mykles et
al., 2002). These S2 fibers exhibit a high degree of
post-synaptic facilitation and are presumably used for sustained postural
control (Mykles et al.,
2002).
Locomotion kinetics
The exercise physiology of invertebrates has received far less attention
than that of vertebrate animals, yet common patterns of function revealed
through comparative studies provide a deeper understanding of the foundational
principles of physiological function. Ghost crabs exhibit remarkable athletic
capabilities that rival those of comparably sized mammals, with maximum
running speeds approaching 4 m s–1 and an aerobic scope of
nearly 12 (Full and Herreid,
1983; Full and Weinstein,
1992; Hafemann and Hubbard,
1969). In the current study, running speed in O. quadrata
increased as a function of mass0.31, meaning that running speed
increases as crabs get larger but then levels off at a maximum of just over 1
m s–1 for crabs of 30 g and larger
(Fig. 4, inset). These values
are similar to those previously reported for O. quadrata
(Blickhan and Full, 1987;
Blickhan et al., 1993),
although maximum running speeds of up to 3–4 m s–1 have
been reported for a sister species of ghost crab, O. ceratophthalma
(Hafemann and Hubbard, 1969).
Running surfaces significantly affect maximum speeds attained by ghost crabs
(Hafemann and Hubbard, 1969;
Pennisi, 2007) and the smooth
surface of our treadmill probably limited maximum speeds attained by our
crabs. Comparably sized rodents and lizards are able to reach speeds of
approximately 3.7 m s–1
(Djawdan and Garland, 1988;
Marsh, 1988;
Marsh and Bennett, 1986b)
(Table 1). Stride frequencies
of the crabs are similar to those of mammals, with 25 g crabs and mice
operating at approximately 6–8 Hz whereas running lizards are able to
operate at higher frequencies of up to approximately 15 Hz
(Marsh, 1988;
Marsh and Bennett, 1986b)
(Table 1). The stride
frequencies of O. ceratophthalma are higher than those of O.
quadrata, with maximum frequencies of 20 Hz, as compared with 8 Hz for
O. quadrata (Burrows and Hoyle,
1973). Stride frequency as a function of body mass in O.
ceratophthalma is biphasic, with the smallest crabs (0.1–5 g)
exhibiting a size-independent frequency of approximately 15 Hz. For larger
crabs (10–50 g), there is a steeper decline in frequency as a
function of body mass than that observed for O. quadrata
(Fig. 4C). These larger O.
ceratophthalma showed a decline in frequency as a function of
mass–0.49 [frequency (Hz) = 38.9 x mass
(g)–0.49; R2=0.83; P<0.0001].
Overall, when compared with similar sized vertebrates, ghost crabs exhibit
slower running speeds but quite similar stride frequencies
(Table 1).
The simple pinnate muscle organization of the extensor and flexor
carpopodite muscles facilitated the estimation of muscle shortening parameters
from running crabs. Based on these measurements, we estimate that in a
mouse-sized crab the extensor muscles have a maximum shortening velocity of
approximately 4 l s–1 (3.8–4.6 l
s–1) (Table 1)
whereas the flexors possess a Vmax of approximately 6
(5.6–7 l s–1)
(Table 1). The mouse soleus,
which is composed of a mixture of type I and IIa fibers, has a
Vmax of approximately 6 l s–1
whereas the exclusively fast muscles of mice and lizards have a
Vmax of approximately 12 and 20 l
s–1, respectively (Askew
and Marsh, 1997; Marsh,
1988; Marsh and Bennett, 1986a)
(Table 1). Overall, the
contractile properties of the muscles used for running by the ghost crabs are
slightly lower than those of a comparably sized mammal but substantially lower
than those of a similar sized reptile. However, the estimates for the lizard
muscles were made at temperatures almost 20 deg. higher than for the mouse or
the crab and these temperatures probably explain the higher values. Skeletal
muscles from diverse species exhibit shortening velocities that differ by more
than 100-fold and the velocities of fast and slow fibers from a single species
frequently differ by up to 10-fold
(Medler, 2002). By comparison,
the relatively minor differences in kinetic properties of muscles driving
terrestrial locomotion in the evolutionarily divergent species examined here
suggests that common design constraints have shaped the organization of these
muscles.
Muscle fiber types
Although the architecture of the extensor and flexor carpopodite is that of
a simple pinnate muscle, the fiber types present in the different regions of
the muscles are complex. It is not currently possible to fit the muscle fibers
in the legs of the ghost crab into discrete fiber type categories of other
crustaceans. One common classification scheme defines one fast and two slow
fiber types [slow phasic (S1) and slow tonic (S2)] based
on different isoforms of the myofibrillar proteins (MHC, P75, TnT, TnI) and on
histochemistry (Medler and Mykles,
2003; Medler et al.,
2004; Mykles,
1985; Mykles,
1988). The MHC protein isoforms identified through SDS-PAGE are
clearly different between lobster muscles and crab muscles, and the gene
sequences from the crab muscles do not offer many clues as to the similarity
to other crustacean MHCs. Overall, all three identified sequences share
80% similarity to each of the identified lobster (Homarus
americanus) MHC isoforms (fast, S1, S2) within the
coding sequence but none of the O. quadrata sequences matches
preferentially with any of the H. americanus sequences. A unique
pattern observed in the mid-region fibers is the apparently invariant
co-expression of two MHC isoforms in single fibers. Single fibers exhibiting
polymorphic, or hybrid, expression of multiple MHC isoforms are not uncommon
but these are often variable in the levels of different isoforms present. In
all of our analyses, the MHC1 and MHC3 are present in
single fibers in a highly consistent 1:1 ratio. The physiological significance
of polymorphic fibers is currently still a matter of debate. Pette and
colleagues interpreted these fibers as being transient in nature, being in the
process of switching from one fiber type to another
(Pette and Staron, 2000;
Pette and Staron, 2001). More
recently, the common occurrence of these fibers has called this interpretation
into question, raising the possibility that these intermediate fibers types
provide a continuum of mechanical properties to the muscles
(Caiozzo et al., 2003;
Medler et al., 2004;
Stephenson, 2001). Indeed,
single fibers containing two MHC isoforms possess contractile properties
intermediate to pure fiber types (Andruchov
et al., 2004; Caiozzo,
2002; Caiozzo et al.,
2003). In the case of the mid-region fibers in the extensor and
flexor carpopodite, it is unclear what advantage might be gained by the
roughly equal expression of two isoforms, rather than expressing a single
isoform. Nevertheless, many types of crustacean fibers co-express multiple
isoforms of other myofibrillar proteins, including TnI and TnT.
In lobster muscles, different isoforms of TnT and TnI provide convenient
markers of fiber type and in ghost crab muscles differences also exist in
terms of fiber type–specific isoform expression. However, the lobster
isoforms do not appear to correspond in a simple way to the ghost crab
isoforms. In lobster muscles, three isoforms of TnT are present, with fast
muscles expressing T2, S1 muscles expressing
T3 and S2 muscles expressing some combination of
T1 and T3. In the leg muscles of the ghost crab, the
mid-region fibers express T3 whereas some proximal and distal
fibers express T2 and some express both T1 and
T2. Overall, the expression of the T1 isoform is much
more restricted than the other two isoforms but when present is always in the
proximal and distal regions of the muscles. Therefore, the ghost crab fibers
expressing the T1 isoform might share similarities with
S2 fibers in other crustaceans. Fewer similarities are shared
between lobster and ghost crab fiber types for TnI isoforms. In the lobster,
five TnI isoforms exist and are expressed in different proportions in
different fiber types. In the crab muscles, we have only identified two
isoforms. Both are expressed in the different fiber types, although the
mid-region fibers tend to have a greater proportion of the I1
isoform (Fig. 10A). Finally,
in lobster and crayfish muscles the 75 kDa protein (P75) is only found in fast
muscle types (Costello and Govind,
1984; Medler and Mykles,
2003; Mykles,
1985; Mykles et al.,
2002). Based on western blots, this protein is present in both the
proximal/distal and mid-region fibers of the ghost crab muscles
(Fig. 11). In the
corresponding leg muscles in lobsters and crayfish, these are composed
entirely of either S1 or S2 fiber types, none of which
express P75 (Mykles et al.,
2002). The presence of P75 in these fibers raises the possibility
that both fiber types represent fast fibers but perhaps with different levels
of contractile speed. This would be similar to the pattern observed in
mammalian muscles, which possess a single slow fiber type and several fast
fiber types (Pette and Staron,
2000; Schiaffino and Reggiani,
1996). Preliminary analyses using ATPase histochemistry are
consistent with this possibility, as acid pre-incubation of muscle sections
leads to differential staining of the proximal and mid-region fibers that both
stain intensely without acid treatment (not shown).
Several of the muscle parameters we observed suggest that these muscles
function as a two-geared system, with the more proximal and distal fiber
bundles functioning for slow, sustained locomotion and the mid-region fibers
being reserved for more explosive power output during sprinting. There are
several indicators that the more proximal and distal fibers are slower
contracting fibers. First, the fibers are shorter than the mid-region fibers
(Fig. 3), which directly
impacts muscle shortening velocity, as velocity is directly proportional to
the number of sarcomeres operating in series
(Josephson, 1975). Second, the
muscles possess longer sarcomeres than the mid-region fibers (6 µm
vs 3.5 µm), a characteristic typical of slow fibers in
crustaceans. Sarcomere length is not simply a convenient descriptor of
shortening velocity but has direct effects on the mechanical properties of the
muscles. Long sarcomere fibers generate greater forces but at the expense of
velocity in comparison with fibers with short sarcomeres
(Josephson, 1975;
Taylor, 2000). Finally, these
fibers possess a distinct MHC isoform not present in the mid-region fibers
(Fig. 7). Although we do not
currently have any direct data about the ATPase activity of the different MHC
isoforms, we can hypothesize that the MHC2 is a slower isoform.
This is consistent with patterns of contractile speed in the different regions
of the flexor muscle in the crab, C. maenas
(Parsons, 1982). In terms of
aerobic capacity, the proximal and distal fibers possess greater mitochondrial
densities than the mid-region fibers. Furthermore, these fibers are
significantly smaller in diameter and possess fiber subdivisions, facilitating
the efficient delivery of oxygen during exercise. This type of muscle
organization is similar to the two-geared system in fish, where the slower
fiber types drive slow sustained swimming and the more powerful fast muscles
power escape responses (Rome et al.,
1988). Physiological measurements support this idea, as slow
locomotion can be sustained by the crabs for over an hour but endurance
capacity is greatly reduced at speeds greater than 30 cm s–1
and sprints can be sustained for only seconds
(Full, 1987;
Full and Herreid, 1983;
Full and Weinstein, 1992).
Exercise leads to rapid accumulation of lactate in ghost crabs but
intermittent exercise allows for prolonged activity and greater distance
capacity than prolonged slow locomotion
(Full, 1987;
Full and Weinstein, 1992;
Weinstein and Full, 1992).
Scale effects and muscle organization
Running ghost crabs exhibit a systematic decline in stride frequency as a
function of increasing body size (Fig.
4B,C). The slope of this decrease is less pronounced in O.
quadrata than in O. ceratophthalma but is similar in magnitude
to the general relationship known for quadrupedal mammals
(Heglund et al., 1974).
Interspecific differences in stride frequencies of running mammals are
associated with systematic shifts in the intrinsic contractile properties of
the skeletal muscles that produce the power to drive locomotion. In
particular, the rate of ATP hydrolysis and shortening velocity of orthologous
isoforms of MHCs from different species are fine-tuned to match the
appropriate rate of intrinsic muscle shortening
(Marx et al., 2006;
Medler, 2002;
Pellegrino et al., 2003;
Reggiani et al., 2000;
Seow and Ford, 1991). Similar
changes in the contractile properties of muscles from individuals of different
size within a single species also exist but the cellular and molecular
mechanisms responsible for these shifts in muscle physiology are poorly
understood. In this context, we found two notable differences in myofibrillar
isoform assemblage in muscle fibers as a function of body size in O.
quadrata. Although we have yet to define precise patterns of expression
correlated with specific sizes of crabs, some general trends are evident. Most
prominently, the expression of MHC2 and TnT1 is more
prevalent in larger crabs than in smaller animals. We also found a potential
difference in the expression of TnI, with lower expression of the
TnI2 isoform in the mid-region fibers of smaller crabs.
Functionally, differences in MHC isoform expression have clear implications
for muscle shortening velocity whereas differences in troponin isoforms may
affect the rates of muscle activation and deactivation
(Schiaffino and Reggiani,
1996). Myofibrillar isoform composition is clearly correlated with
physiology in other decapod crustacean muscles
(Koenders et al., 2004;
Mykles et al., 2002). We
propose that subtle shifts in contractile properties may stem from the
continuum of myofibrillar assemblage known to exist in crustacean muscle
fibers (Medler et al., 2004;
Mykles et al., 2002).
Developing lobster claw muscles exhibit a gradual shift in myofibrillar
isoform expression, as the muscles differentiate into specialized fast and
slow fibers over a period of months
(Medler et al., 2007). Further
studies are needed to understand precisely how these differences influence the
running capability of ghost crabs.
|
Footnotes |
|---|
* These authors contributed equally to this work 
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Andruchov, O., Andruchova, O., Wang, Y. and Galler, S.
(2004). Kinetic properties of myosin heavy chain isoforms in
mouse skeletal muscle: comparison with rat, rabbit, and human correlation with
amino acid sequence. Am. J. Physiol. Cell Physiol.
287,C1725
-C1732.
Askew, G. N. and Marsh, R. L. (1997). The effects of length trajectory on the mechanical power output of mouse skeletal muscle. J. Exp. Biol. 200,3119 -3131.[Abstract]
Atwood, H. L. (1976). Organization and synaptic physiology of crustacean neuromuscular systems. Prog. Neurobiol. 7,291 -391.[CrossRef][Medline]
Blickhan, R. and Full, R. J. (1987). Locomotion
energetics of the ghost crab II. Mechanics of the centre of mass during
walking and running. J. Exp. Biol.
130,155
-174.
Blickhan, R., Full, R. J. and Ting, L. (1993). Exoskeleton strain: evidence for a trot-gallop transition in rapidly running ghost crabs. J. Exp. Biol. 179,301 -321.[Abstract]
Blough, E. R., Rennie, E. R., Zhang, F. and Reiser, P. J. (1996). Enhanced electrophoretic separation and resolution of myosin heavy chains in mammalian and avian skeletal muscles. Anal. Biochem. 233,31 -35.[CrossRef][Medline]
Burrows, M. and Hoyle, G. (1973). The mechanism
of rapid running in the ghost crab, Ocypode ceratophthalma. J. Exp.
Biol. 58,327
-349.
Caiozzo, V. J. (2002). Plasticity of skeletal muscle phenotype: mechanical consequences. Muscle Nerve 26,740 -768.[CrossRef][Medline]
Caiozzo, V. J., Baker, M. J., Huang, K., Chou, H., Wu, Y. Z. and
Baldwin, K. M. (2003). Single-fiber myosin heavy chain
polymorphism: how many patterns and what proportions? Am. J.
Physiol. Regul. Integr. Comp. Physiol.
285,R570
-R580.
Costello, W. J. and Govind, C. K. (1984). Contractile proteins of fast and slow fibers during differentiation of lobster claw muscle. Dev. Biol. 104,434 -440.[CrossRef][Medline]
Covi, J. A., Belote, J. M. and Mykles, D. L. (1999). Subunit compositions and catalytic properties of proteasomes from developmental temperature: sensitive mutants of Drosophila melanogaster. Arch. Biochem. Biophys. 368, 85-97.[CrossRef][Medline]
Djawdan, M. and Garland, T. (1988). Maximal running speeds of bipedal and quadrupedal rodents. J. Mammal. 69,765 -772.[CrossRef]
Full, R. J. (1987). Locomotion energetics of
the ghost crab.1. Metabolic cost and endurance. J. Exp.
Biol. 130,137
-153.
Full, R. J. and Herreid, C. F. (1983). Aerobic response to exercise of the fastest land crab. Am. J. Physiol. 244,R530 -R536.[Medline]
Full, R. J. and Weinstein, R. B. (1992). Integrating the physiology, mechanics and behavior of rapid running ghost crabs: slow and steady doesn't always win the race. Am. Zool. 32,382 -395.
Gruhn, M. and Rathmayer, W. (2002). Phenotype plasticity in postural muscles of the crayfish Orconectes limosus Raf.: correlation of myofibrillar ATPase-based fiber typing with electrophysiological fiber properties and the effect of chronic nerve stimulation. J. Exp. Zool. 293,127 -140.[CrossRef][Medline]
Gunzel, D., Galler, S. and Rathmayer, W. (1993). Fiber heterogeneity in the closer and opener muscles of crayfish walking legs. J. Exp. Biol. 175,267 -281.[Abstract]
Hafemann, D. R. and Hubbard, J. I. (1969). On the rapid running of ghost crabs (Ocypode ceratophthalma). J. Exp. Zool. 170,25 -32.[CrossRef]
Heglund, N. C., Taylor, C. R. and McMahon, T. A.
(1974). Scaling stride frequency and gait to animal size-mice to
horses. Science 186,1112
-1113.
James, R. S., Altringham, J. D. and Goldspink, D. F. (1995). The mechanical-properties of fast and slow skeletal-muscles of the mouse in relation to their locomotory function. J. Exp. Biol. 198,491 -502.[Medline]
Johnson, L. K., Dillaman, R. M., Gay, D. M., Blum, J. E. and
Kinsey, S. T. (2004). Metabolic influences of fiber size in
aerobic and anaerobic locomotor muscles of the blue crab, Callinectes
sapidus. J. Exp. Biol. 207,4045
-4056.
Josephson, R. K. (1975). Extensive and intensive factors determining performance of striated muscle. J. Exp. Zool. 194,135 -154.[CrossRef][Medline]
Josephson, R. K. (1993). Contraction dynamics and power output of skeletal-muscle. Ann. Rev. Physiol. 55,527 -546.[CrossRef][Medline]
Koenders, A., Lamey, T. M., Medler, S., West, J. M. and Mykles, D. L. (2004). Two fast-type fibers in claw closer and abdominal deep muscles of the Australian freshwater crustacean, Cherax destructor, differ in Ca2+ sensitivity and troponin-I isoforms. J. Exp. Zool. 301A,588 -598.[CrossRef]
Marsh, R. L. (1988). Ontogenesis of contractile
properties of skeletal-muscle and sprint performance in the lizard
Dipsosaurus dorsalis. J. Exp. Biol.
137,119
-139.
Marsh, R. L. and Bennett, A. F. (1986b).
Thermal dependence of sprint performance of the lizard Sceloporus
occidentalis. J. Exp. Biol.
126, 79-87.
Marx, J. O., Olsson, M. C. and Larsson, L. (2006). Scaling of skeletal muscle shortening velocity in mammals representing a 100,000-fold difference in body size. Pflugers Arch. 452,222 -230.[CrossRef][Medline]
Medler, S. (2002). Comparative trends in
shortening velocity and force production in skeletal muscles. Am.
J. Physiol. Regul. Integr. Comp. Physiol.
283,R368
-R378.
Medler, S and Hulme, K. (2009). Power output from striated muscles performing cyclical contractions: patterns and constraints. Comp. Biochem. Physiol. A (in press).
Medler, S. and Mykles, D. L. (2003). Analysis
of myofibrillar proteins and transcripts in adult skeletal muscles of the
American lobster Homarus americanus: variable expression of myosins,
actin and troponins in fast, slow-twitch and slow-tonic fibres. J.
Exp. Biol. 206,3557
-3567.
Medler, S., Lilley, T. and Mykles, D. L.
(2004). Fiber polymorphism in skeletal muscles of the American
lobster, Homarus americanus: continuum between slow-twitch (S-1) and
slowtonic (S-2) fibers. J. Exp. Biol.
207,2755
-2767.
Medler, S., Lilley, T. R., Riehl, J. H., Mulder, E. P., Chang, E. S. and Mykles, D. L. (2007). Myofibrillar gene expression in differentiating lobster claw muscles. J. Exp. Zool. 307A,281 -295.
Mykles, D. L. (1985). Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages. J. Exp. Zool. 234, 23-32.[CrossRef][Medline]
Mykles, D. L. (1988). Histochemical and biochemical characterization of two slow fiber types in decapod crustacean muscles. J. Exp. Zool. 245,232 -243.[CrossRef][Medline]
Mykles, D. L., Medler, S., Koenders, A. and Cooper, R.
(2002). Myofibrillar protein isoform expression is correlated
with synaptic efficacy in slow fibres of the claw and leg opener muscles of
crayfish and lobster. J. Exp. Biol.
205,513
-522.
Parsons, D. W. (1982). The leg flexor muscle of Carcinus. I. Innervation and excitatory neuromuscular physiology. J. Exp. Zool. 224,157 -168.[CrossRef]
Parsons, D. W. and Mosse, R. L. (1982). The leg flexor muscle of Carcinus. II. Distribution of muscle fiber types. J. Exp. Zool. 224,169 -175.[CrossRef]
Pellegrino, M. A., Canepari, M., Rossi, R., D'Antona, G.,
Reggiani, C. and Bottinelli, R. (2003). Orthologous myosin
isoforms and scaling of shortening velocity with body size in mouse, rat,
rabbit and human muscles. J. Physiol.
546,677
-689.
Pennisi, E. (2007). Crab's downfall reveals a
hole in biomechanics studies. Science
315, 325.
Pette, D. and Staron, R. S. (2000). Myosin isoforms, muscle fiber types, and transitions. Microsc. Res. Tech. 50,500 -509.[CrossRef][Medline]
Pette, D. and Staron, R. S. (2001). Transitions of muscle fiber phenotype profiles. Histochem. Cell Biol. 115,359 -372.[Medline]
Rathmayer, W. and Maier, L. (1987). Muscle fiber types in crabs: studies on single identified muscle fibers. Am. Zool. 27,1067 -1077.
Reggiani, C., Bottinelli, R. and Stienen, G. J. M.
(2000). Sarcomeric myosin isoforms: fine tuning of a molecular
motor. News Physiol. Sci.
15, 26-33.
Rome, L. C. and Lindstedt, S. L. (1997). Mechanical and metabolic design of the muscular system in vertebrates. In Handbook of Physiology. Comparative Physiology, Section 13, Vol. 13, pp.1587 -1651. Bethesda, MD: American Physiological Society.
Rome, L. C., Funke, R. P., Alexander, R. M., Lutz, G., Aldridge, H., Scott, F. and Freadman, M. (1988). Why animals have different muscle fibre types. Nature 335,824 -827.[CrossRef][Medline]
Schiaffino, S. and Reggiani, C. (1996).
Molecular diversity of myofibrillar proteins: gene regulation and functional
significance. Physiol. Rev.
76,371
-423.
Seow, C. Y. and Ford, L. E. (1991). Shortening
velocity and power output of skinned muscle fibers from mammals having a
25,000-fold range of body mass. J. Gen. Physiol.
97,541
-560.
Silverman, H., Costello, W. J. and Mykles, D. L. (1987). Morphological fiber type correlates of physiological and biochemical properties in crustacean muscle. Am. Zool. 27,1011 -1019.
Stephenson, G. M. (2001). Hybrid skeletal muscle fibres: a rare or common phenomenon? Clin. Exp. Pharmacol. Physiol. 28,692 -702.[CrossRef][Medline]
Taylor, G. M. (2000). Maximum force production:
why are crabs so strong? Proc. Biol. Sci. B
267,1475
-1480.
Tse, F. W., Govind, C. K. and Atwood, H. L. (1983). Diverse fiber composition of swimming muscles in the blue crab, Callinectes sapidus. Can. J. Zool. 61, 52-59.
Weinstein, R. B. and Full, R. J. (1992). Intermittent exercise alters endurance in an eight-legged ectotherm. Am. J. Physiol. Regul. Integr. Comp. Physiol. 31,R852 -R859.
Wray, W., Boulikas, T., Wray, V. P. and Hancock, R. (1981). Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118,197 -203.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
