Marked load-bearing ability of Mytilus smooth muscle in both active and catch states as revealed by quick increases in load
1 Department of Physiology, School of Medicine, Teikyo University,
Itabashi-ku, Tokyo 173-8605, Japan
2 Department of Electronic Engineering, Shibaura Institute of Technology,
Minato-ku, Tokyo 108-8548, Japan
* Author for correspondence (e-mail: sugi{at}med.teikyo-u.ac.jp)
Accepted 12 February 2004
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Summary |
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Key words: Mytilus edulis, smooth muscle, catch state, load-bearing ability, series elastic component, isotonic lengthening, parallel hypothesis
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Introduction |
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There are two different hypotheses to account for the catch mechanism. One
is the linkage hypothesis, in which the catch state is associated with a
marked decrease in dissociation rate of actinmyosin linkages between
the thick and thin filaments (Lowy and
Millman, 1963). The other is the parallel hypothesis, which
assumes, in addition to the actinmyosin linkages responsible for active
force development, formation of linkages interconnecting the
paramyosin-containing thick filaments
(Johnson et al., 1959
;
Heumann and Zebe, 1968
). The
parallel hypothesis is based on electron microscopic evidence that a marked
aggregation of the thick filaments takes place during the catch state
(Heumann and Zebe, 1968
;
Gilloteaux and Baguet, 1977
;
Hauk and Achazi, 1987
). It has
been pointed out, however, that the thick filament aggregation may be an
artifact resulting from glutaraldehyde fixation
(Miller, 1968
). In fact,
Bennett and Elliott (1989
), who
used techniques of quick-freezing and freeze-substitution, observed no thick
filament aggregation in the ABRM fibres during the catch state. By contrast,
using the same techniques of quick-freezing and freeze-substitution, Takahashi
et al. (2003
) found that
linkages interconnecting the thick filaments are associated with the catch
state. Thus, the mechanism underlying the catch state still remains
obscure.
Since the physiological function of the catch state is to resist externally applied forces, its physiological characteristics may best be understood by studying the load-bearing ability of the ABRM. The present experiments were undertaken to examine mechanical responses of the ABRM fibres to quick increases in load applied at various states. It will be shown that the load-bearing ability of the ABRM fibres is several times larger than that of skeletal muscle fibres, indicating the presence of the load-bearing system other than the actomyosin system.
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Materials and methods |
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Transducers
The displacement transducer was a differential transformer (ME Commercial
Co., Tokyo, Japan; modulation frequency, 5.5 kHz) with an aluminium lever
pivoted on bearings. The distal part of the lever dipped into the experimental
solution, and the preparation was connected at a point 3 cm distant from the
pivot. The compliance of the lever at the point of attachment of the
preparation was 0.5 µm g1. The force transducer was a
strain gauge (U-gauge, Shinko, Tokyo, Japan) with the compliance of 1 µm
g1 and the natural frequency of oscillation of 150
Hz.
General procedure
The experimental arrangement is illustrated in
Fig. 1. The lever of the
displacement transducer was initially fixed in position by stops 1 and 2. The
preparation was made to contract actively with a supramaximal concentration of
acetylcholine (ACh, 103 mol l1), and at
various times after the onset of force development, the load on the
preparation was quickly increased by withdrawing stop 2 electromagnetically,
so that the preparation was lengthened under a load imposed by the loading
spring. The lengthening of the preparation under a large load was limited to
10% of L0 by another stop 3, to avoid damage to the
preparation due to a large lengthening, and also to avoid the resting force
development during lengthening. The maximum isometric force
P0 ranged from 6 to 25 g.
The preparation was first subjected to a quick increase in load during
generation of the maximum ACh-induced isometric force P0.
After recording the resulting fibre lengthening, the preparation was restored
to its original length and ACh was removed to put the preparation into the
catch state, which was established 35 min after the ACh removal
(Sugi et al., 1999). The
preparation was then subjected to quick increases in load at various force
levels Px. The experiments were also performed, in which
quick increases in load were applied to the preparation in the catch state
without preceding quick increases in load at P0, with
similar results. In some experiments, quick increases in load were applied
during the early phase of ACh-induced isometric force development. In some
experiments, the length and force changes of the preparation were recorded
using an oscilloscope. The length and force changes were complete in 5 ms.
The length and force changes were recorded on an ink-writing oscillograph. After each experiment, the preparation was relaxed by 5-HT (106 mol l1), and kept for 510 min before it was again stimulated by ACh. All experiments were done at 1823°C
High-speed cinematography of the segmental length changes of the preparation
To examine whether the mechanical response of the preparation is associated
with uniform length changes along the entire length of the preparation, a
number of carbon particles were firmly attached to the preparation and the
lengths of 56 segments between the particles were measured on
videotapes taken at 240 frames s1 with a high-speed video
system (FASTCAMrabbit 1, Photoron, Tokyo, Japan;
Kobayashi et al., 1998) during
the experiments.
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Results |
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The initial elastic extension of the preparation (SEC) is thought to
be due to extension of the series elastic component
(Tameyasu and Sugi, 1976
;
Sugi and Tsuchiya, 1979
),
while the subsequent two phases of isotonic lengthening reflect
characteristics of load-bearing structures in the ABRM fibres. We measured the
amplitude of the early isotonic lengthening
L by extrapolating
the late steady lengthening back to the moment of quick load changes
(Fig. 2, inset). As the catch
force falls gradually with time, the force level Px at
which a quick increase in load was applied during the catch state, ranged from
0.2 to 0.6P0.
Marked load-bearing ability of the ABRM fibres in both the active and the catch state
The load-bearing ability of the ABRM fibres was found to be extremely
large, both during generation of the maximum ACh-induced active force and
during the catch state. As shown in Fig.
3A, the preparation generating the maximum active force
P0 could bear a load up to
1015P0 for more than 3060 s without being
lengthened rapidly. The marked load-bearing ability contrasts with that of
tetanized vertebrate skeletal muscle fibres, which are unable to bear a load
above 1.82P0 and are lengthened rapidly (`give';
Katz, 1939;
Sugi and Tsuchiya, 1981
). The
marked load-bearing ability of the ABRM fibres was well maintained during the
catch state though the catch forces Px were much smaller
than P0. The preparation could bear a load up to
1530Px (corresponding to
1015P0, or approximately 100150 kg
cm2) (Fig.
3B); the marked load-bearing ability was still observed even when
the catch force was reduced to 0.2P0.
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If, on the other hand, the load applied to the ABRM fibres exceeded 1015P0 (or 1530Px), they were lengthened rapidly by 10% of L0 within 1 s after the load application, so that the lever, to which the preparation was hooked, came in contact with stop 3, which was fixed in position (see Fig. 1). As the result, the load was supported by stop 3 but not by the preparation, and the subsequent force changes in the preparation showed the time course of stress relaxation, i.e. decay of the force in the preparation after completion of lengthening (Fig. 4A). The rapid ABRM fibre lengthening under a large load is thought to correspond to `give' in the vertebrate skeletal muscle fibres. After showing `give', the preparation could still develop full isometric force P0 when again stimulated with ACh, indicating that the `give' was associated with rapid slippage of linkages in the contractile system but not with any irreversible damage to the preparation.
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Limited load-bearing ability of the ABRM fibres during the development of isometric force
When the load on the ABRM fibres was increased quickly during the early
phase of ACh-induced force development (force level
Px<0.5P0), they were easily made to
lengthen rapidly under small loads, as indicated by the appearance of stress
relaxation of the force in the preparation immediately after completion of
fibre lengthening (Fig. 4B).
The `give' of the preparation during the early phase of force development took
place with loads above 1.52P0 or
34Px, which were comparable to those causing `give'
in skeletal muscle fibres (Katz,
1939; Sugi and Tsuchiya,
1981
). This indicates that the linkages producing the early active
force development can be made to slip rapidly with loads much smaller than
those causing the rapid slippage of the linkages responsible for production of
the maximum active force or the catch force. When the force developed above
0.5P0, the load-bearing ability increased abruptly with
increasing force towards P0, though the time course was
not studied in detail.
Dependence of the parameters of the mechanical response on the magnitude of quick increases in load
The parameters of the mechanical response of the ABRM fibres to quick
increases in load are shown in Fig.
5 asfunctions of load P. All the data points were
obtained on one and the same preparation, and similar results were obtained
from 12 other preparations examined. In
Fig. 5A, the amount of
extension of the SEC (SEC, expressed as percentage of
L0) is plotted against the magnitude of load (P
expressed as a fraction or multiple of P0 or
Px). Despite a large variation in the value of
Px, all data points obtained during the catch state fell
on the same
SEC versus P curve, The value of
SEC for a
given amount of P was smaller during the catch state (open circles)
than during the maximum active force generation (filled circles), indicating
that the SEC is stiffer during the catch state than during the maximum active
force generation.
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In Fig. 5B, the amplitude of
the early isotonic lengthening L is plotted against
P. As the late steady fibre lengthening under large loads was
terminated by stop 3 in 2030 s after the load change, the velocity of
steady isotonic lengthening (and therefore the value of
L)
could be determined accurately only under loads <15P0.
The data points in the catch state also fell around the same curve. As with
the
SEC versus P relationship, the value of the
L for a given amount of P was smaller during the
catch state than during the maximum active force generation, indicating that
the ABRM fibres are less extensible during the catch state (open circles) than
during the maximum active force generation (filled circles).
Fig. 5C shows the relationship
between the velocity V of the late steady isotonic lengthening and
P. The value of V increased with increasing P, and
the curve of V versus P appeared to be similar both during the
maximum active force generation (filled circles) and during the catch state
(open circles).
Segmental length changes of the ABRM fibres following quick increases in load
Measurement of the segmental length changes of the preparation following a
quick increase in load indicated that the isotonic lengthening always took
place uniformly along its entire length, as shown in
Fig. 6. Even when the
preparation exhibited `give', i.e. rapid fibre lengthening under a large load
(see Fig. 4), all fibre
segments were uniformly lengthened (accuracy of measurement <5%),
indicating that `give' was associated with uniform fibre lengthening but not
with any localized lengthening of the weakest fibre segment. Similar results
were obtained with three other preparations examined
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Discussion |
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During the ACh-induced active contraction of the ABRM fibres, the maximum
shortening velocity Vmax is largest during the isometric
force development, and is reduced to about onethird (from
0.25L0 s1 to
0.08L0 s1) when the force reaches
P0 (Tameyasu and Sugi,
1976
). This may be due to a slowing of cycling of
actinmyosin linkages during the course of contraction, or
alternatively, to a special force-bearing system, which is gradually built up
during the course of active force development. When the active force is
maximum, the force-bearing structure may be fully built up to produce a
load-bearing ability far greater than that expected from actinmyosin
linkages; the reduction of Vmax at P0
may result either from an increased internal resistance against shortening due
to the force-bearing structure and/or from a decrease in the number of
actinmyosin linkages. When the catch state is established, the force
may only be maintained by linkages of the load-bearing system.
Physiological role of the load-bearing system
The build-up of the load-bearing structure during the active force
development, as revealed in the present work, can be understood well by
considering how a typical catch muscle, for example the adductor muscle of a
bivalve, works in its natural environment. When the animals are attacked by
natural enemies, they rapidly close their shells by active shortening of the
adductor muscle, and as soon as the shell is closed the adductor muscle
resists against external forces acting to open the shell. To be able to do
this, the force-bearing structure should be built up during the period of
active muscle shortening produced by actinmyosin sliding. After the
shell is closed, the adductor muscle may gradually go into the catch state, in
which the marked load-bearing ability is well maintained only by the
load-bearing structure, with an extremely small rate of energy consumption.
This idea is supported by the reports that actomyosin ATPase activity is
absent in the ABRM during the catch state
(Güth et al., 1984;
Galler et al., 1999
).
Relation with previous studies
The isotonic lengthening of ABRM fibres was studied by Sugi and Tsuchiya
(1979), but they only focused
attention on early isotonic fibre lengthening with decreasing velocity, and
overlooked the late isotonic lengthening. During the early isotonic
shortening, i.e. during phase 2 (Fig.
2), the velocity of lengthening decreases markedly, and in the
subsequent late isotonic lengthening the ABRM fibres can bear very large loads
while being lengthened extremely slowly (Figs
2 and
3). This marked decrease in the
lengthening velocity in early isotonic lengthening suggests that the build-up
of the load-bearing structure is accelerated when the fibres are lengthened
under a large load.
According to the two-component model
(Hill, 1938), a muscle
consists of the SEC and the contractile component (CC) connected in series.
Jewell and Wilkie (1960
)
showed that the SEC in a muscle originates partly from connections of
experimental apparatus, and partly distributes along the muscle length. The
extension of the SEC in the experimental apparatus can now be minimized by
using single fibres or small fibre bundle preparations, and by reducing
compliance of the experimental apparatus. In single skeletal muscle fibres,
the extension of the SEC under P0 is about 1% of the fibre
length L0, corresponding to about 10 nm per
half-sarcomere. The SEC in skeletal muscle fibres is therefore interpreted to
largely originate from the elasticity of actinmyosin linkages
(Huxley and Simmons, 1971
). In
the ABRM fibres, the extension of the SEC under P0 is
about 2% of L0
(Tameyasu and Sugi, 1976
;
Sugi and Tsuchiya, 1979
).
Based on the very long lengths of thick and thin filaments in the ABRM
(Gilloteaux and Baguet, 1977
;
Squire, 1981
), the functional
and structural unit in the ABRM, corresponding to the half-sarcomere in
skeletal muscle, is very long, being of the order of 10 µm. The extension
of the SEC at P0 is therefore too large to be explained by
extension of actinmyosin linkages. Since the SEC has been shown to
distribute uniformly along the entire length of the ABRM fibres
(Sugi and Tsuchiya, 1979
), it
may originate from some elastic structures uniformly distributed along the
fibre length, and is uniformly extended by forces generated by the CC.
The forceextension curve of the SEC with forces
<P0 in the ABRM was studied by Lowy and Millman
(1963) and Tameyasu and Sugi
(1976
). They reported that the
curve obtained during the maximum active force generation was the same in
shape as that obtained during the catch state, provided the catch tension was
relatively high (
0.5P0). This was interpreted as
evidence that both the active and catch forces are produced by the same
contractile system, i.e. actinmyosin linkages. Recently, however, Sugi
et al. (1999
) have shown that
the SEC in the ABRM fibres becomes definitely stiffer in the late phase of the
catch state, in which the catch force decays below 0.5P0,
which is consistent with the presence of a force-bearing structure other than
the actomyosin system. In the present study, the forceextension curves
of the SEC for forces >P0 were obtained by applying
quick increases in load, and the SEC has also been shown to be definitely
stiffer during the catch state than during the maximum active force generation
(Fig. 5A). This is consistent
with the idea that the ABRM fibres contain linkages of both the actomyosin and
the load-bearing systems during the maximum active force generation, while
they contain only linkages of the latter during the catch state.
The same argument would apply for the L versus P
relationship (Fig. 5B); the
result that the increase of
L with increasing P is
less marked in the catch state than during the maximum active force generation
(Fig. 5B) may be taken to
indicate that linkages associated with the load-bearing system can be made to
slip less readily under large loads than actinmyosin linkages. During
the late slow isotonic lengthening, on the other hand, the V versus P
relation appeared to be the same during both the active force generation and
the catch state (Fig. 5C). This
might result from the fact that only linkages of the load-bearing system are
responsible for the extremely slow isotonic lengthening of the ABRM fibres,
while actinmyosin linkages are dissociated during the period of early
isotonic lengthening.
Based on the linkage hypothesis, on the other hand, Butler et al.
(1998,
2001
) suggest that the catch
state results from conversion of cycling actinmyosin linkages into
non-cycling ones caused by dephosphorylation of twitchin, a high molecular
mass protein known to occur in the ABRM
(Siegman et al., 1997
).
However, their hypothesis explains neither the large load-bearing ability of
non-cycling linkages nor the rapid development of large load-bearing ability
that should be coupled with rapid dephosphorylation of twitchin.
Possible structural basis of the load-bearing system
The extensive aggregation of the thick filament in the ABRM during the
catch state has been reported by many authors
(Heumann and Zebe, 1968;
Gilloteaux and Baguet 1977
;
Hauk and Achazi, 1987
).
Although the marked thick filament aggregation reported by them is claimed to
be an artefact due to glutaraldehyde cross-linking of lysine residues on the
thick filament, such a cross-linking may take place only when the distance
between the thick filaments gets closer in the catch state. In fact, ABRM
fibres quickly frozen in the catch state exhibit abundant thick filament
interconnections (Takahashi et al.,
2003
). The decreased distance between the thick filaments caused
by the formation of the thick filament interconnections might result in the
thick filament aggregation, if the ABRM is fixed in the catch state with
glutaraldehyde.
Although the arrangement of twitchin on the thick filament of the ABRM is unknown, it seems possible that it is twitchin that interconnects the thick filament to build up the force-bearing system. To clarify the arrangement of twitchin in the ABRM thick filaments, we are currently performing high-resolution scanning electron microscopy of the thick filaments in quick-frozen ABRM fibres, in the hope of proving that the thick filament interconnections are actually responsible for the marked load-bearing ability.
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References |
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