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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Standard solutions for skinned fiber experiments

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
F = <IT>A</IT><SUB>R</SUB> ⋅ [exp (−<IT>k</IT><SUB>R</SUB> ⋅ <IT>t</IT>)] − <IT>A</IT><SUB>C</SUB> ⋅ [exp (−<IT>k</IT><SUB>C</SUB> ⋅ <IT>t</IT>)]
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
F = <IT>A</IT><SUB>1</SUB> ⋅ {exp [−<IT>k</IT><SUB>r1</SUB> ⋅ (<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)]} + <IT>A</IT><SUB>2</SUB> ⋅ {exp [−<IT>k</IT><SUB>r2</SUB> ⋅ (<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)]}
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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


View larger version (18K):
[in this window]
[in a new window]
 
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.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Parameters of twitch contractions in intact fibers and twitchlike contractions in skinned fibers at 10°C

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.


View larger version (14K):
[in this window]
[in a new window]
 
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).

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Influence of sarcomere stability on rates of relaxation in intact and skinned fibers at 10°C

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).


View larger version (10K):
[in this window]
[in a new window]
 
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.


View larger version (18K):
[in this window]
[in a new window]
 
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.


View larger version (17K):
[in this window]
[in a new window]
 
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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adams, S. R., J. P. Y. Kao, and R. Y. Tsien. Biologically useful chelators that take up Ca2+ upon illumination. J. Am. Chem. Soc. 111: 7957-7968, 1989.

2.   Blinks, J. R., R. Rudel, and S. R. Taylor. Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. (Lond.) 277: 291-323, 1978[Abstract].

3.   Brenner, B. Technique for stabilizing the striation pattern in maximally calcium-activated skinned rabbit psoas fibers. Biophys. J. 41: 99-102, 1983[Abstract].

4.   Chase, P. B., and M. J. Kushmerick. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys. J. 53: 935-946, 1988[Abstract].

5.  Claflin, D. R., D. L. Morgan, D. G. Stephenson, and F. J. Julian. The intracellular Ca2+ transient and tension in frog skeletal muscle fibres measured with high temporal resolution. J. Physiol. (Lond.) 475: 319-325.

6.   Edman, K. A. P., and F. W. Flitney. Laser diffraction studies of sarcomere dynamics during "isometric" relaxation in isolated fibres of the frog. J. Physiol. (Lond.) 329: 1-20, 1982[Medline].

7.   Fabiato, A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. In: Methods in Enzymology, Biomembranes, edited by S. Fleischer, and B. Fleischer. Orlando, FL: Academic, 1998, vol. 157, p. 378-417.

8.   Goldman, Y. E. Measurement of sarcomere shortening in skinned fibers from frog muscle by white light diffraction. Biophys. J. 52: 57-68, 1987[Abstract].

9.   Gulati, J., and R. J. Podolsky. Isotonic contraction of skinned muscle fibers on a slow time base. J. Gen. Physiol. 78: 233-257, 1981[Abstract].

10.   Guth, K., and J. D. Potter. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J. Biol. Chem. 262: 13627-13635, 1987[Abstract/Free Full Text].

11.   Hou, T.-T., J. D. Johnson, and J. A. Rall. Parvalbumin content and Ca2+ and Mg2+ dissociation rate correlated with changes in relaxation rate of frog muscle fibres. J. Physiol. (Lond.) 441: 285-304, 1991[Abstract].

12.   Huxley, A. F., and R. M. Simmons. Mechanical transients and the origin of muscular force. Cold Spring Harbor Symp. Quant. Biol. 37: 669-680, 1972.

13.   Johnson, J. D., Y. Jiang, and M. Flynn. Modulation of Ca2+ transients and tension by intracellular EGTA in intact frog muscle fibers. Am. J. Physiol. 272 (Cell Physiol. 41): C1437-C1444, 1997[Abstract/Free Full Text].

14.   Julian, F. J., and R. L. Moss. Effects of calcium and ionic strength on shortening velocity and tension development in frog skinned muscle fibres. J. Physiol. (Lond.) 311: 179-199, 1981[Abstract].

15.   Kaplan, J. H., and G. C. R. Ellis-Davies. Photolabile chelators for the rapid photorelease of divalent cations. Proc. Natl. Acad. Sci. USA 85: 6571-6575, 1988[Abstract].

16.   Lannergren, J., and A. Arner. Relaxation rate of intact striated muscle fibres after flash photolysis of a caged calcium chelator (diazo-2). J. Muscle Res. Cell Motil. 13: 630-634, 1992[Medline].

17.   Mulligan, I. P., and C. C. Ashley. Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo-2. FEBS Lett. 255: 196-200, 1989[Medline].

18.   Palmer, R. E., S. J. Simmet, I. P. Mulligan, and C. C. Ashley. Skeletal muscle relaxation with diazo-2: the effect of altered pH. Biochem. Biophys. Res. Commun. 181: 1337-1342, 1991[Medline].

19.   Patel, J. R., G. M. Diffee, X. P. Huang, and R. L. Moss. Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle. Biophys. J. 74: 360-368, 1998[Abstract/Free Full Text].

20.   Rosenfeld, S. S., and E. W. Taylor. Kinetic study of calcium binding to regulatory complexes from skeletal muscle. J. Biol. Chem. 260: 252-261, 1985[Abstract/Free Full Text].

21.   Wahr, P. A., and J. A. Rall. Role of calcium and cross bridges in determining rate of force development in frog muscle fibers. Am. J. Physiol. 272 (Cell Physiol. 41): C1664-C1671, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 274(6):C1608-C1615
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society