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
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
* Author for correspondence (e-mail: smedler{at}lamar.colostate.edu)
Accepted 7 July 2003
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Summary |
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Key words: skeletal muscle, myosin heavy chain, actin, isoform, lobster, Crustacea, Arthropoda, Homarus americanus
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Introduction |
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In many cases, fiber distribution in crustaceans is correlated with the
type of motor innervation a muscle receives, but there is considerable
variability in this pattern. Crustaceans possess motor nerves with a continuum
of properties from purely phasic to purely tonic, with many forms being
intermediate between these two extremes
(Atwood, 1976). Phasic
motoneurons are large, fire in brief bursts, are less fatigue resistant and
form thin filiform endplates (Atwood,
1976
; Bradacs et al.,
1997
). Tonic motoneurons are smaller, are active for prolonged
periods, are fatigue resistant and have large and varicose terminals
(Atwood, 1976
;
Bradacs et al., 1997
). In
addition, inhibitory neurons that modulate the neuromuscular responses are
generally present (Atwood,
1976
). Some muscles receive innervation predominantly from either
phasic or tonic motor neurons, while other fibers are controlled by synapses
from both nerves (Atwood,
1976
). For example, many of the deep abdominal muscles receive
only phasic motor axons, while the more superficial abdominal muscles receive
purely tonic efferents (Atwood,
1976
). Muscles in lobster claws, however, contain fibers that
receive input from both nerve types, as well as some fibers that only receive
input from the phasic or the tonic motor nerve
(Atwood, 1976
;
Lang et al., 1980
;
Costello and Govind, 1983
).
Unlike mammalian muscles, which are often composed of a mosaic of fiber
types, crustacean muscles are often anatomically segregated, and the different
muscle types are correlated with the type of excitatory innervation that they
receive (Mykles,
1985a,b
;
Neil et al., 1993
;
Mykles et al., 2002
). We have
recently shown that myofibrillar isoform composition is correlated with
synaptic efficacy in the claw and leg opener muscles of lobsters and crayfish
(Mykles et al., 2002
). In
these muscles, S1 and S2 fibers form a segregated
continuum along the length of the muscle. Proximal fibers are generally
S2 and show greater post-synaptic facilitation than the
S1 fibers that are located more distally. Similarly, Neil et al.
(1993
) demonstrated that the
superficial abdominal flexor muscle in the Norway lobster, Nephrops
norvegiucus, is comprised of S1 and S2 fibers that
form distinct anatomical bundles from the medial to lateral positions. The
correlation between muscle phenotype and innervation, combined with the
distinct anatomical segregation of muscle types, makes crustacean systems
desirable models for studying basic muscle function
(LaFramboise et al.,
2000
).
Here, we extend our understanding of the molecular diversity associated
with muscle phenotype among skeletal muscles in the American lobster
Homarus americanus. Our primary focus is the different fiber types
that form the claw closer muscles. Adult claws are dimorphic, with a large
crusher claw and a slender cutter claw. The crusher claw is invested with slow
(S1) muscle fibers, while the cutter claw contains predominantly
fast muscle fibers (Mykles,
1985a,b
,
1997
). This asymmetry is
established in early juvenile stages and persists throughout the life of the
animal (reviewed by Govind et al.,
1987
; Govind,
1992
). In the present study, our objective was to more precisely
characterize fully differentiated adult fast and slow claw muscles at the
molecular level of organization. We extend our understanding of these muscles
in terms of the MHC protein isoforms and the mRNA isoforms present in the
fibers.
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Materials and methods |
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Muscle samples from the claws were taken from different regions of the closer muscles identified as central, ventral or distal (Fig. 1). In some cases, single fibers were harvested for analysis, while at other times a larger population of fibers was taken from a single region. Muscle samples from the tail were always a bundle of fibers.
|
Analysis of myofibrillar proteins
For analysis of myofibrillar isoform assemblages and western blotting,
SDS-PAGE was performed using a discontinuous gel system as described in Mykles
(1985b). Briefly, 10%
separating gels (37.5:1 acrylamide:N,N'-methylenebisacrylamide)
were used to separate approximately 4-6 µg of myofibrillar proteins using a
Mini-Protean II gel system (Bio-Rad, Hercules, CA, USA) and were stained with
Coomassie blue or silver (Wray et al.,
1981
). Protein concentrations were determined empirically from
silver-stained gels. For western blots, samples were separated as described
but, instead of staining the proteins, they were electophoretically
transferred to a PVDF (polyvinylidine fluoride) membrane. Following transfer,
the membrane was blocked with 2% non-fat milk in Tris-buffered saline (TBS: 20
mmol l-1 Tris-HCl, pH 7.5, 500 mmol l-1 NaCl) overnight
and then incubated for 1 h with polyclonal antibodies raised in rabbits
against purified P75, troponin T (TnT) or troponin I (TnI) at 1:20 000
dilutions of antiserum in 0.05% Tween in TBS (TTBS;
Sohn et al., 2000
;
Mykles et al., 2002
). After
several washes in TTBS, blots were incubated with biotinylated anti-rabbit IgG
(1:5000) for 1 h followed by avidin/biotinylated horseradish peroxidase
complex (ABC reagent; Vectastain, Vector Labs, Burlingame, CA, USA; 1:1000
dilution in TTBS) and chemiluminescent detection
(Covi et al., 1999
).
MHCs from different muscle samples were resolved according to the methods
of Talmadge and Roy (1993),
with the modification that 0.1% ß-mercaptoethanol was added to the upper
buffer (Blough et al., 1996
;
LaFramboise, 2000). Samples were separated on 8% resolving gels (50:1
acrylamide:N,N'-methylenebisacrylamide) on 15 cm gels (Bio-Rad
Protean II) at 200 V for at least 48 h. Rabbit MHC (Sigma, St Louis, MO, USA)
was loaded as a standard on each gel. The gel apparatus was packed in ice and
run in a refrigerated cabinet at 4-5°C. Gels were stained with Coomassie
blue or silver (Wray et al.,
1981
). The relative amounts of specific proteins in bands from
stained gels and blots were determined using scanning densitometry (NIH Image
1.62).
Isolation and sequencing of cDNA encoding lobster slow MHC and
P75
A cDNA encoding lobster S1 MHC was obtained by the methods of
Cotton and Mykles (1993).
Briefly, a cDNA library was made by Invitrogen from mRNA purified from lobster
crusher claw muscle and cloned into a pcDNAII plasmid vector. Cells were grown
for 8 h on LB ampicillin plates, transferred to dried
isopropyl-1-thio-ß-D-galactoside (IPTG)-soaked nylon membranes
and grown overnight on fresh LB ampicillin plates. The cells were then lysed
with 5% SDS and the proteins were fixed to the membranes by microwaving.
Membranes were then blocked with 10% non-fat milk and incubated with
polyclonal anti-MHC, and the bound antibodies were detected with a Vectastain
kit as described by Mattson and Mykles
(1993
). Clones that showed a
positive reaction with the anti-MHC antibodies were selected for sequencing.
Both strands of the slow MHC cDNA were sequenced by Davis Sequencing (Davis,
CA, USA) using SP6 and T7 primers, followed by sequence-specific primers.
A cDNA encoding approximately 1500 bp of lobster muscle P75 was obtained as
described above but from a cDNA library made from fast muscle cDNA
(Cotton and Mykles, 1993).
Positive clones were selected as described above, but the library was screened
with the polyclonal anti-P75 antibody. A single strand of DNA was sequenced
from the SP6 primer, followed by sequence-specific primers. The complementary
strand from the T7 primer did not yield any usable sequence. More information
about this cDNA will be published once we obtain the full sequence. The
sequence for the fast MHC was selected from the same library and has been
published previously (Cotton and Mykles,
1993
).
RT-PCR of myofibrillar protein mRNAs
Total RNA from lobster muscles was isolated using TRIZOL reagent
(Invitrogen). Tissues (approximately 150 mg) were homogenized in 2 ml TRIZOL
using a hand-held glass homogenizer until the tissue was completely
homogenized. Alternatively, single muscle fibers were homogenized in 1 ml
TRIZOL using the same protocol. Insoluble materials were removed by
centrifugation at 12 000 g for 10 min at 4°C. Chloroform
(0.2 ml per 1 mlTRIZOL reagent) was added to each supernatant following a 5
min incubation at room temperature. Samples were shaken by hand for 30 s,
allowed to sit at room temperature for 5 min and then centrifuged at 12 000
g for 10 min at 4°C. RNA in the aqueous phase was
precipitated by the addition of isopropanol (0.5 ml per 1 ml TRIZOL reagent)
followed by incubation at room temperature for 10 min. RNA was collected by
centrifugation at 12 000 g for 10 min at 4°C and then
washed with 75% ethanol. After air-drying, samples were dissolved in water and
stored at -80°C.
RNA samples were treated with DNase (Invitrogen) for 15 min at room
temperature to remove any genomic DNA contaminating the samples. First-strand
cDNA was synthesized from total RNA using SuperScript II RNase H-reverse
transcriptase (Invitrogen) using oligo(dT) primers. The reaction contained 2.5
µg of oligo(dT) 12-18, 2.5 mmol l-1 dNTP, 1x First-Strand
Buffer, 5 mmol l-1 dithiothreitol (DTT), 2.5 units of RNase
inhibitor, 1-2 µg of RNA and 200 units of SuperScript II RNase H-reverse
transcriptase. MHC primers (fast or slow) were synthesized from the fast and
S1 MHC sequences so that the reverse primers complemented a
sequence in the 3'-UTR, while the forward primers complemented a
sequence in the open reading frame (Table
1; Fig. 2). The
fast MHC primers amplified a 315 bp product, while the S1 MHC
primers amplified a 453 bp product. P75 primers were synthesized based on the
partial sequence of the P75 cDNA and yielded a 176 bp product
(Table 1). Plasmid DNA
containing the specific cDNA sequences for the fast MHC, S1 MHC or
P75 were used as template for PCR to optimize reaction conditions. The actin
primers were designed to amplify -actin from lobster skeletal muscles
as previously described, yielding a 401 bp product
(Table 1;
Koenders et al., 2002
; GenBank
accession no. AF399872). Under these conditions, the primer pairs were found
to be highly specific, amplifying single bands of the expected product
lengths. Hot start ExTaq (Takara, Otsu, Shiga, Japan) was added to tubes
containing 1 µmol l-1 of each primer and 2 µl of the
first-strand cDNA reaction in each PCR reaction (20 µl total volume). PCR
amplification consisted of denaturation of the template and activation of
ExTaq (95°C for 120 s) followed by amplification of the target cDNA (35
cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s
and extension at 72°C for 30 s). Products were separated on a 1% agarose
gel and visualized with ethidium bromide staining. For figures, products from
the individual reactions (fast MHC, S1 MHC and P75) were combined
before separation on agarose gels.
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|
Real-time PCR was used to quantify the levels of fast MHC, S1
MHC and actin mRNA from different muscle tissues. The above protocol was used
for the generation of cDNA from isolated RNA samples. Single cDNA samples were
divided for use in separate reactions to measure the copy numbers of each of
the above target sequences. Thus, at least three distinct transcripts were
monitored for each sample and served as internal controls. The Light Cycler
DNA Master SYBR Green I reaction mix for PCR (Roche Molecular Biochemicals,
Indianapolis, IN, USA) was used for the amplification of target cDNA with a
Cepheid Smart Cycler Instrument (Yu and
Mykles, 2003). PCR master mix containing 1x Light Cycler DNA
Master SYBR Green I, 2.5 mmol l-1 MgCl2, 0.5 µmol
l-1 of each primer and 5 µl of the first-strand cDNA reaction
were added to 25 µl sample tubes. PCR amplification consisted of
denaturation of template and activation of the HS Taq (95°C for 5 min)
followed by amplification of the cDNA target (30 cycles of denaturation at
95°C for 15 s, annealing at 60°C for 6 s and extension at 72°C for
20 s). A standard curve was constructed for each sequence using purified
plasmid DNA containing known amounts of cDNA as template in the PCR reactions.
Plasmids containing specific sequences were purified from using QIAprep
minipreps (Qiagen, Valencia, CA, USA). Copy numbers of plasmid DNA containing
inserts were calculated based on the molecular mass of the plasmid and then
converting to number of copies based on Avagodro's number
(Li and Wang, 2000
). A
standard curve was constructed by plotting the number of copies as a function
of the threshold cycle where product began to accumulate exponentially. The
melting temperature, which is a measure of the GC content and length of the
product, was used to identify the specificity of the PCR product. In addition,
reaction products were separated on 1% agarose gels to verify product size.
The cycle threshold from reactions containing unknown amounts of cDNA was
converted to number of copies with the standard curves. The number of
transcript copies was normalized to the amount of tissue (mg) used for RNA
isolation.
Statistical analyses
A one-way analysis of variance (ANOVA) was used to compare the number of
copies of myofibrillar mRNA (fast MHC, S1 MHC and actin) calculated
from real-time PCR. Values were log-transformed because of a high level of
variability among samples and to correct for the correlation between mean and
variance in these data (Neter et al.,
1990). Pair-wise post-ANOVA comparisons were made using a
Bonferroni test, with an experiment-wise error rate of 0.05. Simple linear
regression was used to examine the correlation between the number of copies of
mRNA for different myofibrillar genes, as well as for the correlation between
TnT1 and S2 MHC from protein gels. Statview 5.0.1 (SAS
Institute Inc., Cary, NC, USA) was used for all statistical analyses.
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Results |
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Characterization of slow MHC and P75 cDNA
A cDNA encoding the C-terminal sequence of the S1 MHC was
isolated from an H. americanus slow muscle library made from mRNA
isolated from crusher claw muscle (Fig.
2;GenBank accession no. AY232598). The cDNA was 1795 bp in length
with an open reading frame of 1523 bp. Thenucleotide sequence had highest
sequence identity with the lobster fast MHC isoform encoding the C-terminal,
or rod, region of the polypeptide (79% in the open reading frame;
Cotton and Mykles, 1993;
GenBank accession no. U03091; Fig.
2). The deduced amino acid sequence had 81% sequence identity (91%
similarity) to the deduced amino acid sequence from the lobster fast MHC
isoform (Fig. 5). In addition,
the deduced amino acid sequence also matched a number of MHC sequences from a
diverse group of animals, including Drosophila melanogaster (473 aa,
78% identity, 89% similarity; GenBank accession no. NM_078863), mollusks
(Loligo peali: 505 aa, 62% identity, 80% similarity; GenBank
accession no. AF042349; Mytilus galloprovincialis: 505 aa, 59%
identity, 79% similarity; GenBank accession no. AJ249993), mammals (Felix
catus: 505 aa, 54% identity, 76% similarity; GenBank accession no.
U51472) and nematodes (Caenorhabditis elegans: 334 aa, 60% identity,
78% similarity; GenBank accession no. Z83107; Toxocara canis: 334 aa,
59% identity, 75% similarity; GenBank accession no. AJ306290).
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Only a partial sequence of the P75 clone was obtained, due to technical difficulties with sequencing. However, one end of the clone was sequenced successfully and yielded enough information to design sequence-specific primers for PCR. This sequence was submitted to GenBank (accession no. AY302591) and the full sequence will be published when available.
Analysis of myofibrillar protein expression by RT-PCR
Expression of fast MHC, S1 MHC, P75 and actin in different
muscles was analyzed by RT-PCR. Single products of 315 bp, 453 bp, 401 bp and
176 bp, respectively, were obtained using oligonucleotide primers specific for
the myofibrillar sequences (Table
1; Figs 6,
7). Fast fibers from the closer
muscles in the cutter claw expressed not only fast MHC but also low levels of
the S1 MHC (Figs 6,
8). The fast fibers from the
deep abdominal muscle did not exhibit any S1 MHC expression (Figs
6,
8). S1 MHC
expression in fast cutter claw fibers was highly variable but, on average, was
about six orders of magnitude less than fast expression
(Fig. 8). Actin expression was
significantly lower than fast MHC expression in the deep abdominal muscles but
not in the fast claw muscles (Fig.
8A,B). In general, actin expression was lower in the fast fibers,
being, on average, about 1000-fold lower in the fast fiber types than in the
S1 fibers (Fig. 8).
In some deep abdominal samples, actin expression was below the threshold of
measurement (Fig. 6B, lane a).
The various slow fibers predominately expressed the S1 MHC and
actin, with actin expressed at higher levels than S1 MHC (Figs
7,
8C). The expression of fast MHC
in the slow fibers was barely detectable by ethidium bromide staining
(Fig. 7), but low levels were
measured in the crusher claw closer muscles using real-time PCR
(Fig. 8). Although fast MHC
expression in S1 fibers was variable, on average fast expression
was 1000-fold less than S1 MHC expression
(Fig. 8). A significant
correlation was observed between actin and S1 MHC expression when
values from both the cutter and crusher muscles were combined [log
S1 MHC=0.0437+0.716(log actin); P<0.0001,
r2=0.537] (Fig.
9). This correlation was not significant when made between
expression levels from the S1 muscles alone (P<0.52,
r2=0.027) but was significant for the fast cutter muscles
alone (P<0.009, r2=0.643).
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Discussion |
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The contractile characteristics of lobster claw muscle fibers, determined
both by fiber type and innervation pattern, can be used to categorize fibers
as fast, intermediate or slow (Costello and
Govind, 1983). Biochemical methods can also be employed to
classify these fibers as fast, S1 or S2, respectively
(Mykles,
1985a
,b
,
1988
;
Silverman et al., 1987
). In
the American lobster, S1 fibers are known to possess higher
myofibrillar ATPase activities than S2 fibers
(Mykles, 1988
). Likewise, for
the Norway lobster, S1 and S2 fibers have different
contractile properties, with S1 fibers contracting more quickly
(Holmes et al., 1999
). These
differences apparently stem from the presence of the two distinct MHC isoforms
as identified previously by partial digestion with
-chymotrypsin
(Neil et al., 1993
) and by
SDS-PAGE in the present study. The amounts of S1 and S2
MHC isoforms in fibers from the distal region of the cutter claw are present
in reciprocal levels, with some fibers possessing a near 1:1 ratio of the
S1 and S2 isoforms
(Fig. 4). Single fibers from
other species under control conditions also contain multiple MHC isoforms,
indicating that these mixtures are not only present in transitional fibers but
represent the normal state of the muscles
(Peuker and Pette, 1997
;
Lutz et al., 1998
;
Stevens et al., 1999
;
Lutz et al., 2001
). In frog
muscle, MHC isoform composition changes along the length of the fiber and is
correlated with characteristic proportions of myosin light chain isoforms
(Lutz et al., 2001
). Further
studies are currently underway to gain a better understanding of the
significant level of polymorphism observed in the lobster muscles.
The relative concentration of the S2 MHC isoform is
significantly correlated with the proportion of the TnT1 isoform
(Fig. 3C), which has been used
to define S2 fibers (Mykles,
1985a,b
,
1988
;
Neil et al., 1993
). While some
fibers contained the S2 MHC isoform exclusively, the maximum amount
of TnT1 isoform was only a 50% mix with the T3 isoform
(Fig. 4C). Mykles
(1988
) reported that the
TnT1 and TnT3 isoforms vary reciprocally in the
S1 and S2 fibers of crabs and lobsters, but it appears
that the maximum amount of the T1 isoform is only about 50% of the
total TnT concentration. The relatively loose correlation between TnT isoform
assemblage and MHC composition (Fig.
4C; r2=0.59) suggests that the S1
and S2 fibers are not completely distinct but probably exist as a
continuum between pure S1 and pure S2 fibers. Consistent
with this interpretation, the proximal fibers of the claw and leg opener
muscles in crayfish and lobsters are S2, while the central part of
the muscles are S1 (Mykles et
al., 2002
). The fibers between the proximal and central fibers
appear to change gradually in their proportion of TnT isoforms, suggesting
that the muscles exist in a continuum
(Mykles et al., 2002
). A
physiological correlate of this continuum is that the more proximal fibers
(S2) show a greater degree of post-synaptic facilitation than the
distal (S1) fibers (Mykles et
al., 2002
). In the superficial abdominal flexors of N.
norvigicus, the medial bundle of fibers is composed of S2
fibers, while the lateral bundle is composed of S1 fibers
(Neil et al., 1993
). While
these two bundles exhibit considerable overlap anatomically, it is unclear
whether individual fibers contain both the S1 and S2 MHC
isoforms.
The fibers of the cutter claw closer muscle can be divided into
anatomically distinct regions characterized by different types of fibers. The
central region of the muscle is composed of fast fibers, while the ventral
edge of the muscle is comprised of S1 fibers
(Mykles, 1985b), a pattern
that is confirmed in the present study. In addition, Mykles
(1985b
) reported that a small
proportion (10-15%) of the slow fibers in the cutter claw closer were
S2 fibers. In the present study, the S2 fibers could be
identified grossly as thin fibers forming a distinct bundle in the ventral
aspect of the distal closer muscle (Fig.
1). This is similar to Mykles' description of a distal group of
fibers in the claw of the land crab, as a wedge-shaped area occupied by
small-diameter fibers (Mykles,
1988
). Lang et al.
(1980
) demonstrated that
fibers in this region of both the cutter and crusher claw muscles possess high
oxidative capacity, consistent with the presence of S2 fibers. As
in a previous study (Mykles,
1985b
), crusher fibers taken from the same region did not appear
to form a distinct bundle and were classified as S1 based on their
myofibrillar isoform profile (not shown).
Analysis of myofibrillar gene expression with RT-PCR
An unexpected finding was the co-expression of fast and S1 MHC
genes in the same muscle tissue and even in single fibers. While the initial
finding from RT-PCR indicated that this co-expression was primarily in the
fast fibers of the cutter claw (Figs
6,
7), quantitative real-time PCR
showed that the S1 fibers also expressed the fast MHC
(Fig. 8). It is unclear whether
the expressed MHC isoforms are present at the protein level, since the fast
and S1 isoforms co-migrate on protein gels. While the closer
muscles of the crusher claw are often considered to be 100% slow by
biochemical methods (Mykles,
1985b,
1997
), analysis of fiber type
by sarcomere length and contractile properties indicates that a small portion
of these fibers are fast (Jahromi and
Atwood, 1971
; Costello and
Govind, 1983
). Some of the expression of the minor MHC in both
fast and slow claw muscles may be due to the presence of a minor population of
such fibers. However, analysis of individual muscle fibers from the fast
region of the cutter claw and from the crusher claw muscles reveals
co-expression within single fibers. A significant number of fibers from
rabbit, rat and frog muscles possess multiple MHC mRNAs, most of which are
`next neighbor' isoforms (Peuker and
Pette, 1997
; Lutz et al.,
1998
; Stevens et al.,
1999
). However, in some cases, MHC mRNA sequences were found in
fibers that did not contain detectable levels of the corresponding proteins
(Peuker and Pette, 1997
;
Stevens et al., 1999
).
Unloading of rat soleus muscle increases the proportion of these mismatched
fibers (Stevens et al., 1999
).
It has been suggested that these discrepancies are evidence for both
transcriptional and translational control of muscle phenotype
(Peuker and Pette, 1997
;
Stevens et al., 1999
). In this
context, it seems likely that the low levels of expression of the unexpected
MHC genes in the lobster may never actually be translated into functional
proteins.
It is also possible that unidentified MHC isoforms hybridized with the
primers designed for the deduced fast and S1 MHC sequences. To
date, only the S1 and fast MHC isoforms have been cloned from the
lobster and it is likely that other isoforms exist. As a case in point, the
sequence encoding the S2 MHC isoform identified in the present
study has yet to be cloned. This protein is clearly co-expressed with the
S1 isoform in a population of slow fibers, and similar expression
of unidentified sequences in other fibers may go undetected or result in
non-specific amplification by one of the primer sets. Recently, the sequences
of several crustacean MHC genes have been identified
(Holmes et al., 2002) but
these were all from the variable region of the myosin head, and the fast and
S1 MHC sequences of the lobster are from the rod region of the
molecule, making direct comparisons impossible.
Each of the fiber types possessed a unique expression pattern with respect
to the MHC isoforms and actin (Fig.
8). Interestingly, the phasic deep abdominal muscles expressed
only the fast MHC isoform, with no S1 MHC detected. These
differences may be related to differences in the type of innervation, since
the deep abdominal muscles are more uniformly phasic in their properties
(Atwood, 1976;
Cooper et al., 1998
), while the
claw closer muscles receive either phasic, tonic or both types of innervation
(Atwood, 1976
;
Lang et al., 1980
;
Costello and Govind, 1983
).
Other crustacean muscles demonstrate definite correlations between muscle
phenotype and innervation (Günzel et
al., 1993
; Mykles et al.,
2002
). Another major difference between fiber types is that the
S1 fibers contained significantly higher levels of actin mRNA than
the fast fibers. Consistent with this, slow fibers have a thin to thick
filament ratio of about six, while fast fiber have a ratio of about three
(Jahromi and Atwood, 1969
,
1971
). A significant
correlation was observed between S1 MHC and actin in all fibers
expressing the S1 MHC isoform. However, this pattern was observed
over a very broad range of isoform expression in fast and slow fibers and was
not significant when examined over the narrower range of actin expressed in
the slow fibers. This correlation and the relationship between the
S2 MHC and TnT1 may reflect a functional linkage between
individual isoforms within a whole myofibrillar protein assemblage. These
isoforms are not typically interchanged in isolation but exist as part of an
entire population of protein isoforms that characterize a particular muscle
type (Schiaffino and Reggiani,
1996
; Mykles,
1997
).
Conclusions
This study extends our understanding of the differences among skeletal
muscle fiber types in crustaceans at a molecular level of organization.
Although fast and S1 MHC isoform expression patterns can be
quantified and correlated with the expression of other myofibrillar proteins,
these isoforms co-migrate in SDS-PAGE gels and cannot be distinguished by
these methods. However, another MHC associated with S2 fibers can
clearly be distinguished from the S1 MHC isoform and coexists with
this isoform in many fibers. The relative concentration of the S2
MHC isoform was positively correlated with the TnT1 isoform, which
has been considered diagnostic for S2 fibers. The gradation between
muscle fiber types like that seen with the S1 and S2
fibers provides an opportunity to examine the determinants of muscle
phenotype, including differences in fiber innervation pattern. Similar MHC
polymorphism has been detected in mammalian and frog muscles in recent years,
but the functional implications of this gradation in muscle phenotype is still
unclear. Further work is needed to identify the gene sequences encoding other
MHC isoforms, such as the S2 MHC. Current studies are focusing on
the correlation between S1 MHC mRNA and S1 and
S2 MHC proteins in superficial abdominal muscles. The methods used
in the present study can also be applied to other questions of muscle
diversity and plasticity in crustaceans. For example, the developing lobster
claw muscles undergo a process of fiber switching during development that
represents a natural model of skeletal muscle plasticity
(Govind et al., 1987;
Govind, 1992
;
Mykles, 1997
). We are using
real-time PCR to examine the changes in myofibrillar gene expression that may
contribute to the fiber switching process in juvenile claw muscles.
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Acknowledgments |
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References |
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