Expression of eight distinct MHC isoforms in bovine striated muscles: evidence for MHC-2B presence only in extraocular muscles
1 Dipartimento di Anatomia e Fisiologia Umana, Università di Padova,
Italy
2 Dipartimento di Scienze Sperimentali Veterinarie, Università di
Padova, Italy
* Author for correspondence (e-mail: masca{at}unipd.it)
Accepted 28 September 2005
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Summary |
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Key words: myosin, MHC isoforms, cattle, skeletal muscles, extraocular muscles, laryngeal muscles, RT-PCR, electrophoresis, immunohistochemistry
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Introduction |
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The inter-species diversity of the expression of the MHC-2B isoforms
between humans and laboratory animals has prompted further studies to assess
the expression in various species of veterinarian or experimental interest,
from monkey to dog, sheep and llama (for a review, see
Reggiani and Mascarello,
2004). In all these species, only three fibre types and three MHC
isoforms have been identified in skeletal muscles, two of them (types 1 and
2A) being very similar to the corresponding ones in rat and mouse and the
third one being similar to 2X. Thus, the 2B fibre type seems to be specific
only for small mammals and marsupials. A recent study suggests that in the
genome of some species, such as the horse, only a pseudogene with a sequence
similar to that of the MHC-2B gene is present
(Chikuni et al., 2004a
). There
are, however, exceptions: in pigs, the 2B isoform is expressed at the protein
level in several skeletal muscles, generally in hybrid 2X/B fibres
(Da Costa et al., 2002
;
Toniolo et al., 2004
), and in
cattle we have recently shown the expression of MHC-2B at the mRNA level in
extraocular muscles (Maccatrozzo et al.,
2004
).
The reliable identification of the presence of an MHC isoform as protein is
often more difficult in large mammals than in laboratory animals because of
the high percentage of hybrid fibres that express two or more isoforms. For
example, in pig skeletal muscles, 2A/X and 2X/B hybrid fibres are abundant in
trunk and limb muscles, whereas the pure 2B fibres are present only in
specialised muscles such as extraocular muscles
(Toniolo et al., 2004). It is
also important to note that antibodies normally used in laboratory animals may
give different results when employed in large animals, making it necessary to
check their specificity by a combination of several independent approaches.
For example, SC-71 monoclonal antibody (mAb), specific for MHC-2A in all
mammalian species studied so far, was not able to distinguish the two types of
fast fibres in bovine muscles (Duris et
al., 2000
). It is likely that the lack of specificity of the SC-71
antibody is due to the sequence similarity between bovine MHC-2X and MHC-2A
(Maccatrozzo et al., 2004
). In
our view (Reggiani and Mascarello,
2004
; Toniolo et al.,
2004
), to identify reliably 2X and 2B MHC in skeletal muscles in
different species, the histochemical and immunohistochemical results need to
be confirmed by (1) MHC gene expression analysis based on RT-PCR and (2)
identification of the corresponding proteins by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Our previous studies on bovine skeletal muscles
(Maccatrozzo et al., 2004)
have shown that the three fibre types identified by m-ATPase staining as type
1, 2A and conventional 2B express slow, 2A and 2X MHC isoforms, respectively,
and that the 2B gene is expressed at the mRNA level only in specific
muscles, such as retractor bulbi and extraocular muscles. The study of
2B expression in these specific muscles has allowed us to sequence a
fragment of the bovine 2B gene. It is still unknown whether the
protein corresponding to the gene coding for MHC-2B is present in bovine
skeletal muscles and, in particular, in bovine specialised muscles such as
extraocular muscles, where the corresponding mRNA is present. The reactivity
of some fibres with the antibody BF-F3 can only be considered as an indication
of the presence of the protein, in view of the above-mentioned limitations in
the reliability of anti-myosin antibodies. In the present study, we aimed to
demonstrate whether MHC-2B protein is present in bovine muscles and to extend
the investigation to laryngeal muscles, a group of muscles where expression of
MHC-2B can be expected. As recently reviewed by Hoh
(2005
), laryngeal muscles and,
in particular, thyreoarytenoideus muscle express, in several species, MHC
isoforms different from those found in the limb skeletal muscles. In rat and
rabbit, thyreoarytenoideus muscle expresses MHC-2B and MHC-Eo (extraocular);
there is also evidence in favour of the presence of MHC-2B in cat
(Hoh, 2005
) and possibly also
in dog (Wu et al., 2000
). In
bovine thyreoarytenoideus muscle, fibres show an unusual histochemical profile
(Mascarello and Veggetti,
1979
) and this suggests the presence of some isoforms different
from those present in limb muscles.
In view of our goal, samples from adult and foetal bovine skeletal muscles and from atrial and ventricular bovine myocardium were analysed by combining immunohistochemistry, RT-PCR and gel electrophoresis to identify all expressed MHC isoforms. The results obtained showed that MHC-2B is present as protein in extraocular muscles but not in laryngeal muscles. The second aim of the study was to describe the contractile properties of bovine single muscle fibres. In this respect, the identification of the electrophoretic bands corresponding to MHC isoforms was a pre-requisite to classify the fibres whose isometric tension and maximum shortening velocity were measured. On the whole, the results provide a complete picture of bovine fibre types with regard to their MHC isoform composition and their contractile properties.
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Materials and methods |
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RNA extraction and RT-PCR
Fragments (100 mg) of the muscle samples to be used for RNA analysis
were immersed as quickly as possible in RNA Later Reagent (Ambion, Austin, TX,
USA). Total RNA was extracted from muscle tissue using TRIZOL® reagent
(Gibco-BRL, Gaithersburg, MD, USA) and reverse-transcribed with Superscript II
protocols (Invitrogen, Life Technologies, Paisley, UK) using a mixture of
random hexamers as primer. The obtained cDNAs were used as template for RT-PCR
analysis. PCR reactions and bovine-specific primers used to amplify MHC-1, 2A,
2X and 2B isoforms were described in a previous study
(Maccatrozzo et al., 2004
).
Primers for MHC-1, 2A and 2X were kindly provided by Prof. K. Chikuni; the
primers for MHC-2B were designed by our group
(Maccatrozzo et al., 2004
);
new primers were prepared for extraocular and neonatal MHC; their sequences
are shown in Table 1. Primers
for neonatal MHC were designed from sequences available in GenBank (accession
number AB090156), whereas for extraocular MHC a first cloning was obtained
using degenerate primers and, from the sequence obtained with degenerate
primers, new specific primers were designed
(Table 1).
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Gel electrophoresis
Muscle samples for protein electrophoresis were solubilized at 90°C for
5 min in Laemmli buffer solution [Tris 62.5 mmol l-1, pH 6.8,
glycerol 10%, SDS 2.3%, ß-mercaptoethanol 5%, with E-64 0.1% and
leupeptin 0.1% (Sigma, St Louis, MO, USA) as antiproteolytic factors].
Two distinct protocols were used to determine the composition of MHC
isoforms in muscle samples. A first protocol was used to separate all MHC
isoforms except the two cardiac isoforms (MHC-ß and MHC-). Samples
were analysed on 9% polyacrylamide slab gels after denaturation in SDS
(SDS-PAGE) with a procedure derived from Blough et al.
(1996
), modifying stacking gel
composition with 29% glycerol. Slabs 18 cm wide, 16 cm high and 1 mm thick
were used. Electrophoresis was carried out at 4°C for 46 h, at100 V for
the first 3 h and at 230 V for the remaining time. Gels were silver stained
(Silver stain plus, Biorad, Hercules, CA, USA). Six bands were separated in
the region of 200 kDa, corresponding, in order of migration rate from the
fastest to the slowest, to MHC-1, MHC-Eo, MHC-2B, MHC-Neo and MHC-2X, which
co-migrated, MHC-Emb and MHC-2A.
A second protocol was used to separate the two cardiac isoforms (MHC-ß
and MHC-). The separation was obtained on 8% polyacrylamide slab gels
after denaturation in SDS (SDS-PAGE) and was based on the procedure described
by Talmage and Roy (1993), modifying run parameters. Electrophoresis was
carried out at 4°C for 40 h, at 70 V for the first 2 h and 170 V for the
remaining time. Gels were silver stained (Bio-Rad Silver stain plus).
The identification of the electrophoretic bands in the gel region corresponding to 200 kDa as distinct MHC isoforms was obtained by comparing RT-PCR results, immunohistochemistry assay and protein electrophoresis, as described in the Results.
RT-PCR and gel electrophoresis on fibres and fibre bundles
Small muscle fragments (<10 mg), bundles of fibres (2-10 fibres) and
single fibres were manually dissected from samples of rectus lateralis (Rl),
retractor bulbi (Rb) and diaphragm (D) muscles in skinning solution (see
below) under a stereomicroscope (10-60x magnification). Fibre bundles
and single fibres were transversally cut into two parts (each of them 2
mm long), one half of which was immersed in 200 µl TRIZOL® reagent and
used for total RNA extraction whereas the other half was immersed in 20 µl
of Laemmli solution to solubilize proteins for MHC electrophoretic
analysis.
RNA was extracted with the same protocol used for larger samples, scaling down the reaction. The cDNA reverse-transcribed from extracted total RNA was used to test the expression of adult MHC isoforms (1, 2A, 2X and 2B), extraocular (Eo) and neonatal (Neo) MHC isoforms. The products obtained with PCR were used as templates in a second round of PCR with the same conditions because of the small amount of starting sample. To assess the quality of the RNA and the efficiency of the RT reaction, a fragment of the bovine ß-actin cDNA was amplified for each sample. From the Laemmli solution containing solubilized proteins, a 3 µl sample was used for electrophoretic separation of MHC isoforms under the condition described above.
Immunohistochemistry
A specialized extraocular muscle (rectus lateralis) was combined with a
skeletal muscle (longissimus dorsi) into a composite block and frozen in
isopentane cooled with fluid nitrogen. Serial sections (10 µm) were cut in
a cryostat and stained according to the protocol described by Maccatrozzo et
al. (2004) with the following
antibodies: monoclonal BF-F3, which is specific for MHC-2B in rat and in pig
(Lefaucheur et al., 2002
;
Toniolo et al., 2004
),
polyclonal anti-MHC-Eo (Sartore et al.,
1987
) and polyclonal anti-MHC-Neo
(Mascarello and Rowlerson,
1992
). The procedures to assess the specificity of the reactions
were described in Maccatrozzo et al.
(2004
).
Single fibre mechanics
Muscle samples from masseter, diaphragm and longissimus dorsi were immersed
in ice-cold skinning solution (see below), divided into small fibre bundles
and stored at -20°C in a mixture of skinning solution and 50% glycerol. On
the day of the experiment, the bundles were removed from the freezer and
repeatedly washed with skinning solution. Single fibres were manually
dissected from fibre bundles under a stereomicroscope (10-60x
magnification). At the end of the dissection, fibres were bathed for 1 h in
skinning solution containing 1% Triton X-100 to ensure complete membrane
solubilization. Segments of 1-2 mm in length were then cut from the fibres,
and light aluminium clips were applied at both ends.
Skinning, relaxing, pre-activating and activating solutions utilized for
mechanical experiments with single fibres were prepared as previously
described (Bottinelli et al.,
1996). Preactivating solution had a composition similar to the
relaxing solution except that EGTA concentration was reduced to 0.5 mmol
l-1, and 25 mmol l-1 creatine phosphate and 300 U
ml-1 creatine phosphokinase were added. Activating solution was
similar to the relaxing solution except for the addition of 5 mmol
l-1 CaCl2, 25 mmol l-1 creatine phosphate and
300 U ml-1 creatine phosphokinase. The pH of all solutions was
adjusted to 7.0 at the temperature at which solutions were used (12°C).
Protease inhibitors [E64 (10 µmol l-1) and leupeptin (40 µmol
l-1)] were present in all solutions.
In each fibre segment, isometric tension (P0) and
unloaded shortening velocity (v0) were measured during
maximal activations at 12°C, pCa=4.8 under the conditions described in
previous studies (Pellegrino et al.,
2003; Toniolo et al.,
2004
). The kinetics of tension redevelopment was measured from the
time course of tension development after a fast (<1 ms)manoeuvre of
shortening to reduce tension to zero and fast re-lengthening back to the
initial length after an interval of 30 ms. Tension redevelopment was recorded
and characterised by the time required to redevelop two-thirds of isometric
tension. Images of each fibre were taken with a camera connected to the
microscope at 360x magnification. Cross-sectional area (CSA) was
calculated from the average of three diameters, spaced at equal intervals
along the length of the fibre segment, assuming a circular shape. Sarcomere
length was determined by counting sarcomeres at several intervals of 30
µm.
Statistical analysis
Data were expressed as means and standard errors. Statistical significance
of the differences between means was assessed by analysis of variance (ANOVA)
followed by the Student-Newman-Keuls test. A probability of less than 5% was
considered statistically significant.
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Results |
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When electrophoretic protocol was applied to extraocular muscles (Rl and
Rb), a high number of bands (up to six) was detected in the region of 200-220
kDa (Fig. 1A). RT-PCR showed
that all four adult MHC isoforms (i.e. 1, 2A, 2X and 2B) were expressed
(Fig. 1B; see also
Maccatrozzo et al., 2004). To
identify other bands detectable in the MHC region, we first analysed immature
bovine muscles. Fig. 1C shows
MHC isoform separation in samples of foetal muscles: two bands, one migrating
above the MHC-2X band and one co-migrating with the MHC-2X band, are clearly
detectable and correspond to neonatal (MHC-Neo) and embryonic (MHC-Emb)
isoforms. As expected (Picard et al.,
1994
), at 120 days after conception no other MHC isoforms are
expressed.
Using a specific protocol (8% gels, see Materials and methods) for
separation of cardiac MHC from MHC-1 or ß/slow, the two cardiac
isoforms were resolved. Fig. 2
shows the two cardiac MHC isoforms in bovine atrial and ventricular
myocardium, which contain
and ß MHCs, respectively
(Gorza et al., 1986
). Among
the muscles examined, a weak band of
MHC was detected only in masseter
(Figs 2,
6), whereas both rectus
lateralis and retractor bulbi muscles showed the presence of
cardiac
MHC isoform (data not show).
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Fig. 3 shows
immunohistochemical staining of a composite block with rectus lateralis and
longissimus dorsi. Serial sections showed that individual fibres of rectus
lateralis were reactive with the antibodies anti-MHC-Neo, anti-MHC-Eo and
anti-MHC-2B (BF-F3), whereas longissimus dorsi was completely negative when
exposed to the same antibodies. It is interesting to note that, in the
extraocular muscle, some fibres react with all three antibodies (asterisks in
Fig. 3),many fibres react with
two of them (circles in Fig. 3) and one fibre reacts only with anti 2B (hash mark in
Fig. 3). Asdescribed in
previous studies (Jacoby et al.,
1990; Lucas and Hoh,
1997
), hybrid fibres are particularly abundant in extraocular
muscles.
The unambiguous identification of electrophoretic bands required the parallel analysis of MHC isoforms at the RNA and protein level in samples with different MHC isoform composition. RNA and protein identification from individual fibres was first validated on diaphragm fibres (N=10) and then applied to bundles of few (2-10) fibres from extraocular muscles, since fibres were very thin in these muscles and did not yield enough material to determine protein and RNA in a single fibre. The analysis of such fibre bundles permitted us to completely identify the relative positions of MHC isoforms in electrophoretic migration. Fig. 4 shows three typical small bundles with increasing numbers of electrophoretic bands. Fibre bundle 10Rb exhibited two electrophoretic bands and two RT-PCR products: MHC-2A, with the slowest electrophoretic migration (see above), and MHC-Eo. Thus, the second band in 10Rb could be identified as MHC-Eo with a migration position above MHC-slow (compare with the larger samples of retractor bulbi, where five bands are visible). The migration position of MHC-Emb was determined in fibre bundle 7Rl. At RNA level, two products corresponding to 2A and Eo were amplified. A third band showed a migration position identical to one of two developmental isoforms (just below MHC-2A; see Fig. 1C). This band was identified as MHC-Emb, because in this sample no MHC-Neo was amplified with RT-PCR. A similar analysis on bundle 7Rb from retractor bulbi confirmed the migration positions of MHC-2A, MHC-Emb, MHC-2X and MHC-Eo. Finally, the analysis of larger samples of retractor bulbi (Rb in Fig. 4), where neonatal, Eo, 2A, 2X, 2B and slow MHC isoforms were expressed, made it possible to determine the migration position of MHC-2B. MHC-2B migration was intermediate between MHC-Eo and MHC-2X, which co-migrated with MHC-Neo (see also Fig. 1C). Thus, the immunohistochemical (Fig. 3) and the electrophoretic (Fig. 4) results both support the presence of MHC-2B protein in rectractor bulbi and rectus lateralis.
Since MHC-2B might be expressed also in laryngeal muscles (see for a review
Hoh, 2005), we extended the
analysis to bovine laryngeal muscles. Results are shown in
Fig. 5. All specialized
laryngeal muscles expressed MHC-1 and MHC-2A, both at the RNA (data not shown)
and the protein level (Fig.
5B). MHC-2X was present in arytenoideus transversus (At) and in
thyreoarytenoideus (Tvr, Tvc, Tvo). MHC-Neo was expressed in rostral
ventricularis portion (Tvr) and in the vocalis portion (Tvo) of
thyreoarytenoideus muscle (see Fig.
5A,B, where the band corresponding to MHC-Neo is superimposed on
the MHC-2X band). MHC-2B and MHC-Eo isoforms were absent in all laryngeal
samples, both at the RNA and the protein level (see
Fig. 5A,B).
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The values of v0 determined in the fibres dissected
from masseter showed a clear bimodal distribution, as can be seen in
Fig. 6. The electrophoretic
analysis on 8% gels showed the presence of MHC- at various proportions
in a group of fibres dissected from masseter. In this way, two groups could be
formed: one containing only MHC-ß/slow and showing lower average
v0 (0.25±0.02 L s-1) and a
second also containing MHC-
and characterised by higher average
v0 (0.668±0.08 L s-1;
P<0.01 compared with the former group). P0 was
not significantly different between the two groups (53.8±6.8
vs 54.5±7.0 mN mm-2), and the same was true for
cross-sectional area (1401±89 vs 1536±182
µm2). Slow fibres (i.e. fibres containing only MHC-ß/slow)
from masseter showed v0 values similar to those isolated
from diaphragm (0.29±0.03 L s-1); this was in
agreement with our previous observations
(Toniolo et al., 2004
) that
fibres with the same MHC isoform composition have similar values of
v0 regardless the muscle of origin. It was thus possible
to form a large homogeneous group of slow fibres, whose parameters
(v0, P0 and CSA) are reported in the
histograms of Fig. 7 together
with other groups of fibres, formed on the basis of their MHC isoform
composition regardless of the muscle of origin.
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The mean CSA values of single fibres grouped according to their MHC isoform composition are shown in Fig. 7A. Slow fibres were much thinner than fast fibres: this was true for both slow fibres from masseter (1401±89 µm2, N=25) and slow fibres from diaphragm (2050±274 µm2, N=13). Fast 2A and 2X fibres were significantly thicker.
Slack sarcomere lengths were not significantly different among fibre types: 2.38±0.04 µm for slow fibres, 2.42±0.08 µm for 2A fibres, 2.44±0.09 µm for 2X fibres. Since fibres were stretched by approximately 20% after mounting, the activation was induced at sarcomere lengths of 2.78±0.04 µm in slow fibres, 2.73±0.08 µm in 2A fibres and 2.78±0.11 µm in 2X fibres.
Fibres with different MHC isoform composition exhibited large diversity in
maximum v0 and, to a lesser extent, in
P0 (see Fig.
7 where statistical comparisons are reported).
v0 values increased from slow to fast 2A and 2X fibres,
with intermediate values in fibres containing MHC- and hybrid 1-2A
fibres. Fast 2X fibres had a v0 value significantly higher
than fast 2A fibres. Series compliance (expressed relative to fibre segment
length) was not significantly different among the fibre groups, ranging from
5.3±1.4% in slow fibres to 6.6±0.9% in 2X fibres.
The time course of tension redevelopment after a manoeuvre of fast shortening and re-lengthening, with an interval of 30 ms, was recorded and characterised by the time required to redevelop two-thirds of isometric tension. Slow fibres showed the longest redevelopment time (1.94±0.16 s), 2X fibres showed the shortest (0.29±0.02 s), and 2A fibres showed an intermediate value (0.60±0.22 s).
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Discussion |
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The procedure adopted for MHC isoform identification was similar to that
utilized in our recent study on pig muscles
(Toniolo et al., 2004). The
specificity of monoclonal antibodies needs to be validated in each species,
and the order of electrophoretic migration of MHC isoforms might change from
species to species (Reggiani and
Mascarello, 2004
). Thus, MHC isoforms were first identified at the
mRNA level, since this allows an unambiguous identification based on the
comparison of base or amino-acid sequences with orthologous isoforms in other
animal species. Then, immunohistochemistry and electrophoresis were employed
on those samples that had been previously analysed with RT-PCR, and the
presence of electrophoretic bands was considered the ultimate proof of the
existence of a protein product. Importantly, electrophoresis could be applied
to determine MHC isoform composition in single muscle fibres that had been
characterised with regard to their contractile properties, and this provided a
functional validation of the MHC identification.
The expression of MHC-2B is of special interest as it represents a
controversial issue. This isoform is abundantly expressed in small rodents,
rabbit and marsupials (Schiaffino and
Reggiani, 1996; Zhong et al.,
2001
). In human muscles, it is present as mRNA but not as protein
(Smerdu et al., 1994
;
Horton et al., 2001
). Recent
studies (Chang and Fernandes,
1997
; Chikuni et al.,
2001
; Toniolo et al.,
2004
) have shown MHC-2B expression in porcine skeletal muscles.
Controversial results have been obtained with gel electrophoresis in bovine
muscles, as two (Totland and Kryvi,
1991
; Jurie et al.,
1995
) or three (Picard et al.,
1999
) fast MHC isoforms have been separated in adult skeletal
muscles. RT-PCR has unambiguously shown that only MHC-2A and MHC-2X are
expressed in trunk and limb muscles of cattle
(Tanabe et al., 1998
;
Chikuni et al., 2004b
). Our
results confirm that only 1, 2A and 2X MHC isoforms are expressed in trunk and
limb muscles (Maccatrozzo et al.,
2004
) but also show (present study) the presence of MHC-2B as mRNA
and protein in specialized extraocular muscles. This finding is clearly in
contrast with the conclusion of Chikuni and co-workers
(Chikuni et al., 2004b
) that a
functional gene coding for MHC-2B is not present in the genome of all
ungulates examined, including the water buffalo (Bubalus bubalis), a
species strictly related to cattle (Bos taurus). Our results are in
accordance with the information recently made available in GenBank showing the
sequence of a gene coding for MHC-2B in the cattle genome (accession number
XM_615303).
Interestingly, our finding that MHC-2B is present in specialized muscles as
mRNA and protein is at variance with the observation that, in some human
muscles, MHC-2B is present as mRNA but not as protein
(Horton et al., 2001). The
lack of a protein in fibres where the corresponding mRNA is present implies a
post-transcriptional regulation that either inhibits the translation of the
protein or induces a prompt degradation, making accumulation in the cytosol or
in the myofibrils impossible. An example of incomplete transcription followed
by degradation is the MHC-2M isoform in human masticatory muscles
(Stedman et al., 2004
). If the
observations by Horton and co-workers raise a question about
post-transcriptional regulation, our finding that MHC-2B expression is
restricted to extraocular muscles raises a question about transcriptional
regulation. Why is expression restricted to such specialized muscles? Several
MHC isoforms are expressed in extraocular muscles, as suggested by published
evidence on various animal species [mouse
(Porter et al., 2001
), rat
(Wieczorek et al., 1985
;
Rubinstein and Hoh, 2000
),
rabbit (Briggs and Schachat,
2002
; Lucas and Hoh,
2003
)]. In particular, MHC-Eo
(Sartore et al., 1987
),
developmental MHCs (Jacoby et al.,
1990
) and
cardiac MHC
(Wieczorek et al., 1985
;
Lucas and Hoh, 2003
) can be
expressed in extraocular muscles. Whether such peculiar expression should be
related to the embryological origin or to the specialized contractile
requirements of extraocular muscles is a matter of speculation. MHC-2B and
MHC-Eo are expressed both in rectus lateralis and retractor bulbi. Whereas
rectus lateralis is involved in a variety of motor tasks, such as fixation,
pursuit and saccades, which might require fibres expressing different MHC
isoforms, retractor bulbi does not have any known motor function. The
mechanisms leading to stabilization of the expression of developmental
isoforms in specialized laryngeal and extraocular muscles are also unknown.
Bovine laryngeal muscles are rather similar to trunk and limb muscles in their
MHC isoform expression, as MHC-Eo and MHC-2B are not present. In
thyreoarytenoideus, a fourth MHC isoform was present and was identified as
MHC-Neo. The expression of an additional MHC isoform is a typical feature of
thyreoarytenoideus as discussed by Hoh
(2005
) and may need further
investigation.
A last `open issue' concerns the presence of MHC- in masseter.
Although this is the first demonstration of the expression of MHC-
in
bovine masseter, the presence of this isoform in masticatory muscles is not
surprising as it has been found in human, rabbit and marsupial masseter
(Bredman et al., 1991
;
Hoh et al., 2000
). As
discussed by Hoh et al.
(2000
), fibres expressing
MHC-
are particularly suited for the diet and the chewing action of
wallaby and kangaroo, where masseter is homogeneously composed of this
isoform. The masticatory function of cattle, i.e. the slow, rhythmic and
long-lasting rumination, seems to fit well with the expression of MHC-slow. It
is likely that for other types of movement, e.g. grasping grass and grazing, a
small group of fast motor units is needed and, in those fast fibres,
MHC-
might replace MHC-2A, which is not expressed in bovine masticatory
muscles.
The second novel result of this study is the characterisation of
contractile properties of single skeletal muscle fibres in relation to their
MHC isoform content. Fibres containing the three major MHC isoforms -
ß/slow/1, 2A, 2X and the less-studied MHC- - have been
characterised. In a previous study (Seow
and Ford, 1991
), only two types of fibres were described - fast
and slow - without any further identification of fast subtypes. Here, five
types (1,
, 1-2A, 2A, 2X) were characterised on the basis of a large
pool of fibres dissected from masseter, diaphragm and longissimus dorsi. Until
now, only one study has measured maximum shortening velocity in skeletal
muscle fibres expressing MHC-
(Sciote and Kentish, 1996
).
Fibres were dissected from rabbit masseter and their v0
was intermediate between slow and fast 2A fibres. Similar conclusions were
reached in a recent study (Andruchov et
al., 2004
) where tension transients after a small and fast
elongation were analysed. Our results indicate that fibres containing
MHC-
are similar to hybrid 1-2A fibres in their speed of shortening,
tension development and, thus, in power output. The functional advantage given
by the presence of MHC-
is not clear (see above), but one might
speculate that fibres expressing MHC-
alone or together with MHC-ß
represent more stable units than hybrid 1-2A, which are considered transition
fibres.
Interestingly, bovine fibres dissected for this study from cows of more
than 400 kg were rather thin (range 1700 µm2 for slow fibres and
5600 µm2 for 2X fibres, after swelling due to chemical skinning)
compared with those dissected from other mammalian species in the same
experimental conditions [see, for example, human fibres
(Bottinelli et al., 1996) and
pig fibres (Toniolo et al.,
2004
)]. Extraocular and laryngeal muscles provided fibres too thin
to be successfully analysed in mechanical experiments. The low values of
diameters of bovine skeletal muscle fibres were observed also by Ford and
co-workers (Seow and Ford,
1991
), who proposed an inverse correlation between isometric
tension and diameters. Actually, we confirmed that values of isometric tension
developed by fast bovine fibres are rather high in comparison with
corresponding fibres from other animal species (see
Fig. 8).
|
In conclusion, our results provide a general and complete description of bovine striated muscles at the fibre level, particularly with regard to specialized muscles such as masseter, extraocular and laryngeal muscles, myosin isoform expression and contractile properties. The most intriguing finding of this study is the evidence of MHC-2B expression at the protein level in those muscles where the corresponding mRNA is present, i.e. extraocular muscles. This finding raises new and unanswered questions about signalling pathways and gene expression regulation concerning the MHC-2B isoform.
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Acknowledgments |
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References |
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