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First published online October 18, 2006
Journal of Experimental Biology 209, 4319-4328 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02501
The catch state of mollusc catch muscle is established during activation: experiments on skinned fibre preparations of the anterior byssus retractor muscle of Mytilus edulis L. using the myosin inhibitors orthovanadate and blebbistatin
Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
* Author for correspondence (e-mail: Stefan.Galler{at}sbg.ac.at)
Accepted 4 August 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: catch muscle, mollusc smooth muscle, Mytilus edulis, skinned muscle preparations, myosin inhibitors, calcium activation
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Many of the
studies on catch were carried out on the anterior byssus retractor muscle
(ABRM) of Mytilus edulis. Catch is characterized by basal levels of
both ATP consumption (Güth et al.,
1984) and cytosolic free Ca2+ concentrations
(Ishii et al., 1989). In
experiments under isometric conditions, catch is indicated by a very slow
force decrease after cessation of stimulation
(Rüegg, 1965).
Furthermore, a substantial force recovery after a quick release of the muscle
length (active state) (Jewell,
1959) is absent. In intact ABRM preparations, catch force is
relaxed by serotonin (5-hydroxytryptamin), which is released from synapses of
specific neurons (Twarog,
1954). Serotonin induces an increase of intracellular cAMP
(adenosine 3 '5 '-cyclic monophosphate)
(Achazi et al., 1974). cAMP
activates protein kinase A, which in turn phosphorylates twitchin
(Siegman et al., 1998;
Butler et al., 2001) resulting
in termination of catch (Siegman et al.,
1997; Siegman et al.,
1998). Twitchin is a mini-titin (for a review, see
Funabara et al., 2005) which
is located on the thick filaments (Siegman
et al., 1998).
In skinned ABRM preparations a catch-like state can be established by
removal of Ca2+ after Ca2+-induced activation at a
moderately acidic pH (pH 6.5-6.8) (e.g.
Rüegg, 1971;
Siegman et al., 1998;
Galler et al., 2005). At this
stage, quick stretches induce a rigor-like force response whereas during
Ca2+ activation the same length change is followed by a delayed
force increase (stretch activation)
(Gagelman et al., 1984).
Similarly, in release experiments a substantial force recovery is present
during Ca2+ activation but not after Ca2+ removal,
indicating the absence of an active state. cAMP
(Cornelius, 1982) or the
catalytic subunit of protein kinase A
(Pfitzer and Rüegg, 1982)
leads to the termination of this catch-like state due to phosphorylation of
twitchin (Siegman et al.,
1997). Catch is also terminated by a moderate alkalisation, e.g.
when the pH is increased from 6.2 to 7.5
(Rüegg, 1964), from 6.5
to 7.7 (Rüegg, 1971) or
from 6.7 to 7.4 (Galler et al.,
2005). The termination of the catch state by moderate alkalisation
seems to be independent of cAMP and protein kinase A
(Höpflinger et al.,
2006).
The molecular basis of catch is still unclear. Two different mechanisms
have been proposed. The first explains catch in terms of myosin heads
(cross-bridges) remaining sustainable attached to actin filaments (myosin head
model) (Lowy et al., 1964).
The very slow force decrease during catch is thought to be due to a very slow
detachment of myosin heads. The second mechanism envisions the formation of
link structures (interconnections), which are different from myosin head
cross-bridges, between myofilaments [alternative linkage model
(Rüegg, 1963;
Rüegg, 1965)]. Recently,
the alternative linkage model was supported by some physiological data
(Galler et al., 1999;
Sugi et al., 1999;
Mukou et al., 2004;
Galler et al., 2005;
Andruchova et al., 2005;
Höpflinger et al., 2006;
Butler et al., 2006). However,
the kind of linkages has not yet been identified.
The time point where catch is developed during an activation-relaxation
cycle is not clear. The catch state becomes obvious after termination of
activation; however, it is possible that catch linkages are established
already during activation. Studies of intact ABRM preparations showed that a
serotonin-sensitive stiffness is present not only in the catch state but also
during active contraction (for a review, see
Rüegg, 1971).
Furthermore, in skinned ABRM preparations it was found that force is reduced
by cAMP at submaximal Ca2+ activations
(Butler et al., 1998;
Siegman et al., 1998). The
cAMP-sensitive force component decreases with increasing activation
(Butler et al., 2006) and it is
absent at maximal Ca2+ activation. The fraction of force that is
sensitive to cAMP was thought to be catch force, since the application of cAMP
was not associated with any significant change of ATPase activity
(Butler et al., 1998). From
these results it was concluded that catch linkages are already established
during Ca2+ activation. Furthermore, the authors assumed that the
number of catch linkages decreases with increasing activation and it reaches
zero at maximal activation (Butler et al.,
2006).
The reliability of these conclusions should be reflected in the context of
the following considerations. (1) The force depression by cAMP at submaximal
Ca2+ activation may not necessarily indicate the presence of catch,
because, in principle, it could also be due to a change in the cross-bridge
kinetics. In particular, a slowing of the myosin head attachment (rate
constant, f) or an acceleration of the myosin head detachment (rate
constant, g) would result in a force depression. This is a
consequence of the two-state model of force generation
(Huxley, 1957), where force is
proportional to f/(f+g). There are in fact some
indications that cAMP slows myosin head attachment, which could be responsible
for the observed force depression: cAMP significantly decelerates both the
delayed force increase following a quick stretch (stretch activation) and the
force rise following a stepwise increase of ATP concentration (caged-ATP)
starting at low-force rigor (Andruchova et
al., 2005). The assumption that cAMP in fact detaches catch
linkages at submaximal Ca2+ activations was substantially
corroborated by the finding of Butler et al.
(Butler et al., 2006) that cAMP
even depresses force after inhibition of the cycling myosin head
cross-bridges. (2) The absence of a change in ATPase activity during
cAMP-induced force depression at submaximal Ca2+ activations does
not unequivocally indicate that the force change must be due to other factors
than a change in cross-bridge kinetics: a decrease in force can be due to a
drop of f and a simultaneous rise of g, which may not
necessarily change the ATPase activity which is proportional to
gf/(f+g). (3) Furthermore, small differences in
ATPase activity are difficult to detect, and thus, the significance of the
available measurements should be carefully considered. A precise determination
of ATPase activity is difficult in experiments where it is measured during the
time course of Ca2+ activation before and after cAMP application
because, as noted by Butler et al. (Butler
et al., 1998) (their fig.
2), the ATPase activity is not constant during prolonged
activation. Moreover, in such types of experiments the ATP cleavage due to the
myosin head power strokes cannot be separated from the ATP cleavage due to the
phosphorylation reaction (transfer of the 
-phosphate of ATP to the
phosphorylation site) stimulated by cAMP. Precise measurements of ATPase
activity are also difficult if preparations, which were thiophosphorylated
before the measurement, are compared with control preparations [figs 4 and 5
of Butler et al. (Butler et al.,
1998)], because the volume of the active muscle preparations is
hard to investigate precisely. (4) The conclusion of Siegman et al.
(Siegman et al., 1998) and
Butler et al. (Butler et al.,
2006) that catch linkages are established only at submaximal but
not at maximal Ca2+ activation is questionable because
Ca2+ removal leads to a catch state after both submaximal and
maximal Ca2+ activation. Moreover, the time course of the force
decay after Ca2+ removal is not apparently different after
submaximal and maximal Ca2+ activation (e.g.
Butler et al., 2001;
Galler et al., 2005;
Butler et al., 2006), and thus,
it seems unlikely that at maximal Ca2+ activation the catch
linkages are established after Ca2+ removal, whereas at
submaximal activations the catch linkages are established in the
presence of Ca2+.
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) by using maximally Ca2+-activated skinned ABRM
preparations and by including rapid changes of the fibre length: as in the
work of Butler et al. (Butler et al.,
2006), we inhibited the active cycling cross-bridges by specific
myosin inhibitors during Ca2+ activation. Orthovanadate
(Herzig et al., 1981;
Dantzig and Goldman, 1985;
Chase et al., 1993;
Jaworowski et al., 1999;
Galler et al., 2005) and
blebbistatin (Kovacs et al.,
2004; Butler et al.,
2006) were chosen as myosin inhibitors. The force remaining after
myosin inhibition was investigated by addressing the following questions. (1)
Is the remaining force sensitive not only to cAMP but also to moderate
alkalisation? (2) Does the remaining force show an increased stiffness/force
ratio as it is known for the rigor or catch state
(Pfitzer and Rüegg, 1982;
Sugi et al., 1999;
Höpflinger et al., 2006)?
(3) Are rectangular length changes followed by rigor-like force responses,
i.e. no delayed force increase following a stretch (stretch activation) or no
substantial force recovery following a quick release (active state)? Our
experimental results provide increasing evidence for the assumption that catch
linkages are established already during Ca2+ activation. This seems
to apply not only for submaximal but also for maximal Ca2+
activations. The implications of these findings for the understanding of the
molecular mechanism of catch are discussed. |
Materials and methods |
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). Briefly, ABRMs of fresh blue mussels (Mytilus
edulis L.; shell length, 4-7 cm), obtained from a local sea-food
supplier, were isolated. The dissected muscle was teased into several bundles
about 0.4 mm wide in artificial seawater containing (in mmol l-1):
490 NaCl, 8 KCl, 10 CaCl2, 15 MgCl2, 1
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes), pH 7.2. These
bundles were exposed first to a sodium skinning solution and then to a
potassium skinning solution both containing saponin
(Cornelius, 1980;
Castellani and Cohen, 1992;
Chick and Stephenson, 1995;
Galler et al., 2005). In
addition to 0.05% (w/v) saponin, the sodium skinning solution contained (in
mmol l-1): 132 sodium propionate, 5 ethylene
glycolbis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 5
Na2H2ATP, 7 MgCl2, 10
3-morpholinopropanesulfonic acid (Mops), 2 dithioerythritol (DTE), 646
sucrose, pH adjusted to 6.9 with KOH. In the potassium skinning solution the
sodium propionate was replaced by potassium propionate. The skinning procedure
took about 30 min in total and was carried out at 20-24°C. Solutions used for the mechanical experiments contained (in mmol l-1): 150 sucrose, 5 EGTA, 5 Na2H2ATP, 5 disodium creatinphosphate, 3 free Mg2+, 1 DTE, 1 NaN3 and 30 i.u. ml-1 creatine phosphokinase. Solutions of pH 6.7 contained 20 mmol l-1 Mops and solutions of pH 7.4 contained 20 mmol l-1 Hepes. The ionic strength was adjusted to 0.20 mol l-1 with KCl, and the pH was adjusted to 6.7 or 7.4 using KOH. The pCa (=-log[Ca2+]free) was adjusted with CaCl2 and measured using a calcium-sensitive electrode (Fluka 21188). The relaxation solution had a pCa of >8 and the activation solution had a pCa of 4.4. Submaximal activation solutions were prepared by mixing relaxation and activation solution in different proportions.
The solutions containing 10 mmol l-1 orthovanadate
(Na3VO4; Vi) were prepared as described in
our previous study (Galler et al.,
2005). Briefly, a stock solution of 300 mmol l-1
Vi (pH 10) was prepared in double-distilled water and boiled until
colourless. After adding the Vi, the pH of skinned fibre solutions
was adjusted to either pH 6.7 or pH 7.4 with HCl. Blebbistatin was dissolved
in DMSO at a concentration of 5 mmol l-1. This stock solution was
added to the activation solution to obtain a final blebbistatin concentration
of 5 µmol l-1. The final DMSO concentration was about 0.1% (v/v)
and did not affect the contractile properties of the skinned ABRM muscle
bundles (Andruchova et al.,
2005).
Experimental setup and procedure
For mechanical experiments, skinned fibre bundles of 100-300 µm diameter
and 2.5-4.9 mm length were used. The bundles were mounted horizontally between
two vertically oriented pins of an isometric apparatus (compliance, 4 µm
mN-1). One pin was attached to a force sensor; the other pin was
attached to a stepping motor. The apparatus and the methods for mechanical
measurements have been described previously
(Galler and Hilber, 1994). A
force sensor, AE 801 (SensoNor, Horten, Norway) with a resonance frequency of
7.5 kHz was used. Rapid changes (about 1-2 ms) in fibre length were
achieved by a feedback-controlled stepping motor based on a Ling vibrator. A
cuvette transporting system provided quick changes of solutions. Small
repetitive rectangular stretches were continuously applied to measure
stiffness. These stretches were about 0.1% of the skinned fibre bundle length
(L0) in amplitude and 20 ms in duration and the frequency
was <0.5 s-1. The stiffness is given by the ratio
F/ L, where F represents the
amplitude of the force spikes and L the length change.
All mechanical experiments were carried out at 22.0-22.5°C. Results are expressed as means ± s.d.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Effect of orthovanadate on maximal Ca2+ activation
Fig. 2A shows the effect of
the inhibitor orthovanadate on force and stiffness of a maximally
Ca2+-activated (pCa 4.4) skinned ABRM fibre bundle. The first and
main part of the experiment was carried out under conditions which are
favourable for the formation of the catch state: pH 6.7 and absence of cAMP.
The exposure to pCa 4.4 induced an increase in force and stiffness to a
plateau. The stiffness/force diagram in
Fig. 2B (grey squares) shows
the increase of these two parameters during the development of the activation.
The relationship resembles that shown in
Fig. 1B where the
Ca2+ concentrations was increased in a stepwise manner, and data
points were obtained at steady-state conditions. When the plateau of
Ca2+ activation was reached, 10 mmol l-1 Vi
were added. This resulted in a pronounced depression of force (values in
Table 1). Stiffness was less
decreased by Vi (open circles of
Fig. 2B) than force, causing an
increased stiffness/force ratio.
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In Fig. 2A the broken line
represents a force trace of a Vi experiment carried out at pH 7.4.
As shown by Höpflinger et al.
(Höpflinger et al., 2006)
the maximal force was similar at pH 7.4 and pH 6.7. However, in comparison
with experiments at pH 6.7 (solid line in
Fig. 2A), 10 mmol
l-1 Vi depressed force significantly more at pH 7.4 (for
values see Table 1). The plot
in Fig. 2C shows the
relationship between stiffness and force during the time course of the
experiment. In contrast to pH 6.7 (Fig.
2B), the stiffness/force relationship during force depression by
Vi (open circles) does not deviate from that during the development
of the Ca2+ activation (grey squares). Thus, the increase in the
stiffness/force ratio present at pH 6.7 was not observed at pH 7.4. A larger
force depression and no deviation of the stiffness/force relationship were
also observed when 100 µmol l-1 cAMP and 2 µmol
l-1 of the phosphatase 2B inhibitor cyclosporine A (CsA) were
present during the whole experiment (Table
1 and stiffness/force plot of
Fig. 2D).
Application of blebbistatin during the catch state
Orthovanadate, at concentrations of 1-10 mmol l-1, has been
shown to not affect the catch force
(Galler et al., 2005).
However, there was no information on the effect of the myosin inhibitor
blebbistatin, and therefore, we examined the effect of blebbistatin on the
catch state. The catch state was induced by Ca2+ removal after
maximal Ca2+ activation. As shown in many other studies (e.g.
Butler et al., 1998;
Höpflinger et al., 2006),
the Ca2+ removal results in an initial rapid relaxation which is
followed by a much slower relaxation a few minutes later. This slow relaxation
phase is thought to represent the catch state. Application of blebbistatin
(1-10 µmol l-1, 2-10 min) during the slow phase of relaxation
had no effect on force (N=4).
Effect of blebbistatin on maximal Ca2+ activation
Fig. 3A shows the effect of
5 µmol l-1 blebbistatin on force and stiffness of a maximally
Ca2+-activated (pCa 4.4) skinned ABRM fibre bundle. The experiment
was carried out under conditions that are favourable for the formation of the
catch state: pH 6.7 and absence of cAMP. Blebbistatin at the concentration of
5 µmol l-1 depressed force at a substantially slower rate than
Vi at a concentration of 10 mmol l-1. Likewise, removal
of blebbistatin led to a much slower force recovery than removal of
Vi (data not shown). The force levels (mean ± s.d.) measured
at a time point when the force decay tended to reach a plateau (about 7-10 min
after blebbistatin application) are given in
Table 1. Like orthovanadate,
blebbistatin also depressed force more than stiffness. The resulting increase
in the stiffness/force ratio can be seen in the stiffness/force plot in
Fig. 3B.
The stretch-induced delayed force increase (stretch activation) and the substantial force recovery following a quick release of fibre length which were present at maximal Ca2+ activation disappeared after force depression by blebbistatin (insets of Fig. 3A).
The broken line in Fig. 3A represents a force trace of a blebbistatin experiment carried out at pH 7.4. In comparison with experiments at pH 6.7 (solid line of Fig. 3A), 5 µmol l-1 blebbistatin depressed force substantially more at pH 7.4. The amount of force depression (mean ± s.d.) about 7-10 min after blebbistatin application is given in Table 1. It can be seen, that force decreased by a greater extent than at pH 6.7. The plot in Fig. 3C shows the relationship between stiffness and force during the time course of a blebbistatin experiment carried out at pH 7.4. As in Vi experiments at pH 7.4, the stiffness/force relationship during force depression by blebbistatin (open circles) also does not deviate from that during the development of the Ca2+ activation (grey squares). Thus, the increase in the stiffness/force ratio present at pH 6.7 was not observed at pH 7.4. A larger force decrease and no deviation of the stiffness/force relationship was also observed in experiments where 100 µmol l-1 cAMP and 2 µmol l-1 CsA were continuously present (Table 1; Fig. 3D).
Effects of moderate alkalization or cAMP after cross-bridge inhibition by Vi or blebbistatin
In addition to the cross-bridge inhibition by Vi at maximal
Ca2+ activation, Fig.
2A (second part) shows the effect of moderate alkalisation on
depressed force. When the pH is changed from 6.7 to 7.4 in the presence of pCa
4.4 and 10 mmol l-1 Vi, the depressed force further
decreased. As seen in Table 2,
the total amount of force depression in these experiments was similar to that
observed in experiments where pH 7.4 was continuously present during the whole
experiment (see also broken line on the
Fig. 2A). The pH change
affected stiffness more than force, resulting in a reversal of the deviation
of the stiffness/force relationship back to that during the development of the
Ca2+ activation (solid circles of
Fig. 2B). Thus, the increase of
the stiffness/force ratio, which was observed during Vi inhibition
was abolished by moderate alkalisation. The depression of force and stiffness
by pH 7.4 was irreversible: a change of pH from 7.4 back to 6.7 did not result
in a recovery of either force or stiffness
(Fig. 2A). However, when the
preparations were activated again with Ca2+ at pH 6.7, the catch
state was re-established after removal of Ca2+ (data not
shown).
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Similar to the Vi experiments (Fig. 2A, second part), a change of pH from 6.7 to 7.4 after force depression by blebbistatin resulted in a further depression of force (Table 2) and in the abolition of the increased stiffness/force ratio. The total amount of force depression was similar to that observed in experiments conducted continuously at pH 7.4 (Table 1 and broken line in Fig. 3A).
Similar to moderate alkalisation, cAMP also depressed force and abolished the increased stiffness/force ratio when applied after cross-bridge inhibition by blebbistatin or Vi at pCa 4.4. The total amount of force depression was similar to that observed in experiments where cAMP was continuously present (Table 1). Furthermore, as shown in Table 2 the force depression by cAMP is similar to that reached by pH 7.4.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Chase et al., 1993). At a
concentration of 10 mmol l-1 the force depression by Vi
is saturated in skinned ABRM preparations
(Galler et al., 2005).
Blebbistatin is a recently discovered specific inhibitor of myosin ATPase
activity (Kovacs et al.,
2004). It is thought to bind to the detached myosin heads with
bound ADP and inorganic phosphate (M.ADP.Pi state) and inhibit the
transition into a force generating state
(Allingham et al., 2005). At a
concentration of 5 µmol l-1 the force depression by blebbistatin
is close to saturation in skinned ABRM preparations
(Butler et al., 2006). Whereas
Vi and blebbistatin inhibit the cross-bridges they seem not to have
any effects on catch linkages. This is suggested by the observation that
application of 1-10 mmol l-1 Vi does not change force
when the catch state is established
(Galler et al., 2005). The
same conclusion can be drawn for blebbistatin, since the present study showed
that 1-10 µmol l-1 blebbistatin does not influence the force
when catch is established after Ca2+ removal following
Ca2+ activation.
Butler et al. (Butler et al.,
2006) had already shown that saturating concentrations of
blebbistatin depress the force of Ca2+-activated ABRM preparations
to intermediate levels. Furthermore, it was found that subsequent addition of
25 mmol l-1 phosphate did not influence force; however addition of
cAMP caused a further substantial force depression. These results suggest that
actively cycling myosin head cross-bridges are totally inhibited by
blebbistatin and, thus, the remaining force should be based on passive catch
linkages which are detached by cAMP. To further corroborate this assumption,
in the present study we investigated the effects not only of cAMP but also of
moderate alkalisation on force remaining after cross-bridge inhibition by
Vi and blebbistatin. In contrast to most of the experiments carried
out by Butler et al. (Butler et al.,
2006), our investigation concerned maximally
Ca2+-activated preparations. We further investigated the
stiffness/force relationship and the force transients following rapid length
changes before and during inhibitor application.
We found that the force remaining after cross-bridge inhibition is
substantially reduced not only by cAMP but also by moderate alkalisation. This
is an important hint for the presence of catch linkages because it is known
that catch (induced by Ca2+ removal after Ca2+
activation) is abolished by these two factors [cAMP
(Cornelius, 1982) and pH
(Höpflinger et al.,
2006)].
We further found that force inhibition by Vi or blebbistatin was
accompanied by an increase of the stiffness/force ratio when compared with
submaximal force levels reached either by partial Ca2+ activation
under steady-state conditions (Fig.
1B) or during the development of the maximal Ca2+
activation (e.g. Fig. 2B). This
is also an important hint for the presence of catch linkages, because a
similar increase is usually observed when catch is induced by either
Ca2+ removal after Ca2+ activation in skinned
preparations (Pfitzer and Rüegg,
1982; Höpflinger et al.,
2006) or removal of acetylcholine in intact preparations
(Pfitzer and Rüegg, 1982;
Sugi et al., 1999). In skinned
skeletal muscle preparations [frog
(Brozovich et al., 1988),
rabbit psoas (Martyn and Gordon,
1992; Chase et al.,
1993)] it was found that application of phosphate or orthovanadate
increases the population of attached but non-force generating cross-bridges.
This cross-bridge population is detectable only with very rapid length changes
(<1 ms) at low temperature (5-13°C) indicating rapid detachments and
reattachments (Chase et al.,
1993; Dantzig and Goldman,
1985). Thus, this cross-bridge population markedly differs from
long-lasting catch linkages. Furthermore, if this cross-bridge population is
really present in ABRM, it may not contribute to our stiffness measurements
which were performed with stepwise stretches (1-2 ms ramps) at
22.0-22.5°C. This assumption is further supported by our observation that
the increased stiffness/force ratio during force depression by Vi
or blebbistatin is abolished by moderate alkalization or cAMP. Thus, the
increased stiffness/force ratio is rather due to the catch linkages and not to
a population of short-living attached but non-force generating
cross-bridges.
Finally, we found that the stretch-induced delayed force increase
disappeared, and the force recovery following a release of the fibre length
was substantially decreased, when the force was depressed by Vi or
blebbistatin. These rigor-like responses are also a strong hint of the
presence of catch linkages, because similar force transients are observed in
ABRM during catch induction either by Ca2+ removal after
Ca2+ activation in skinned preparations (Gagelmann et al., 1984;
Cornelius, 1982;
Galler et al., 2005;
Butler et al., 1998) or by
removal of acetylcholine in intact preparations
(Pfitzer and Rüegg, 1982;
Cornelius, 1980;
Cornelius, 1982).
All these results strongly suggest that the force component that remains after cross-bridge inhibition by Vi or blebbistatin is largely due to catch linkages. The observation that the force did not fall to zero, but only to low levels (<10% of maximal force) when orthovanadate or blebbistatin and pH 7.4 or cAMP were applied concomitantly may be mainly because exposure times to the various factors was too short.
Are catch linkages also present at the maximum of Ca2+ activation?
A change in pH from 6.7 to 7.4
(Höpflinger et al., 2006)
or addition of cAMP (Butler et al.,
1998; Andruchova et al.,
2005) does not affect the force of maximally activated skinned
ABRM preparations. At first glance, this finding suggests that during maximal
Ca2+ activation no catch linkages are established. However, it
could also be that catch linkages are established but they are not under
tension, so that their detachment (by pH 7.4 or cAMP) cannot be detected by
measuring stiffness with short length changes (about 0.1%
L0). A detachment of such catch linkages would also not
result in a change of isometric force. Our experiments clearly showed that
catch force appears when myosin inhibitors are applied to maximally
Ca2+-activated skinned ABRM preparations. Therefore, unlike Siegman
et al. (Siegman et al., 1998)
or Butler et al. (Butler et al.,
2006), we conclude that catch linkages are present also during
maximal Ca2+ activation. However, the catch linkages are not under
tension under isometric conditions as long as the force does not decline due
to myosin inhibitors (or by the removal of Ca2+) (see below).
Mechanism of catch
The fact that catch linkages are present already during Ca2+
activation has an important implication on the molecular mechanism underlying
the catch phenomenon. Skinned ABRM fibre bundles are able to shorten both at
maximal and submaximal Ca2+ activation with remarkable velocity
(Butler et al., 1998). Firmly
attached catch linkages would substantially prohibit shortening independently
of their nature and location. The contradiction, however, can be resolved, if
catch linkages behave like a ratchet. That is, the catch linkages exhibit a
high resistance when the fibre is stretched and a low resistance when the
fibre shortens. Based on these considerations we interpret important
experimental findings around the catch phenomenon as follows.
When considering catch experiments under isometric conditions it appears
that the state of catch is developed only after a decrease, but never during
an increase, of force. This applies for intact muscles where catch is
established after a force decrease resulting from cessation of stimulation
(Rüegg, 1971). This also
applies for experiments with skinned fibres where catch is established by a
force decrease due to one of the following experimental procedures: (1)
Ca2+ removal following Ca2+-activated contraction
(Rüegg, 1971;
Siegman et al., 1998;
Galler et al., 2005); (2)
application of myosin inhibitors to Ca2+-activated preparations
(Butler et al., 2006) (this
study); or (3) stepwise ATP increase in the high-force rigor at basal
Ca2+ concentration (Galler et
al., 1999; Butler et al.,
2001).
That the appearance of catch properties requires a decrease of force can be
seen in the present study. At about 30% of maximal force a clear stretch
activation response was present if this force was reached by partial
Ca2+ activation starting at relaxing conditions. However, if the
same force was reached by depressing the force during maximal Ca2+
activation, using myosin inhibitors, a rigor-like response was present,
indicating catch. Likewise, an extra-stiffness indicating catch-linkages
(increased stiffness/force ratio) could also be observed only during a force
decrease, i.e. after Ca2+ removal following Ca2+
activation (Pfitzer and Rüegg,
1982; Höpflinger et al.,
2006) or after myosin inhibition during Ca2+ activation
(this study). By contrast, during the force increase at ongoing
Ca2+ activation this extra stiffness was absent.
These observations can be explained using the ratchet model. During Ca2+ activation, cycling cross-bridges generate a pulling force. This force produces an intrinsic shortening (including a short filament sliding) and a stretch of series elastic components. The catch linkages are progressively (and slowly) established during Ca2+ activation, but they do not appreciably resist to the intrinsic shortening because, (1) the movement is in the low resistance direction and (2) the shortening is small. The catch linkages will not be stressed in the high resistance direction as long as the force rises or remains at a plateau. Under this condition the catch linkages have no contribution to stiffness (investigated with length changes of only 0.1% Lo) and isometric force. The situation changes when force decreases as a result of Ca2+ removal or myosin inhibition. The force decrease is associated with a reversal of the intrinsic shortening, which is driven by the stretched series elastic components. The reversal of the intrinsic shortening represents an internal stress which puts the catch linkages under tension because they are pulled in the high resistance direction. With decreasing force the number of catch linkages under tension increases. However, obviously not all catch linkages are put under tension; some of them remain unstrained. They only become visible if they are stretched during stiffness measurements. These unstrained catch linkages are responsible for the extra stiffness.
Since a force decrease seems to be necessary for putting the catch linkages
under tension, it seems at first glance surprising that cAMP is able to
depress force at stable plateau values during submaximal Ca2+
activations. This finding might be explained by the dual effects of cAMP on
both catch linkages and cross-bridge kinetics
(Andruchova et al., 2005).
Thus, the force decrease is probably initialized by the cAMP-dependent change
of cross-bridge kinetics (e.g. drop of f); it is then augmented,
because force is not sustained by the catch linkages which are detached by
cAMP. The initial force decrease due to a change in cross-bridge kinetics may
occur only at submaximal but not at maximal Ca2+ activations. It is
possible that f is in the order of g only at submaximal
activations, but f is much larger than g at maximal
Ca2+ activation. Consequently a change in f would affect
the ratio f/(f+g) (force) only at submaximal, but
not at maximal, activation levels.
In contrast to our interpretation, Butler et al.
(Butler et al., 2006) assumed
that the whole cAMP-sensitive force component is catch force. This assumption
and the dependency of the cAMP-sensitive force component on the grade of
activation resulted in the hypothesis that actively cycling myosin head
cross-bridges and catch linkages compete for a common binding site
(Butler et al., 2006). As long
as changes in the cross-bridge kinetics due to cAMP cannot be ruled out (see
Introduction) these conclusions are debatable.
The molecular interpretation of catch reported here is compatible with the
myosin head model of catch where the catch linkages are represented by the
myosin head cross-bridges remaining attached to the actin filaments. If this
model is true, it must be specified by assuming that the attached myosin heads
exhibit ratchet properties. However, recent findings make the myosin head
model of catch unlikely: cAMP (Siegman et
al., 1998) or moderate alkalisation induce relaxation of catch
force but do not accelerate myosin head detachment
(Andruchova et al., 2005;
Höpflinger et al., 2006).
However, orthovanadate, phosphate and 2,3-butanedione monoxime (BDM)
accelerate myosin head detachment but do not influence the catch state
(Galler et al., 2005;
Butler et al., 2006).
Therefore, it is more likely that catch is based on link structures other than
the myosin head cross-bridges.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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