Fiber polymorphism in skeletal muscles of the American lobster, Homarus americanus: continuum between slow-twitch (S1) and slow-tonic (S2) fibers
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
* Author for correspondence (e-mail: smedler{at}lamar.colostate.edu)
Accepted 18 May 2004
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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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Like mammalian muscles, crustacean fibers are categorized into several
different fiber types based on a number of criteria. Broadly, crustacean
fibers are distinguished on the basis of structural characteristics (Jahromi
and Atwood, 1969,
1971
;
Atwood, 1976
;
West, 1997
), ATPase
histochemistry (Ogonowski and Lang,
1979
; Silverman et al.,
1987
), specific myofibrillar isoform assemblages (Mykles,
1985a
,b
,
1988
,
1997
) and the contractile
properties of the fibers (Holmes et al.,
1999
). Currently, at least three different crustacean fiber types
are recognized: fast, slow-twitch (S1) and slow-tonic
(S2). In lobsters (H. americanus), fast fibers comprise
the majority of the muscle mass in the cutter claw closer and in the deep
abdominal flexor muscles. S1 fibers are the exclusive fiber type in
the crusher claw closer and are present in the claw and leg opener muscles, in
the ventral musculature of the cutter claw closer and in the superficial
flexors and extensors of the tail. S2 fibers are present in the
distal fibers of the cutter claw closer, claw and leg openers, as well as the
superficial abdominal flexors and extensors. Previous studies have
demonstrated that the superficial abdominal muscles of decapod crustaceans are
comprised of both S1 and S2 fibers
(Fowler and Neil, 1992
;
Neil et al., 1993
).
In the current study, we focused on S1 and S2 fibers
from the superficial abdominal muscles in the lobster as a model to study the
continuum among muscle fiber types. We recently identified single fibers from
the lobster claw closer that were polymorphic, expressing variable levels of
both S1 and S2 MHC, as well as other myofibrillar
isoforms (Medler and Mykles,
2003). In addition, we demonstrated that most lobster muscles
co-expressed multiple MHC genes in muscles that had previously been viewed as
distinct in their fiber types. These trends suggest that lobster muscle
fibers, like the muscles of mammals and other vertebrates, often represent a
point in a continuum of muscle phenotypes. While MHC is the most important
determinant of the contractile properties of a muscle
(Schiaffino and Reggiani,
1996
), different fiber types exhibit whole assemblages of distinct
myofibrillar isoforms (Schiaffino and
Reggiani, 1996
; Mykles,
1997
). Because of the importance of these non-MHC proteins to
muscle function, we examined the relative proportion of other myofibrillar
isoforms (Tm, TnT, TnI) and how they were correlated in different fibers. For
the present study, we obtained partial cDNAs for previously unidentified
S2 MHC and tropomyosin (Tm) isoforms and used sequence-specific PCR
primers to measure the relative expression of S1 and S2
isoforms using real-time PCR. In addition, we used SDS-PAGE and western
blotting to identify different isoforms of MHC, troponin T (TnT) and troponin
I (TnI) in small muscle bundles and single muscle fibers. Together, these data
provide information about the level of polymorphism present in crustacean
muscle fibers and the correlation among different myofibrillar isoforms, at
both the protein and mRNA levels.
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Materials and methods |
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Isolation and sequencing of cDNAs encoding slow-tonic (S2) MHC and Tm
A cDNA encoding the 3' end of the coding region and 3' UTR of
an MHC-encoding gene was obtained using the 3'-RACE system (Invitrogen
Inc., Carlsbad, CA, USA) and a gene-specific forward primer designed to a
conserved region of the MHC sequence, as based upon the sequences of lobster
fast and slow (S1) sequences
(5'-GAAGGCTAAGAAGGCCATGGTTGA-3')
(Medler and Mykles, 2003).
Briefly, RNA was isolated from samples of superficial flexor muscles with
TRIZOL (see Analysis of myofibrillar protein mRNAs). First-strand cDNA
synthesis reactions contained 100 ng total RNA and 1 µl of 10 µmol
l-1 Invitrogen Inc. adaptor primer (Cat. No. 10542-017):
5'-GGCCACGCGTCGACTAGTAC(T)17-3'. The other conditions of the
first-strand synthesis reaction were the same as described below (see Analysis
of myofibrillar protein mRNAs). Muscles used for RNA isolation were
prescreened with high-resolution SDS-PAGE to ensure that the muscle samples
were exclusively expressing the S2 MHC (see Analysis of
myofibrillar proteins). PCR was carried out on approximately 10 ng cDNA using
a universal amplification primer (Invitrogen Inc.; Cat. No. 18382-010) as the
reverse primer and the MHC-specific forward primer. PCR products were
identified by separating samples on 1% agarose gels and staining the DNA with
ethidium bromide. The PCR yielded two distinct products, one of approximately
1500 bp and the other of approximately 1000 bp. Both PCR products were
purified by gel purification (Qiagen, Valencia, CA, USA) and cloned into the
pCR 2.1 TOPO plasmid using TOPO TA cloning kit (Invitrogen Inc.). Transformed
bacteria were grown overnight on pre-warmed MacKonkey ampicillin plates and
were selected based on the inability to metabolize lactose. The presence of
PCR inserts in the transformed colonies was confirmed by PCR using vector
primers (M13 reverse and T7). Several positive clones containing inserts
ranging from approximately 1 kb to 2 kb were selected for sequencing by Davis
Sequencing (Davis, CA, USA). Of these clones, two were found to be a
previously unidentified MHC that was presumed to code for an MHC protein in
S2 muscle fibers. Both strands of the MHC cDNA were ultimately
obtained using a combination of vector primers and internal sequence-specific
primers (Davis Sequencing).
A smaller cDNA insert yielded a DNA sequence for a previously unidentified
tropomyosin cDNA bearing high identity to the fast and slow (S1)
tropomyosin sequence previously identified in the lobster
(Mykles et al., 1998). It was
later discovered that the last 12 bp on the 3' end of the forward primer
designed to amplify the MHC sequence shared 100% sequence identity to a region
of the Tm sequence. This sequence similarity may stem from the fact that both
the tail region of the MHC and the Tm proteins form coiled-coil structures. A
full sequence was obtained by PCR using the universal amplification primer and
a forward primer designed to the 5' end of the published S1
Tm sequence. This PCR product was cloned into the pCR 2.1 TOPO plasmid
(Invitrogen Inc.) as described above. Both strands of this cDNA were obtained
by sequencing with T7 and M13 vector primers and sequence-specific internal
primers (Davis Sequencing).
Analysis of myofibrillar proteins
Muscle bundles used for protein analyses were processed according to the
methods of Mykles (1985b).
Briefly, muscles were glycerinated in ice-cold buffer containing 20 mmol
l-1 Tris-HCl (pH 7.5), 50% glycerol, 100 mmol l-1 KCl, 1
mmol l-1 EDTA and 0.1% Triton X-100 for 2-3 h with stirring. Single
fibers or fiber bundles were removed from the muscle and solubilized in 250
µl of SDS sample buffer containing 62.5 mmol l-1 Tris-HCl (pH
6.8), 12.5% glycerol, 1.25% SDS and 1.25% ß-mercaptoethanol. Muscle
samples were left in this solution overnight at room temperature with
occasional vortexing. Individual freeze-dried fibers were placed directly in
50 µl SDS sample buffer and homogenized with a hand-held pestle that fitted
directly into the 0.5 ml microcentrifuge tube.
For analysis of myofibrillar isoform assemblages, 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 the prepared samples using a Mini-Protean 3 gel system
(Bio-Rad, Hercules, CA, USA). Samples were then electrophoretically
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) for at
least 1 h and then incubated for at least 1 h with polyclonal antibodies
raised in rabbits against purified TnT or 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 antirabbit IgG
at 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 3) 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).
Analysis of myofibrillar protein mRNAs
RNA was isolated using TRIZOL reagent (Invitrogen Inc.). Fibers were
homogenized in 1 ml TRIZOL using a hand-held glass homogenizer until the fiber
was completely homogenized. Insoluble materials were removed by centrifugation
at 12 000 g for 10 min at 4°C. Chloroform (0.2 ml per 1 ml
TRIZOL 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 in 75% ethanol. After
air-drying, samples were dissolved in water and quantified by measuring
absorbance at a wavelength of 260 nm. The samples were then stored at
-80°C. The samples were then treated with DNase (Invitrogen Inc.) for 15
min to remove any genomic DNA still in the samples. First-strand DNA was
synthesized from total RNA using Moloney murine leukemia virus (M-MLV) reverse
transcriptase (Invitrogen Inc.) using oligo (dT) primers. The 20 µl
reaction contained 2.5 µg 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 RNA and 200 units of MMLV reverse
transcriptase.
PCR of MHC and Tm isoforms was performed using sequence-specific primers
that amplified the specific isoform of interest. Forward primers were designed
to anneal to regions of the open reading frame, while reverse primers annealed
to unique non-coding regions of the 3' UTR (Figs
1,
2;
Table 1). Since the 3'
UTRs of the fast and S1 isoforms are thought to be the same
(Mykles et al., 1998), it is
possible that the S1 primers also amplified the fast Tm isoform.
However, this possibility is unlikely since all of the fibers used in the
current study were from slow muscles. Nonquantitative PCR was used to
demonstrate the specificity of the primers, and purified plasmids containing
the cDNA of interest were used as positive and negative controls. 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 stained with
ethidium bromide. For figures, products from the individual reactions
(S1 and S2 MHC, or S1 and S2 Tm)
were combined before separation on agarose gels.
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Real-time PCR was used to quantify the levels of S1 MHC,
S2 MHC, S1 Tm and S2 Tm. The above protocol
was used for the generation of cDNA from the isolated RNA. These samples were
then amplified with primers specific to each isoform using The Light Cycler
DNA Master SYBR Green I reaction mix (Roche Molecular Biochemicals,
Indianapolis, IN, USA) and Cepheid Smart Cycler Instrument
(Medler and Mykles, 2003;
Yu and Mykles, 2003
). A master
reaction mix consisting of 1x Light Cycler DNA Master SYBR Green I, 2.5
mmol l-1 MgCl2, 0.5 mmol l-1 of each primer
and 0.8 mmol l-1 dNTP was prepared before adding 5 µl of the
first-strand cDNA to yield a total reaction volume of 25 µl. PCR
amplification consisted of denaturation of template and activation of HS Taq
at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 15
s, annealing at 60°C for 6 s, and extension at 72°C for 30 s. A
standard curve was constructed using purified plasmid with known amounts of
cDNA template. Copy numbers of the plasmid were calculated based on the
molecular mass of the plasmid and then converting to number of copies based on
Avogadro's number (Li and Wang,
2000
). The standard curves were created through serial dilutions,
which were then plotted as a function of threshold cycle at which products
began to accumulate exponentially. The product specificity was confirmed in
two ways. First, the melting temperature was determined and used as a measure
of G-C content. Second, samples were electrophoretically separated on 2%
agarose gels to determine product size. The cycle threshold from reactions
containing unknown amounts of cDNA was converted to number of copies using the
established standard curves. The isoforms could then be correlated based on
total amount of expression or based upon percent of expression relative to
total expression of all isoforms coding for the same gene.
For the comparison of S2 MHC expression among different muscle
types, the number of copies of S2 MHC mRNA was standardized to the
total RNA in the samples, as previously described
(Medler and Mykles, 2003). For
the smaller samples from the superficial extensors and flexors, the amount of
total RNA was too low to quantify accurately. For each of these samples, the
amount of S1 MHC mRNA relative to the amount of S2 MHC
mRNA from the same sample was used to assess the relative expression of these
two genes. The same procedure was used for the S1 and S2
Tm mRNA measurements. Through use of these ratios, the S1 and
S2 expression levels for both the MHC and Tm genes served as
internal standards for each other.
Statistical analyses
A one-way analysis of variance (ANOVA) was used to compare the relative
number of copies of myofibrillar mRNA and protein calculated from real-time
PCR and western blots, respectively. Expression levels for the S2
MHC isoform in different muscles were log-transformed because of high levels
of variability among samples and to correct for the correlation between mean
and variance in these data (Neter et al.,
1990). The arcsin transformation is often used when comparisons
are made among variables that are proportions
(Neter et al., 1990
), but we
found that this transformation did not affect our comparisons. Consequently,
the relative proportions of mRNAs and proteins were not transformed. 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 relative number of copies of mRNA for
different myofibrillar genes, as well as for the correlations between TnT and
TnI isoforms. 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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A cDNA encoding the entire sequence of the S2 Tm was isolated
from H. americanus superficial flexor muscle using the same PCR-based
cloning approach as used to obtain the S2 MHC sequence
(Fig. 2; GenBank accession no.
AY521627). The cDNA was 1526 bp in length with an ORF of 855 bp and a 3'
UTR of 612 bp. The nucleotide sequence had highest sequence identity with the
lobster fast and slow (S1) Tm isoforms previously reported
(Mykles et al., 1998). The
sequence shared 97% identity with the S1 Tm sequence in the ORF,
differing only at the 3' end of the ORF (bp 833-stop codon) and in the
3' UTR. The sequence was 92% identical to the fast Tm, differing at both
the 5' (bp 174-299) and 3' (bp 833-stop codon) ends of the
ORF.
The deduced amino acid sequences for the three tropomyosin isoforms
(S1, fast and S2) are shown in
Fig. 3. As reported previously
(Mykles et al., 1998), the
fast and S1 sequences are identical except in the region comprising
residues 39-80, resulting in 95% identity. The S2 and S1
isoforms were identical except in five residues at the C-terminal end of the
molecule (residues 269-284), resulting in 98% identity between these two
proteins.
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Expression of S2 MHC in different muscles
The S2 MHC gene was found to be expressed in each of the muscles
examined, with the highest expression being in the superficial muscles of the
abdomen. Intermediate expression was observed in the slow (S1)
muscles of the claw, with the lowest expression being observed in the fast
muscles of the claw and abdomen (Fig.
4). The average copy number per µg total RNA in the superficial
muscles was five times higher than in the crusher claw muscles
(9.58x107 vs 1.91x107), 147 times
higher than in the cutter claw muscles (9.58x107 vs
6.51x105) and more than 500 times higher than in the deep
abdominal flexors (9.58x107 vs
1.85x105).
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Coexpression of the S1 and S2 MHC and Tm in superficial abdominal muscles
Bundles of fibers and single fibers alike were found to coexpress the
S1 and S2 isoforms in single samples. This coexpression
was evident for both the MHC and Tm isoforms. PCR with sequence-specific
primers for these genes clearly demonstrated this coexpression, and
simultaneous amplification with purified plasmid as template demonstrated that
the primers were highly specific for each isoform
(Fig. 5). In essentially every
sample analyzed, co-expression of the S1 and S2 isoforms
for both MHC and Tm was observed. Overall, the relative expression of the
S1 and S2 MHC isoforms was significantly correlated with
the S1 and S2 isoforms of Tm, although this relationship
was non-linear (Fig. 6). The
relative expression of the MHC isoforms ranged from nearly 100% of the
S1 isoform to nearly 100% of the S2 isoform
(Fig. 6). By contrast, the
S2 Tm isoform represented about 90% of Tm expression, even in
fibers expressing the S1 MHC isoform almost exclusively
(Fig. 6).
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|
Protein isoforms in different fibers
Relative amounts of protein isoforms were measured by SDS-PAGE and western
blotting, followed by densitometry of the gels and X-ray films exposed during
chemiluminescent detection of western blots. Protein isoforms for MHC, TnT and
TnI in single fibers and bundles were present in a number of different
combinations. These combinations followed predictable patterns that
represented a continuum from what could be described as `pure' S1
to `pure' S2 fibers (Fig.
7). While multiple MHC isoforms were observed in muscle bundles,
all of the single fibers analyzed were found to contain detectable levels of
only single isoforms (Fig. 7).
TnT1 was found to be highest in S2 fibers, while
TnT3 was the predominant TnT isoform in S1 fibers (Figs
7,
8,
9). TnT2 is only
expressed in fast fibers (Mykles,
1985a,
1997
). TnI1 was a
minor isoform observed in only a few samples and was not correlated with
either TnT isoform (Fig. 9A).
TnI2 was the main TnI isoform in S2 fibers (Figs
7,
8C), and TnI2
content was inversely correlated with the proportion of TnT3 in the
fibers (positively correlated with TnT1;
Fig. 9B). TnI3
content was found in reciprocal levels to TnI2, being the main
isoform in S1 fibers (Figs
7,
8D), and was positively
correlated with the proportion of TnT3 (negatively correlated with
TnT1; Fig. 9C).
TnI4 was a minor isoform in the fibers and was found primarily in
S1 fibers, being positively correlated with TnT3
concentration (Figs 7,
9D).
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Major characteristics of S1 and S2 fibers
Fibers were classified as S1, S2 or intermediate
based on MHC protein isoforms as identified by SDS-PAGE
(Fig. 7). Based on this
classification, several characteristic patterns were observed
(Fig. 8). First, S1
fibers expressed a higher proportion of the S1 MHC mRNA,
intermediate fibers contained slightly lower but non-significantly different
levels, and S2 fibers expressed a significantly lower proportion of
the S1 MHC mRNA (more S2 MHC mRNA;
Fig. 8A). Second, slow fibers
contained TnT1, TnT3 or a combination of both isoforms.
The S1 fibers possessed a higher proportion of TnT3,
S2 fibers contained significantly less TnT3 (more
TnT1), while samples containing both isoforms were intermediate in
the proportion of TnT1 and TnT3. Finally, lobster slow
muscles contained varying amounts of TnI1, TnI2,
TnI3 and TnI4 (Fig.
9). S1 fibers primarily contained TnI3,
while the S2 fibers mainly contained TnI2. Intermediate
samples contained intermediate values for both TnI2 and
TnI3 (Figs 8C,D,
9).
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Discussion |
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In addition, we report the entire sequence for a previously unidentified Tm
gene that shares high sequence identity (more than 90% in the ORF) to the fast
and S1 Tm from lobster muscles
(Mykles et al., 1998). Mass
spectroscopy indicated that a third Tm isoform existed in some proportion in
slow fibers (Mykles et al.,
1998
) and this is presumably that unidentified isoform (the
predicted mass by mass spectroscopy is 32 884, and the estimated mass based on
the deduced amino acid sequence is 32 877). The fast and S1
isoforms are presumably produced from alternative splicing of exons in the
5' end of the mRNA, while the rest of the sequence including the
3' UTR is formed from the same exons
(Mykles et al., 1998
;
Fig. 2). The S2 Tm
sequence is identical to the S1 sequence, except that a different
exon apparently comprises the 3' end of the ORF and 3' UTR
(Fig. 2). The deduced amino
acid sequence shows that the S1 and S2 isoforms differ
by only five amino acids from residues 269-284, a region of the molecule
important for head to tail overlap between sequential Tm molecules in the thin
filament, as well as for TnT binding (Cho
and Hitchcock-DeGregori, 1991
;
Perry, 2001
). Alternative
splicing of exons is used to generate different Tm isoforms in both vertebrate
and invertebrate species (Basi et al.,
1984
; Cho and
Hitchcock-DeGregori, 1991
;
Perry, 2001
), and alternate
exons in the C-terminal end of the molecule in particular have direct effects
on contractile function (Cho and
Hitchcock-DeGregori, 1991
;
Perry, 2001
). Physiological
studies are needed to characterize the functional consequences of the
alternate Tm isoforms in lobster muscle.
We used the sequence divergence in the 3' UTR of the two isoforms to design reverse PCR primers to distinguish expression levels of the S1 and S2 isoforms. The relative expression of the S1 and S2 Tm isoforms was positively correlated with the expression of the S1 and S2 MHC isoforms, confirming that the newly identified sequence is an S2 isoform of Tm. Interestingly, the S2 isoform was expressed to a greater extent than the S1 isoform, amounting to 90% or more in fibers that predominantly expressed the S1 MHC isoform (Fig. 6).
Crustacean fiber types
Crustacean muscle fibers exist as a number of different fiber types, as
identified by histochemistry, protein composition, ultrastructure and
contractile properties (Jahromi and Atwood,
1969,
1971
;
Ogonowski and Lang, 1979
;
Mykles,
1985a
,b
,
1988
,
1997
;
Silverman et al., 1987
;
West, 1997
). In previous
studies, S1 fibers have been distinguished from S2
fibers based on differences in myofibrillar isoform composition,
histochemistry (Mykles, 1988
;
Fowler and Neil, 1992
;
Neil et al., 1993
), sarcomere
length (Neil et al., 1993
),
myofibrillar isoforms (Mykles,
1988
; Neil et al.,
1993
), post-synaptic facilitation
(Mykles et al., 2002
) and
in vitro shortening velocity
(Holmes et al., 1999
). The
results of the current study are consistent with previous studies in several
respects. MHC isoforms were identified based on different migration distances
between the S1 and S2 isoforms, with the S1
MHC having greater electrophoretic mobility
(Fig. 7;
Medler and Mykles, 2003
).
Interestingly, we did not identify any single fibers with both MHC proteins,
even though all of the samples examined had a mixture of S1 and
S2 MHC mRNA. Samples containing both MHC proteins were small
bundles containing several fibers (Fig.
7, lanes e-g). In addition, the S2 Tm isoform was found
to be the predominant isoform, even in fibers that would be classified as
S1 based on other myofibrillar isoforms. In a previous study, mass
spectroscopy data indicated that S1 fibers from the lobster claw
muscles contained approximately an equal mixture of the S1 and
S2 Tm at the protein level
(Mykles et al., 1998
). It is
possible that the S1 fibers of the claw and those of the
superficial abdominal extensors are not strictly equivalent but that the
fibers from the abdomen are more S2 in nature. Consistent with
previous studies (Mykles,
1985a
,b
,
1988
;
Neil et al., 1993
;
Medler and Mykles, 2003
),
S1 fibers were found to predominantly possess the TnT3
protein isoform, while S2 fibers had a higher level of the
TnT1 isoform (Figs
7,
8,
9). In previous studies,
S2 fibers were never found that possessed more than
50% of the
TnT1 isoform, with the rest being composed of TnT3
(Mykles, 1988
;
Medler and Mykles, 2003
). In
the current study, two fibers were found to contain 100% of the
TnT1 isoform (Fig.
7A,B). Moreover, we found that the relative amounts of
TnT1 and TnT3 existed in a continuum, from fibers with
100% of TnT1, to fibers with both isoforms, to fibers with 100% of
TnT3 (Figs 7,
8,
9). Previous studies have
reported differences in TnI isoforms between S1 and S2
fibers (Mykles,
1985a
,b
,
1988
;
Neil et al., 1993
), but in the
current study we define these differences more precisely. TnI2 was
found to be the predominant protein isoform in S2 fibers, while
TnI3 was the predominant isoform in S1 fibers. In
addition, TnI4 became more abundant in S1 fibers that
had little TnT1 (Figs
7,
9D). Isoforms associated with
fast fibers (TnI1 and TnI5) were absent or extremely
rare in both S1 and S2 fibers. The low levels of
TnI1 observed in a few muscle bundles
(Fig. 9A) were unexpected,
although a previous study reported some levels of TnI1 in slow
muscles of the Norway lobster (Neil et
al., 1993
). While S1 and S2 fibers could be
distinguished based on the characteristic presence of the above isoforms, many
intermediate fibers and bundles possessing a blend of isoforms formed a
continuum from pure S1 to pure S2 fibers (Figs
5,
6,
7,
8,
9).
Hybrid muscle fibers
Recent advances in the methodology used to identify different skeletal
muscle fiber types have led to greater resolution of muscle phenotype in terms
of the MHC isoforms present and copies of different mRNAs in individual fibers
(Pette et al., 1999;
Stevenson, 2001; Caiozzo,
2002
; Caiozzo et al.,
2003
). Through these methods, a number of studies of several
different mammalian and amphibian species have found that fibers expressing
more than one MHC isoform are more common than once thought
(Peuker and Pette, 1997
;
Lutz et al., 1998
;
Nguyen and Stephenson, 2002
;
Stevens et al., 1999
;
Lutz et al., 2001
;
Caiozzo et al., 2003
). In
fact, recent studies have demonstrated that these hybrid fibers can represent
a higher proportion of fibers than the `pure' fibers that were once thought to
represent the general state of muscle fibers
(Caiozzo et al., 2003
;
reviewed by Stephenson, 2001
).
We recently found that single fibers of the claw closer muscle in the lobster
possess both S1 and S2 MHC isoforms
(Medler and Mykles, 2003
).
However, in the current study we found that while single MHC isoforms were
almost always identified at the protein level, essentially all of the fibers
and bundles expressed a mixture of both S1 and S2 MHC
mRNA, suggesting that the MHC protein isoforms may be regulated through
translational (Jefferson and Kimball,
2001
; Bolster et al.,
2003
) or post-translational control mechanisms. It is also
possible that low levels of MHC proteins went undetected in the silver
staining procedure, although samples with 30% or more of the S1 MHC
mRNA had no detectable amounts of the corresponding proteins
(Fig. 8A). While MHC isoforms
have received the greatest attention, additional factors including other
myofibrillar proteins also contribute to muscle function and should not be
overlooked (Bottinelli, 2001
;
Clark et al., 2002
). For
example, mutations of TnT associated with hypertrophic cardiomyopathy lead to
significant increases in unloaded shortening velocity in vitro
(Sweeney et al., 1998
), and
alternative splice variants of TnT in dragonfly flight muscles are associated
with significant differences in muscle power output and flight performance
(Fitzhugh and Marden, 1997
;
Marden et al., 1999
,
2001
). In the present study,
mixtures of the S1 and S2 Tm mRNA were observed in all
of the fibers and bundles examined, and isoforms of TnT and TnI proteins were
found to exist in intermediate ratios between the extremes of the pure
S1 fibers to pure S2 fibers. Collectively, these
findings are consistent with other recent observations suggesting that muscle
fiber types in the lobster are not discrete entities but represent a continuum
of fiber types (Mykles et al.,
2002
; Medler and Mykles,
2003
). Intermediate phenotypes were regularly observed in single
fibers, ruling out the possibility that fiber polymorphism was an artifact of
multiple different fibers within a single sample (Figs
5,
6,
9). More broadly, the presence
of intermediate fiber types in an invertebrate species indicates that mixed
phenotype fibers are not a phenomenon limited to mammals and other vertebrates
but are probably characteristic of skeletal muscles in general.
While we did not examine the anatomical correlates of fiber heterogeneity,
a number of studies have reported differences in muscle composition along the
length of fibers. Edman et al.
(1985) observed differences in
shortening velocity along the length of frog muscle fibers, and a recent study
of toad muscle demonstrated that strain patterns vary along the length of the
toad muscle fascicles (Ahn et al.,
2003
). These differences may be related to the variability in MHC
and myosin light chain content that has been reported along the length of
muscle fibers in Rana pipiens
(Lutz et al., 2001
), as well
as to mechanical factors such as connective tissue properties and muscle
architecture (Ahn et al.,
2003
). In birds, MHC isoforms are asymmetrically distributed
within single muscle fibers, with neonatal isoforms being concentrated at the
ends of the fibers (Bandman and Rosser,
2000
). In future studies, we will examine differences that may
exist at the protein and mRNA level along the length of the lobster
fibers.
Continuum among fiber types
Two compelling patterns are apparent from the results of this study. As
discussed above, fibers were found to contain a mixture of mRNA and protein
isoforms, verifying the existence of hybrid fibers that are intermediate
between what might be called pure fiber types. More importantly, these hybrid
fibers existed along a continuum from pure S1 to pure S2
fibers. This continuum was observed not only in the expression of MHC (Figs
6,
7,
8) but also for Tm
(Fig. 6) and TnT and TnI (Figs
7,
8,
9). At each end of the
continuum, a characteristic myofibrillar assemblage of isoforms exists that
typifies the pure S1 and S2 fibers. However, expression
of these isoforms exists as a gradient from high expression to low expression
and, in the span between the two extremes, fibers exist that possess isoforms
characteristic of both the S1 and the S2 fibers. Recent
studies have drawn attention to the commonplace of hybrid muscle fibers, and
the recognition that fibers are not discrete entities but represent a whole
range of subtly different phenotypes challenges our current understanding of
fiber types in skeletal muscles
(Stephenson, 2001;
Caiozzo et al., 2003
). What
are the factors that influence muscle phenotype and allow for a continuum of
physiological properties? In crustaceans, muscles are often controlled by
multiple motor neurons (Atwood,
1976
; Mykles et al.,
2002
), and this complexity probably contributes to fiber
heterogeneity. Claw and leg opener muscles of crayfish and lobsters exhibit a
continuum of phenotypes from S1 to S2 fibers that is
correlated with the synaptic efficacy. Proximal and distal fibers are
innervated by endplates that produce larger excitatory postsynaptic potentials
(EPSPs) than the central fibers (Mykles et
al., 2002
). In clear correlation with synaptic properties, the
proximal and distal fibers possess the TnT1 isoform associated with
S2 fibers, while the central fibers are the S1 phenotype
(Mykles et al., 2002
). This
pattern supports the hypothesis that the physiology of the excitatory motor
neuron has a direct effect on muscle phenotype and that gradations in neuronal
activity may produce a gradation in muscle phenotype. Additionally, skeletal
muscles are multinucleate cells, and recent studies have suggested that
myonuclear domains may differ with respect to the genes that are expressed
(Newlands et al., 1998
;
Allen et al., 1999
). A study of
transcriptional activity in cultured skeletal muscle fibers showed that gene
expression occurred in pulses and that nuclei within a muscle cell were not
active simultaneously (Newlands et al.,
1998
). Over the next several years, studies will reveal the
contribution of these domains to the existence of hybrid muscle fibers and,
more generally, the physiological relevance of single fiber polymorphism.
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Acknowledgments |
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