Determinants of relaxation rate in skinned frog skeletal
muscle fibers
Philip A.
Wahr1,
J. David
Johnson2, and
Jack. A.
Rall1
Departments of 1 Physiology and
2 Medical Biochemistry, The Ohio State
University, Columbus, Ohio 43210
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ABSTRACT |
The influences of sarcomere uniformity and
Ca2+ concentration on the kinetics
of relaxation were examined in skinned frog skeletal muscle fibers
induced to relax by rapid sequestration of
Ca2+ by the photolysis of the
Ca2+ chelator, diazo-2, at
10°C. Compared with an intact fiber, diazo-2-induced relaxation
exhibited a faster and shorter initial slow phase and a fast phase with
a longer tail. Stabilization of the sarcomeres by repeated releases and
restretches during force development increased the duration of the slow
phase and slowed its kinetics. When force of contraction was decreased
by lowering the Ca2+
concentration, the overall kinetics of relaxation was accelerated, with
the slow phase being the most sensitive to
Ca2+ concentration. Twitchlike
contractions were induced by photorelease of
Ca2+ from a caged
Ca2+ (DM-Nitrophen), with
subsequent Ca2+ sequestration by
intact sarcoplasmic reticulum or
Ca2+ rebinding to caged
Ca2+. These twitchlike responses
exhibited relaxation kinetics that were about twofold slower than those
observed in intact fibers. Results suggest that the slow phase of
relaxation is influenced by the degree of sarcomere homogeneity and
rate of Ca2+ dissociation from
thin filaments. The fast phase of relaxation is in part determined by
the level of Ca2+ activation.
muscle relaxation; caged calcium; caged calcium
chelator
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INTRODUCTION |
MECHANICAL RELAXATION IN skeletal muscle fibers can be
divided into four phases. First, there is an initiation phase beginning after the last stimulus during which force remains approximately constant. The second phase consists of a slow, almost linear decay in
force during which the sarcomeres remain isometric. This slow phase is
followed by a shoulder during which the sarcomeres begin to move and
then followed by a fast, exponential phase in which sarcomere movements
are amplified (6). The factors that determine the rates of the slow and
fast phases have not been well established. This is in large part due
to the difficulty in gaining access to the interior of the fiber while
maintaining the ability to produce a physiologically rapid decrease in
Ca2+. Skinned fiber preparations
allow access to the contractile apparatus but eliminate the
Ca2+ sequestration ability of the
fiber that is essential for physiological relaxation. Therefore,
studies of relaxation have been largely limited to the use of intact
fibers. Some factors that have been suggested to affect the rate of
relaxation are the rate of Ca2+
removal from the sarcoplasm (11, 16),
Ca2+ dissociation from troponin C
(TnC) (13), and sarcomere motion (12).
This paper describes two methods of producing relaxation in skinned
fibers that result in rates similar to those seen in intact fibers.
First, fibers were mechanically skinned with the sarcoplasmic reticulum
(SR) left intact. Ca2+ released by
photolysis of the caged Ca2+
DM-Nitrophen (15) was subsequently sequestered by the SR. This procedure results in a force transient similar to a twitch. Second, photolysis of the caged Ca2+
chelator diazo-2 (1) was used to rapidly reduce the
Ca2+ level in actively contracting
skinned fibers, thus mimicking the relaxation seen from a tetanus. This
second method was then used to investigate the roles of
Ca2+ and sarcomere stability on
the rates of relaxation.
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MATERIALS AND METHODS |
Skinned and intact fibers.
Fibers from the tibialis anterior muscle of the frog
Rana temporaria were mechanically
skinned on a cooled (~10°C) stage under a dissecting microscope
as previously described (21). Fibers that sustained visible damage to
the myofibrils during skinning were discarded. Aluminum T-clips were
attached to the ends of the skinned fibers, and the fiber ends were
fixed in a 25% glutaraldehyde solution to provide a firm, minimally
compliant attachment to the experimental apparatus (4). If the protocol
required the destruction of the SR, the fibers were soaked for 30 min
in relaxing solution containing 1% Triton X-100. Electron micrographs
of fibers after Triton X-100 treatment indicated complete destruction
of all intracellular membranes (M. Yamaguchi, P. A. Wahr, and J. A. Rall, unpublished observations). Fibers were used within a few hours of
being skinned and remained in dissecting solution until transfer to the
experimental chamber.
Single fibers with intact membranes were prepared and isometric force
was measured as described by Hou et al. (11).
Experimental apparatus.
The experimental apparatus has been described in detail previously
(21). Briefly, skinned fibers were mounted by the T-clips in one of
three 325-µl chambers milled in a spring-mounted aluminum block
between a Cambridge force transducer (Cambridge Technologies, series
400, Watertown, MA) and either a small motor (Cambridge Technology,
model 372) for rapidly shortening the fiber or a stationary hook.
Solutions were changed by manually lowering the block containing the
chambers, sliding a new chamber underneath the fiber, and then raising
the block. Small aluminum inserts were used with the caged compounds to
decrease the volume of the chambers to 50-100 µl. The striation
spacing of the fiber was adjusted to 2.4 µm by measuring the
first-order diffraction pattern from a 5-mW HeNe laser directed through
the fiber.
The output of a Lumonics frequency-doubled ruby laser (model QSR2,
~300 mJ at 347 nm, pulse duration of 30 ns) was directed onto the
fiber from above by means of a fused silica reflecting prism and
cylindrical condensing lens mounted above the chamber. In addition,
glass microscope slides were placed between the prism and the lens to
attenuate the laser energy to ~100 mJ at the fiber. Fine adjustment
of the fiber position was accomplished by noting the burn pattern
produced by the laser on a piece of ZAP-IT paper (Kentek, Pittsfield,
NH) placed just above the fiber. The spot size at the position of the
fiber was focused by the condensing lens to 2 mm × 1 cm. Thus the
fiber was exposed to ~0.5 J/cm2.
A large motion artifact caused by exposing the T-clips to the laser
pulse was prevented by placing an adjustable aluminum mask above the
fiber such that the entire fiber length was exposed but the T-clips
were protected.
Temperature was monitored by a small (0.009 in. diameter) thermocouple
(type IT-23, Physitemp, Clifton, NJ) placed near the fiber by tying it
to one of the mounting hooks. The temperature for all experiments was
10°C. The output of the force transducer was recorded on a Nicolet
digital oscilloscope (model 2090-III, Madison, WI) for later analysis.
Solutions.
The bathing solutions for the preparation of skinned fibers were
prepared according to a computer program developed by R. Godt (Medical
College of Georgia), whereas the solutions containing caged compounds
were prepared using a program developed by Fabiato (7). The components
of these solutions are given in Table 1. Dissecting and relaxing solutions were prepared from stocks and refrigerated. Caged compound solutions were made up in stock solutions containing all ingredients except creatine phosphokinase (CPK) and the
caged compound and stored frozen. Diazo-2 (Molecular Probes, Eugene,
OR) was dissolved at high concentration in a small amount of the stock
and later diluted to the appropriate concentration. DM-Nitrophen
(Calbiochem, La Jolla, CA) was weighed out and added to the stock
solution as a powder. These solutions were kept frozen in aliquots of
~200 µl until the time of the experiment. These aliquots were
prepared frequently in small amounts and used within several days. The
dissociation constants for Ca2+
and Mg2+ from DM-Nitrophen at
10°C utilized in these studies were 21.0 nM and 14.1 µM,
respectively, as determined previously (21). Shortly before the
experiment, the solution containing the caged compound was thawed and
~1 mg/ml CPK was added just before use. Care was taken to avoid
exposing solutions containing caged compounds to excessive light to
prevent degradation of the caged compound.
General caged compound protocol.
Fibers were mounted in a chamber filled with relaxing solution. The
sarcomere length was set at 2.4 µm, and the fiber was transferred to
a chamber containing the caged compound solution. The fiber was soaked
until a plateau in the force trace was reached or, if no force was
developed, for a minimum of 2 min. The fiber was removed from the
chamber and then flashed in air. The temperature was noted, and the
fiber returned to relaxing solution. Thus the fibers were allowed to be
in air for <3 s. Fibers were allowed a 5-min rest in relaxing
solution before the next flash.
During force development before the laser pulse, the fiber was released
by the motor to slack length for 10 ms once every 4 s and then
restretched to its original length. This technique has been shown to
help preserve striation uniformity (3, 8).
Data analysis.
Twitches and twitchlike contractions were fit to the equation
where
kR and
AR are the rate
and amplitude of the relaxing phase and
kC and
AC are the rate
and amplitude of the force-producing phase, F is force, and
t is time. The fast,
exponential phase of relaxation from tetani and tetanus-like
contractions was fit to the equation
where
kr1 and
kr2 are the rate
constant of the fast phase of relaxation, to evenly spaced points along
the curve from the first 500 ms following the shoulder. Because the
shoulder is fairly broad,
to was used as an
estimate of its position
(tshoulder) for
analytical purposes.
The slow phase of relaxation was evaluated by fitting a single
exponential to the force trace from
1/4tshoulder
to tshoulder. Any
artifact produced by the laser pulse was thus excluded from the fit.
The average force from DM-Nitrophen contractions given before and after
each diazo-2 contraction is defined as
Fmax. Finit is defined as the force
developed in diazo-2 solution immediately preceding flash photolysis.
In the diazo-2 experiments, the fibers were generally submaximally
activated and thus the initial force, Finit, was usually less than
Fmax.
Fpre is used to denote the force
developed by the preceding maximal contraction induced by photolysis of
DM-Nitrophen. These parameters are illustrated in Fig.
2A.
Results are presented as means ± SE. Student's
t-test and a paired
t-test were used for determination of
significance. A significance level of
P < 0.05 was accepted.
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RESULTS |
Twitchlike and twitch contractions.
Twitchlike contractions were induced in skinned fibers and
compared with twitch contractions in intact fibers. Figure
1A shows the time course of a twitch and tetanus in an intact fiber. Mechanical skinning of the fibers leaves the SR able to take up
Ca2+ released on photolysis of
DM-Nitrophen. In the tetanus-like experiments described
below, the amount of
Ca2+ released on photolysis
exceeded the Ca2+-sequestering
capacity of the SR. Therefore, to produce a transient force similar to
a twitch, it was required that the
Ca2+ released on photolysis be
reduced. This reduction in Ca2+
release was accomplished by reducing the fraction of DM-Nitrophen bound
with Ca2+ by decreasing the
Ca2+ concentration in the
DM-Nitrophen solution (with an increase in the
Mg2+ concentration to prevent the
MgATP concentration from decreasing, see twitchlike SR solution in
Table 1). Flash photolysis of DM-Nitrophen under these conditions
produced a twitchlike contraction, as shown in Fig.
1B (trace marked
Triton).
Destruction of the SR by soaking in Triton X-100 abolished these
twitchlike contractions and produced a forceful, tetanus-like
contraction with little relaxation (Fig. 1B, trace marked +Triton). This result
indicates that Ca2+ sequestration
by the SR was indeed responsible for the relaxation.

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Fig. 1.
Twitch and twitchlike contractions in intact and skinned fibers. Twitch
or twitchlike responses are compared with tetanus or tetanus-like
responses from the same fiber. A:
intact single fiber. B: sarcoplasmic
reticulum (SR)-induced relaxation, which is abolished by destruction of
SR with 1% Triton X-100 for 30 min (fiber activated in twitchlike
solution SR, free Mg2+
concentration = 0.19 mM, see Table 1).
C: relaxation induced by
Ca2+ rebinding to
Mg · DM-Nitrophen. Fiber was activated in twitchlike
solution DM (high Mg2+-DM, free
[Mg2+] = 0.19 mM) or
in tetanus-like solution (low
Mg2+-DM, free
[Mg2+] = 36 µM, see
Table 1). D: twitchlike and twitch
responses from
A-C
normalized to peak force to show similarity in kinetics.
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The 500-fold-greater affinity of DM-Nitrophen for
Ca2+ over
Mg2+ also can be exploited to
produce similar twitchlike force transients in the absence
of SR (i.e., following treatment with Triton X-100). If a significant
portion of DM-Nitrophen is bound to
Mg2+ at rest,
Ca2+ released on photolysis can
then be rebound by the portion of unphotolyzed DM-Nitrophen with bound
Mg2+ at a rate determined by the
Mg2+ off rate from DM-Nitrophen.
Thus, with high Mg2+ and low
Ca2+ concentrations, the
incomplete photolysis of DM-Nitrophen produces an increase in the
Ca2+ concentration, followed by
exchange of the released Ca2+ with
the Mg2+ on the unphotolyzed
portion. This transient increase in the
Ca2+ level leads to a transient
force response. By reducing the total Ca2+ in the DM-Nitrophen solution
from 1.0 to 0.15 mM (see twitchlike DM solution in Table 1), flash
photolysis produces a transient, twitchlike contraction (Fig.
1C, trace marked high
Mg2+-DM) of ~30% of the force
produced by maximum Ca2+ release
from DM-Nitrophen (Fig. 1C, trace
marked low Mg2+-DM). The twitch
and twitchlike traces have been superimposed and normalized to peak
force in Fig. 1D. The parameters of
fits to these contractions are given in Table
2.
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Table 2.
Parameters of twitch contractions in intact fibers and twitchlike
contractions in skinned fibers at 10°C
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Although both types of twitchlike responses in the skinned fibers are
similar to one another, they are smaller and slower than the twitch
response in the intact fiber. The similarity of the rate of twitchlike
relaxation, kR,
for both SR- and Mg · DM-Nitrophen-induced relaxation
in skinned fibers suggests that the same process may be responsible for
limiting the twitchlike relaxation rate. It might be argued that this
relaxation is limited by the Mg · DM-Nitrophen rebinding of released Ca2+.
However, destruction of the SR abolished twitchlike contractions in the
intact SR fibers, indicating that the SR
Ca2+ uptake was producing the
Ca2+ transient in these fibers. It
is unlikely that both the SR Ca2+
uptake and Mg · DM-Nitrophen rebinding of released
Ca2+ occur with the same rate.
Therefore, it is unlikely that the slower twitchlike relaxation
observed in skinned fibers is due to a slow
Ca2+ exchange with
Mg2+ on DM-Nitrophen. Rather, the
SR Ca2+ uptake and the
Ca2+ exchange with
Mg2+ on DM-Nitrophen are both
likely to be fast compared with the observed relaxation rates.
Characterization of diazo-2 relaxation.
Diazo-2-induced relaxation was produced in fibers where the SR was
destroyed to ensure a uniform Ca2+
activation throughout the fiber. An example is shown in Fig. 2A, in
which the parameters of the fit of the contraction and relaxation are
described. The magnitude of relaxation produced by photolysis of
diazo-2 was limited to ~70% of the maximum force produced by
photolysis of 2 mM DM-Nitrophen. This limitation probably is due to the
fact that the dissociation constant for
Ca2+ binding on photolysis
decreases ~105 for DM-Nitrophen
(15) but only increases ~30-fold for diazo-2 (1). Photolysis of
diazo-2 produced nearly complete relaxation in fibers activated at pCa
5.8 to produce 75.5 ± 4.3% (n = 5) of Fmax. The diazo-2-induced
relaxation displayed the same phases as seen during relaxation from a
tetanus in intact fibers as shown by the example in Fig.
2B in which relaxations from skinned
and intact fibers are compared.

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Fig. 2.
Comparison of relaxation in skinned and intact fibers.
A: an example of a maximum
DM-Nitrophen-induced contraction and a diazo-2-induced relaxation from
the same fiber with method of analysis illustrated.
tshoulder and
kshoulder, Time
and rate of slow (shoulder) phase of relaxation;
kr1 and
kr2, fast-phase
rate constants; Fpre, force developed by preceding maximal
contraction; Finit, initial force;
Ffinal, final force.
B: an example of diazo-2-induced
relaxation from high initial force superimposed on relaxation from an
electrically stimulated intact fiber (1.3-s tetanus).
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The main differences in the relaxations of intact and skinned fibers
are the faster initial decline of relaxation,
kshoulder, and
the shorter time to the shoulder,
tshoulder,
observed in skinned fibers (Fig. 2B
and Table 3). Because the
tshoulder in
intact fibers correlates positively with sarcomere homogeneity (12), the present result is probably an indication of an increased amount of
inhomogeneity in the sarcomeres of the skinned fiber. Another feature
is that the initial rate of relaxation after the shoulder, kr1, in a skinned
fiber (with sarcomere stabilization, see Table 3) is similar to that
observed in the intact fiber. Finally, there is a protracted slow tail
in the skinned fiber trace that results in a crossover with the intact
fiber. This result occurs because
kr2, although
similar in magnitude in intact and skinned fibers, is, relative to
kr1, a much
smaller component of the intact fiber relaxation. In summary, relative
to intact fibers, diazo-2-induced relaxation in skinned fibers is
initially faster due to a shorter tshoulder and
faster kshoulder,
probably because of increased sarcomere inhomogeneity but, finally,
slower due to a larger relative magnitude of
kr2.
Effect of sarcomere stabilization on relaxation.
The sarcomere pattern in skinned fibers deteriorates with activation.
To minimize this effect, fibers were repeatedly released and
restretched during contraction. This procedure has been shown to
stabilize the sarcomeres during contraction (3). When this procedure to
stabilize sarcomere uniformity was not used, the kinetic parameters of
the subsequent relaxation were significantly increased (Table 3). The
parameters of the slow phase,
kshoulder and
tshoulder, are
~1.7- to 2-fold faster than in fibers subjected to the stabilization
protocol, which in turn are 1.8-fold faster than in the intact fibers.
The fast phase parameters,
kr1 and kr2, which with
stabilization are not significantly different from intact fibers, are
also increased when sarcomere stabilization is not employed. Thus it is
inferred that the relaxation rates are influenced by sarcomere
uniformity, i.e., more uniform sarcomeres lead to slower relaxation
rates.
Effect of altered
Ca2+ level on
relaxation.
The amount of force produced before photolysis of diazo-2 varied with
the level of Ca2+ in the diazo-2
solution, and this force level was taken as a measure of the level of
Ca2+ activation of the thin
filament. Figure 3 compares the
diazo-2-induced relaxation from high (1.0 Finit/Fmax)
and low (0.2 Finit/Fmax) force. Plots of the kinetic parameters of relaxation vs. relative force
are shown in Figs. 4 and
5. It is apparent from Figs. 4 and 5 that
the overall rate of relaxation is faster from low force. Both
the kshoulder
(Fig. 4A) and the
tshoulder (Fig.
4B) are slowed with increased
Ca2+-activated force. Also,
kshoulder is 6- to 15-fold more sensitive to the relative
Ca2+-activated force level
[150.2 ± 31.7 s
1/Frel
(where Frel = Finit/Fmax),
Fig. 4A] than the fast-phase rate constants
(kr1 = 28.6 ± 11.3 s
1/Frel,
Fig. 5A;
kr2 = 9.4 ± 3.3 s
1/Frel,
Fig. 5B). It is important to note
that the absolute values of these parameters
(kshoulder,
tshoulder,
kr1, and
kr2) were not
dependent on the diazo-2 concentration in the range of 1-4 mM.
This result suggests that the Ca2+
sequestration rate is not limiting the overall rate of relaxation. These results are in agreement with the hypothesis that the slow phase
is dominated by the rate of Ca2+
removal from TnC, whereas the rate of the fast phase is dominated by
the rate of cross-bridge detachment and is therefore relatively insensitive to Ca2+. It must be
remembered, however, that fibers soaked in a high-pCa solution require
a much longer activation time to reach a steady-state force than fibers
exposed to a lower pCa. It is possible that these differences in
activation times might lead to an increased level of sarcomere disorder
under low-force conditions, which would cause an increase in the
overall rate of relaxation (Table 3). However, this prediction would be
contrary to the reported result that lower activation levels lead to
decreased sarcomere disorder (8, 9, 14).

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Fig. 3.
Comparison of diazo-2 relaxation from high and low
Ca2+-activated force. In the trace
marked high force, fiber was activated in a pCa 5.0 solution and
induced to relax by photolysis of diazo-2 from
Finit/Fmax
of 1.0 to
Finit/Fmax
of 0.67 (where Finit is force
developed in diazo-2 solution immediately preceding flash photolysis
and Fmax is average force from
DM-Nitrophen contractions given before and after each diazo-2
contraction). In the low-force trace, a different fiber was activated
in a pCa 6.0 solution and induced to relax from
Finit/Fmax
of 0.2 to 0 force. Force traces have been normalized.
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Fig. 4.
Kinetics
(kshoulder,
A) and duration
(tshoulder,
B) of the slow phase of relaxation
induced by photolysis of diazo-2 vs. relative force. Solid lines are
least squares linear regression fits to data; 95% confidence intervals
are indicated by dashed lines. A:
kshoulder, with
slope = 150.2 ± 31.7 s 1/Frel,
y-intercept = 158.2 ± 22.5 s 1,
r = 0.807, n = 14. B:
tshoulder, with
slope = 111.8 ± 12.5 ms/Frel
(where Frel = Finit/Fmax),
y-intercept = 1.0 ± 8.9 ms,
r = 0.933, n = 14.
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Fig. 5.
Kinetics
[kr1
(A) and
kr2
(B)] of the fast phase of
relaxation vs. relative force. Solid lines are least squares linear
regression fits to data; 95% confidence intervals are indicated by
dashed lines. A:
kr1, with slope = 28.6 ± 11.3 s 1/Frel,
y-intercept = 40.5 ± 8.0 s 1,
r = 0.347, n = 14. B:
kr2, with slope = 9.4 ± 3.3 s 1/Frel,
y-intercept = 11.0 ± 2.3 s 1,
r = 0.408.
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DISCUSSION |
The transition from the slow to the fast phase of relaxation occurs
when the sarcomeres cease to be isometric (6). This transition
indicates that the kinetics of cross-bridge detachment is dependent on
the amount of mechanical strain on the individual cross bridges. This
factor is not present in solution studies of the actomyosin ATPase. For
this reason, a major goal of this study was to develop a method of
producing relaxation in skinned fibers with kinetics similar to those
seen in intact fibers. This goal was accomplished through the use of
diazo-2 to rapidly (within a few milliseconds) decrease the
Ca2+ level in skinned fibers (17,
18). Because excessive sarcomere movement is expected to accelerate the
kinetics of the slow phase of relaxation and shorten its duration, it
was necessary to minimize end compliance in the fiber through the use
of glutaraldehyde fixation of the ends to characterize slow-phase
relaxation (4).
Because Ca2+ sequestration by
diazo-2 is considerably faster than by intact SR and because the rate
of Ca2+ removal may be a
determinant of the relaxation rate (11, 16), a potential concern is
that the relaxation induced by photolysis of diazo-2 is qualitatively
different from that in the intact fiber. It must be remembered,
however, that in intact fibers the fall in
Ca2+ concentration precedes and is
considerably faster than that of force relaxation following both
twitches and tetani (2, 5). Therefore, the rate of
Ca2+ sequestration cannot be the
sole determinant of the rate of relaxation but may influence the
overall relaxation rate. Consequently, the increased rate of
Ca2+ sequestration by diazo-2 over
the SR may serve to increase the overall rate of relaxation but should
not alter the fundamental kinetic pathways. Indeed, relaxation in
response to diazo-2 is qualitatively similar, in both slow and fast
phases, to relaxation in intact fibers (see Fig. 2), indicating that
the mechanism of relaxation is likely to be qualitatively similar for
both the intact and skinned fibers. Thus the kinetics of relaxation can be studied in skinned fibers. This preparation has the advantage of
having a fiber interior that is accessible to manipulation of factors
thought to play a role in determining the rate of muscle relaxation.
However, the rates of relaxation observed in skinned and intact fibers
have quantitative differences. The relaxation in response to diazo-2
photolysis in skinned fibers is initially faster but later slower and
not as complete as that seen in intact fibers. Differences in the
kinetics of relaxation in skinned fibers compared with intact fibers
may be due to changes in the end compliance, contractile filament
lattice spacing, and/or sarcomere homogeneity of the fibers
produced by skinning.
Determinants of the time to shoulder during relaxation.
The previously published force records of diazo-2-induced relaxation in
skinned frog fibers lack the rate-limiting slow phase of relaxation
characteristic of intact muscle (17, 18). In contrast to the previous
study, care was taken in the present work to ensure a firm, minimally
compliant attachment to the apparatus by glutaraldehyde fixation of the
fiber ends. This technique has allowed the observation of the slow
phase of relaxation in all force traces examined here. Thus it appears
that the fiber must be held at least partially isometric during
relaxation for the slow phase to occur. The requirement of sarcomere
isometry for the occurrence of the slow phase is in agreement with the
observed loss in sarcomere isometry at the time of the transition from the slow to the fast phase (6). This observation leads to the prediction that an increase in sarcomere homogeneity should result in a
delayed appearance of the shoulder that has been observed in intact
frog fibers (12). This prediction also is confirmed here in skinned
fibers by the observation that fibers that were subjected to a rapid
release and restretch during force development, which has been shown to
improve sarcomere homogeneity (3, 8), exhibited decreased kinetics and
longer slow phase durations than fibers that were not subjected to this
protocol (see Table 3). Although the lower relative force
(Finit/Fmax)
observed without the shortening protocol (0.73 vs. 0.91, Table 3) is
expected to have an effect on the
tshoulder (see
Fig. 4B), this cannot be the sole
cause of the shorter duration seen without the shortening protocol. The
predicted
tshoulder for
sarcomere stabilized fibers at a
Finit/Fmax
of 0.73 (Fig. 4B) is 81 ms, which is
longer than the value of 49.3 ms (Table 3) observed without the
sarcomere stabilization technique. Likewise, the
tshoulder
observed in intact fibers (176.8 ms, Table 3) also is considerably
longer than predicted from Fig. 4B
(111 ms at 1.0 F/Fpre). These
results provide convincing evidence that
1) the duration of the slow phase of
relaxation is dependent on the ability of the sarcomeres to remain
isometric and 2) sarcomeres are less
homogeneous in skinned than in intact fibers. Furthermore, the inverse
correlation of the rate (Fig. 4A)
and duration (Fig. 4B) of the slow
phase indicates that the loss of isometric sarcomeres can play a major
role in accelerating the overall rate of relaxation.
Modulation of the slow phase of relaxation by
Ca2+
concentration.
The slow phase of relaxation in skinned fibers is 6-15 times more
sensitive to Ca2+ than the fast
phase. Also, the
kshoulder is
inversely correlated with the duration of this phase,
tshoulder.
Because it is conceivable to have a slow
kshoulder of
short duration, the correlation of these two parameters is not
necessarily expected. As the level of
Ca2+ activation is reduced,
kshoulder
increases (Fig. 4A) and
tshoulder decreases (Fig. 4B). Thus the slow
phase of relaxation is at least in part determined by the level of
Ca2+ activation. Because none of
the components of relaxation is affected by variation of the
Ca2+ buffering capacity (in the
range of diazo-2 from 1 to 4 mM), these results suggest that the fibers
relax more slowly when relative force development is high because
Ca2+ dissociates more slowly from
TnC. This conclusion is consistent with the suggestion that the
affinity of TnC for Ca2+ increases
as the number of bound cross bridges increases (10, 20). This increased
affinity of Ca2+ for TnC would
lead to a decreased dissociation rate of
Ca2+ from TnC and thus a longer
slow phase and slower relaxation. Consistent with this suggestion,
Johnson et al. (13) recently have shown that as the rate of
Ca2+ sequestration is increased in
intact frog skeletal muscle fibers, the overall rate of relaxation
increases until it approaches a rate similar to the rate of
Ca2+ removal from purified whole
troponin. Thus Ca2+ removal from
troponin appears to be an important rate-limiting step in muscle
relaxation.
Fast phase of relaxation.
The fast phase of relaxation in response to photolysis of diazo-2 in
skinned fibers is well fit by a double exponential equation, whereas in
the intact fiber it is more monoexponential. The rates without
sarcomere stabilization observed here at 10°C (Table 3) are in
reasonable agreement with published results of relaxation induced by
photolysis of diazo-2 in frog skinned fibers at 12°C, where rates
of 42 s
1
(kr1) and 12 s
1
(kr2) have been
reported (18). With repeated releases and restretches of the fiber to
stabilize the sarcomeres, the diazo-2-induced fast phase rate constants
from high force (0.91 Finit/Fpre)
correspond closely with those observed in intact fibers (Table 3).
The appearance of two rates suggests that relaxation occurs from at
least two distinct states of the cross-bridge cycle or, alternatively,
along two distinct biochemical pathways. Both
kr1 and
kr2 increase with
a decrease in Ca2+ activation
(Fig. 5). However, these fast-phase
Ca2+ dependencies are fairly weak
in contrast to the slow-phase rate, kshoulder, which
is strongly dependent on Ca2+
concentration (Fig. 4A). Recent
results with skinned mammalian psoas fibers also show that both
kr1 and
kr2 increase with
a decrease in Ca2+ activation
(19). The mechanism by which increases in
Ca2+ concentration and force could
lead to a decrease in the fast phase of relaxation cannot be determined
by the experiments performed here. However, the results are consistent
with the interpretation of this effect as a change in the cross-bridge
detachment rate mediated by the removal of
Ca2+ from TnC.
Twitchlike contractions.
The twitchlike contractions induced by photolysis of DM-Nitrophen in
skinned fibers with intact SR provide a potentially useful model for
the study of SR function and relaxation in situ. The kinetics of
contraction and relaxation are similar but somewhat slower than those
observed in the intact fiber (Table 2). These differences may be due to
differences in end compliance in skinned and intact fibers. Another
possibility is that the slower kinetics of relaxation may relate to the
fact that parvalbumin diffuses out of skinned fibers. Parvalbumin is
known to accelerate the rate of relaxation in frog skeletal muscle by
about twofold (11). Its absence may explain the slower relaxation rate
in skinned fibers. Nonetheless, the overall relaxation rate from a
twitch in skinned fibers with functional SR of 10.3 s
1 (Table 2) is similar to
the fast phase rate of 13.0 s
1 observed in skinned
fibers induced to relax by diazo-2 photolysis (Table 3, with
stabilization). This similarity suggests that the same mechanisms
underlie relaxation in twitches and tetani.
In summary, the duration of the slow phase of relaxation increases as
the amount of Ca2+ activation is
increased. The data are consistent with the hypothesis that the rate
and duration of the slow phase are determined in part by the rate of
Ca2+ dissociation from the thin
filaments. The slow phase kinetics is faster and the shoulder appears
sooner with a decrease in sarcomere homogeneity. This observation
agrees with the hypothesis that the shoulder is caused by a mechanical
disruption of the sarcomere spacing. The fast phase of relaxation is in
part determined by the level of
Ca2+ activation. The mechanism of
this determination is unclear but is consistent with a decreased rate
of cross-bridge detachment at high
Ca2+ concentrations. The data are
consistent with greater cross-bridge formation resulting in an increase
in affinity of TnC for Ca2+, which
produces a slower overall rate of relaxation and a delay in the
appearance of the shoulder.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Tien-Tzu Hou for help with the intact fiber experiments
and Dr. Mamoru Yamaguchi for taking the electron micrographs of Triton
X-100-treated fibers.
 |
FOOTNOTES |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-20792 and by the American
Heart Association, Ohio Affiliate.
Address for reprint requests: J. A. Rall, Dept. of Physiology, The Ohio
State Univ., 1645 Neil Ave. Columbus, OH 43210.
Received 30 June 1997; accepted in final form 11 March 1998.
 |
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