Non-cross-bridge calcium-dependent stiffness in frog muscle fibers

M. A. Bagni, B. Colombini, P. Geiger, R. Berlinguer Palmini, and G. Cecchi

Dipartimento di Scienze Fisiologiche, Università degli Studi di Firenze, 50134 Florence, Italy

Submitted 7 November 2003 ; accepted in final form 21 January 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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At the end of the force transient elicited by a fast stretch applied to an activated frog muscle fiber, the force settles to a steady level exceeding the isometric level preceding the stretch. We showed previously that this excess of tension, referred to as "static tension," is due to the elongation of some elastic sarcomere structure, outside the cross bridges. The stiffness of this structure, "static stiffness," increased upon stimulation following a time course well distinct from tension and roughly similar to intracellular Ca2+ concentration. In the experiments reported here, we investigated the possible role of Ca2+ in static stiffness by comparing static stiffness measurements in the presence of Ca2+ release inhibitors (D600, Dantrolene, 2H2O) and cross-bridge formation inhibitors [2,3-butanedione monoxime (BDM), hypertonicity]. Both series of agents inhibited tension; however, only D600, Dantrolene, and 2H2O decreased at the same time static stiffness, whereas BDM and hypertonicity left static stiffness unaltered. These results indicate that Ca2+, in addition to promoting cross-bridge formation, increases the stiffness of an (unidentified) elastic structure of the sarcomere. This stiffness increase may help in maintaining the sarcomere length uniformity under conditions of instability.

intact muscle fiber; static stiffness; tension inhibitors; titin


TENSION DEVELOPMENT in intact skeletal muscle fibers after stimulation is preceded by an increase of fiber stiffness that begins during the latent period and continues throughout the whole rise in both twitch and tetanic contractions (6, 8, 12). Previous work (3, 5) has shown that a small portion of the muscle stiffness increase arises from a sarcomere structure(s), outside the cross bridges, whose stiffness increases upon stimulation. This non-cross-bridge stiffness contributes very little to the stiffness of the muscle fiber at moderate or high tension; however, it represents the whole muscle stiffness increase occurring during the latent period and a substantial fraction at very low tension. The presence of this stiffness was demonstrated by studying the force response to fast ramp stretches and hold, applied to a single muscle fiber at various tension levels during a twitch or a tetanus. It was found that force, after the fast transient synchronous with the stretch, settled to a steady level greater than the isometric tension preceding the stretch, until relaxation or until the fiber was returned to the original length. Because of this characteristic, the excess of tension with respect to isometric tension was referred to as static tension, whereas the ratio between static tension and stretch amplitude, measured at sarcomere level, was termed static stiffness. Experiments made on tetanic contractions in Ringer containing 1–6 mM 2,3-butanedione monoxime (BDM), an agent that strongly inhibits cross-bridge formation (4, 14) without altering static stiffness (5), showed that the structure responsible for static stiffness behaves like an Hookean elasticity located in parallel with cross bridges (3). Interestingly, in both twitch and tetanic contractions, static stiffness development followed a characteristic time course distinct from that of tension and roughly similar to that of internal Ca2+ concentration. For this reason we speculated that static stiffness increase could be due to a sarcomere structure(s) whose stiffness increases after the stimulation in a Ca2+-dependent way. The experiments reported here were performed to investigate this possibility. We measured the static stiffness in single intact frog muscle fibers under various conditions in which isometric tension was inhibited either by reducing the Ca2+ release by the sarcoplasmic reticulum or directly by inhibiting actomyosin interaction. In addition to further investigating the effects of BDM, which confirmed previous measurements, we analyzed the static stiffness in the presence of Dantrolene, deuterium oxide (2H2O), methoxyverapamil (D600), and hypertonic solutions. All of these agents inhibit twitch tension, but they have a different action mechanism: Dantrolene (9, 13, 20, 25), 2H2O (1, 23), and D600 (10, 19) all depress force development, mainly by reducing the Ca2+ release, whereas BDM (4, 14) and hypertonic solution (22) inhibit tension generation, mainly by affecting actomyosin interaction so as to reduce cross-bridge formation with little or no effect on Ca2+ release. The comparison of static stiffness in fibers in which similar degrees of force inhibition were obtained with and without inhibition of Ca2+ release allowed us to isolate the effects of intracellular Ca2+ on static stiffness.

The results confirm that static stiffness is unaffected by BDM even at concentrations that strongly reduced twitch tension. The same effect was obtained by bathing the fiber with hypertonic solutions. On the contrary, 2H2O, Dantrolene, and D600 all depressed both tension and static stiffness. These findings suggest that static stiffness is modulated by intracellular Ca2+ concentration.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Frogs (Rana esculenta) were killed by decapitation followed by destruction of the spinal cord. Single intact fibers, dissected from the tibialis anterior muscle, were mounted by means of aluminum foil clips (11) between the lever arms of a force transducer and an electromagnetic motor in a thermostatically controlled chamber provided with a glass floor for ordinary and laser light illumination. The temperature was maintained constant at 14°C (±0.2°C). Single stimuli of alternate polarity, 0.5-ms duration, and 1.5 times threshold strength were applied transversely to the muscle fiber by means of platinum-plate electrodes. Tension was measured by means of a capacitance force transducer (natural frequency between 40 and 60 kHz) similar to that previously described (15). Sarcomere length changes were measured by using a striation follower device (16) in a fiber segment (1.2–2.5 mm long) selected for striation uniformity in a region as close as possible to the force transducer. This eliminated the effects of tendon compliance on stiffness measurements, allowing us to attribute the results directly to the sarcomere structure. Resting sarcomere length was set at ~2.1 µm.

After a test of fiber viability and a measurement of the isometric tetanic tension (P0), the experiments were made on twitch responses evoked in Ringer solution and in a series of test solutions containing one of the following agents: 1) BDM at a concentration of 2.5 mM, 2) Dantrolene at a concentration of 6.25 µM, and 3) D600 at a concentration of 20 µM. Experiments were also made in 2H2O Ringer (98% of water substituted with 2H2O) and in hypertonic solution at 1.4 normal tonicity (T), obtained by adding 50 mM NaCl to the normal Ringer. Experiments in 2H2O were made after waiting for the equilibration time (~20 min) to allow a complete exchange of 2H2O for water in the fiber. The responses in D600 were obtained in normal Ringer during the force recovery from the paralysis induced by exposing the fiber, loaded with D600, to high-potassium solution, as described previously (10, 19). We also evaluated the effects of nitrate Ringer (92 mM NaCl substituted by NaNO3) on fibers perfused with 6.25 µM Dantrolene to verify the possibility of reversing the Dantrolene effect on static stiffness with a Ca2+ release potentiator (21, 24). Experiments were made on twitch contractions because 1) the great and fast stretches necessary to measure the static stiffness easily damaged fibers developing the full tetanic tension; and 2) tension inhibitors, especially those acting by depressing Ca2+ release, have a greater effect on twitch than on tetani. As judged by light microscopy observations and by the sarcomere length signals from the striation follower, activated fibers in all test solutions did not develop any particular sarcomere nonhomogeneity upon stretching. The fibers survived after hours of experiments with stretches and fully recovered the isometric twitch tension when returned to normal Ringer, with the exception of the recovery after the exposure to D600, which was not always complete (see also Ref. 19). Resting fiber length, fiber cross-sectional area, and resting sarcomere length (L0) were measured under ordinary light illumination by using a x10 or x40 dry objective and x25 eyepieces. The normal Ringer solution had the following composition (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 3 phosphate buffer at pH 7.1. BDM Ringer, Dantrolene Ringer, and D600 Ringer were obtained by adding the appropriate amount of each agent to the normal Ringer solution. Force, fiber length, and sarcomere length signals were measured with a digital oscilloscope (4094 Nicolet), and data were stored on floppy disks and transferred to a personal computer for further analysis.

Static stiffness measurements. Static stiffness was measured by applying ramp-shaped stretches (amplitude 20–40 nm/hs and duration 0.6–0.7 ms) to one fiber end and measuring the force response at the other end. The short stretch duration was used to reduce as much as possible cross-bridge cycling during the stretch itself. Usually, three records were taken for each measure: 1) isometric, 2) isometric with stretch, and 3) passive response to the stretch. The isometric and passive responses were subtracted from the isometric record with stretch to obtain the subtracted trace on which measurements were made. In principle, the subtraction should have been made with the isometric and passive tension trace at the stretched length, rather than at resting length; however, because the lengthening is so small (2–4%), it can be assumed that the effect on both twitch tension and static stiffness is negligible. By subtracting the isometric record, we could always measure the static tension on a flat baseline, even when the stretch was applied on tension rise or relaxation. By subtracting the passive response, we corrected for the resting tension and stiffness of the relaxed fiber. This correction was, however, usually negligible on the experiments reported here, which were all made at ~2.1 µm of sarcomere length. The ratio between the static tension and the sarcomere elongation represents the static stiffness of the sarcomere. To describe the time course of the static stiffness development after the activation, we applied stretches in fibers at rest and at different times after the stimulus on both tension rise and relaxation.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 shows the force response to a fast stretch applied on the rise of a twitch and illustrates the method used to measure the static tension. The subtracted trace shows that, after the fast transient, the force settled to an almost steady level, higher than isometric, until relaxation. This level represents the static tension, which corresponds, in this case, to 0.13 P0. The static stiffness obtained by dividing the static tension by the sarcomere elongation (30 nm/hs) was 0.0043 P0·nm–1·hs. Considering that at 14°C the stiffness of a fully activated fiber at tetanus plateau is ~0.2 P0·nm–1·hs (2), it is clear that the static stiffness represents a very small fraction (~2.1% in this example) of the total stiffness of the fully activated fiber (a complete discussion on the static stiffness properties is reported in Ref. 3).



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Fig. 1. Force response to a stretch applied on the rise of a twitch in normal Ringer solution. The stretch was applied 14 ms after the stimulus when force was 0.14 times the peak twitch force (stretch amplitude, 30 nm/hs; stretch duration, 720 µs).

 
Superimposed fast time-base force responses to stretches applied to a fiber bathed in normal, BDM, and Dantrolene Ringer are reported in Fig. 2. As expected from the higher active tension developed at the time of the stretch, the force transient elicited in normal Ringer was much greater than that in BDM; however, after the transient, the tension settled to the same static level in both cases. The force transient in Dantrolene was similar to that in BDM, but the static tension was 50% smaller.



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Fig. 2. Comparison of force responses to stretch in normal, 2,3-butanedione monoxime (BDM) and Dantrolene Ringer at a fast time base. Top traces: sarcomere length (dashed line, BDM; solid line, Dantrolene Ringer). Bottom traces: subtracted force responses (dotted line, BDM; thin solid line, normal Ringer; thick solid line, Dantrolene). Stretch (amplitude, 27 nm/hs; duration, 680 µs) was applied on the rising phase of twitch contraction, 18 ms after the stimulus on the same fiber bathed in normal Ringer, BDM Ringer (2.5 mM), and Dantrolene Ringer (6.25 µM). The tension developed before the stretch in BDM and Dantrolene Ringer was 19 and 26%, respectively, of that developed in normal Ringer. Traces show that the static tension is not related to the force transient amplitude. Isometric tension levels before the stretch have been superimposed for all records (thin dashed line) to show more clearly the differences among the responses. The sarcomere length trace in normal Ringer is not reported because it was practically identical to that in BDM Ringer. Arrows indicate static tension.

 
The effects of various tension inhibitors on twitch tension and static stiffness time courses are reported in Fig. 3. In all solutions, static stiffness development preceded tension, reaching the peak value 10–12 ms after the stimulus, when twitch tension was still very low (in normal Ringer, ~5% of the twitch peak), and falling to zero before the twitch peak. As shown in Fig. 2, static stiffness was almost unaffected by BDM but was greatly reduced by D600 (Fig. 3A). Both agents strongly reduced tension. BDM slightly decreased the fall of static stiffness during relaxation. Similar to D600, both Dantrolene (Fig. 3B) and 2H2O (Fig. 3C) inhibited tension and static stiffness. With respect to normal Ringer, 2H2O slightly slowed down the time course of static stiffness. Similar to BDM, hypertonicity (Fig. 3D) reduced tension and slightly slowed down the static stiffness time course but did not alter its peak value.



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Fig. 3. Effects of BDM (2.5 mM), D600 (20 µM), Dantrolene (6.25 µM), 2H2O, and hypertonic solution (1.4 T) on twitch tension and static stiffness time courses. Dashed lines and filled symbols represent tension; solid lines and open symbols represent static stiffness. Squares, normal Ringer; inverted triangles, BDM; triangles, Dantrolene; circles, D600; pentagons, 2H2O; and diamonds, hypertonic solution. Tension was greatly reduced by all agents, but static stiffness was inhibited only by D600, Dantrolene, and 2H2O. DAN, Dantrolene; NR, normal Ringer.

 
Figure 4 shows the results of an experiment in which we tested the effect of a solution containing both BDM and Dantrolene. The addition of BDM to a fiber bathed in Dantrolene Ringer produced a further strong inhibition of twitch force but almost no effect on static stiffness. The effects of Dantrolene and BDM on tension were cumulative, whereas those on static stiffness were not, in agreement with the idea that static stiffness is affected only when Ca2+ release is affected.



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Fig. 4. Static stiffness time course in normal Ringer (squares) and in the presence of both Dantrolene (3.125 µM) and BDM (2.5 mM) (inverted triangles). Addition of BDM to the fiber bathed in Dantrolene (triangles) further inhibited tension but had no significant effect on static stiffness.

 
The effects of nitrate Ringer on a fiber bathed in Dantrolene are reported in Fig. 5. Nitrate almost completely reversed the effect of Dantrolene on static stiffness, again in agreement with the idea that static stiffness is Ca2+ modulated.



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Fig. 5. Effect of nitrate Ringer on static stiffness and tension in a fiber bathed in Dantrolene (6.25 µM). Squares, normal Ringer; triangles, Dantrolene; hexagons, nitrate Ringer plus Dantrolene. Nitrate almost completely reversed the effect of Dantrolene on static stiffness and slightly prolonged its time course. NO3, nitrate.

 
Figure 6 summarizes the effects on tension and static stiffness of all the agents tested in all the experiments performed. It is clear that the inhibitory effects of D600, Dantrolene, and 2H2O on twitch tension are accompanied by the inhibition of static stiffness, whereas BDM and hypertonic solutions inhibit tension but not static stiffness.



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Fig. 6. Data represent tension and stiffness in the presence of the various tension-inhibiting agents, and values are expressed as means ± SE relative to normal Ringer values. Narrow columns represent tension; wide columns represent static stiffness; n = 4 experiments for D600, Dantrolene, and BDM; n = 5 experiments for 2H2O and hypertonicity. Changes in static stiffness induced by BDM and hypertonicity are not statistically different (P > 0.05) from changes in normal Ringer. Hyper, hypertonicity.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
From the data reported in previous works (3, 5), we concluded that static stiffness is caused by the stiffening of some unknown elastic structure(s) of the fiber upon activation, rather than by cross-bridge activity. Several observations led us to this conclusion. First, the presence of a residual constant tension after the transient elicited by the stretch (the static tension) was not expected from the mechanism of the quick force recovery, which should bring the tension down to the level preceding the stretch in a few milliseconds (11). In addition, static tension increased linearly in the whole range of amplitudes tested (up to 40 nm/hs), even when the stretch was great enough (>12 nm/hs) to forcibly detach cross bridges. The absence of correlation between static stiffness and sarcomere length and the independence of the static stiffness from stretching speed are further characteristics suggesting a non-cross-bridge nature of the static tension. Finally, our data showed that static tension was established during the stretch itself, even when stretch duration was <400 µs. This makes unlikely the possibility that static stiffness could be caused by the formation of new cross bridges promoted by the stretch, because their attachment kinetics should have been extraordinarily fast.

Because the time course of static stiffness in both twitch or tetanic contractions was similar to the internal Ca2+ concentration, we suggested that the static stiffness could be modulated by Ca2+. The clarification of this point is the principal aim of this article. Static stiffness was measured in the presence of a series of tension inhibitors acting through either the inhibition of Ca2+ release or a direct inhibition on cross-bridge formation. Our data show that the agents tested, all of which inhibit twitch tension, can be grouped into two groups regarding their effect on static stiffness. The first group, including BDM and hypertonic solutions, has no effect on static stiffness, whereas the second group, including Dantrolene, 2H2O, and D600, inhibits static stiffness as well as tension.

As reported in the literature, the main effect of BDM on frog skeletal muscle at the concentrations used here is a direct inhibition of actomyosin interaction, reducing the number of attached cross bridges (4, 14) without affecting Ca2+ release (14). Hypertonic solutions, at the tonicity used here (1.4 T) on frog muscle, have an effect similar to that of BDM, mainly altering cross-bridge formation without affecting Ca2+ release (21).

The other group of agents has a different action mechanism. Dantrolene (9, 13, 20, 25), 2H2O (1, 23), and D600 (10, 19) all inhibit tension generation mainly by reducing Ca2+ release. 2H2O has an additional effect on cross-bridge formation and kinetics (7) that further increases tension inhibition. It is likely that this is the reason for the strongest inhibitory effect of 2H2O on tension (see Fig. 6). In summary, all of the agents tested substantially decreased twitch tension; however, only those reducing Ca2+ release reduced the static stiffness at the same time. These effects are consistent with previous data showing the similarity between the intracellular Ca2+ time course and the static stiffness time course and are consistent with the hypothesis that static stiffness is Ca2+ dependent. The observation reported in Fig. 5 that the depressant effects of Dantrolene on static stiffness can be reversed by the Ca2+ release potentiator nitrate further supports this idea.

The results reported here do not give further information about the structure responsible for the static stiffness; however, they show that Ca2+, in addition to promoting cross-bridge formation, also increases the stiffness of some unknown sarcomere structure. It is possible that this effect is due to a Ca2+-dependent titin-actin interaction (17) or to a Ca2+-dependent change in titin elasticity (26). This second possibility is strongly suggested by recent work (18) in which Ca2+ effects on titin properties were studied both at the molecular level and on skinned fibers. In agreement with our data on intact fibers, the results showed that Ca2+ decreased the persistence length of the elastic PEVK titin segment and increased the titin-based force response to stretch in skinned fibers. The Ca2+-dependent increase of passive stiffness could be important in maintaining the sarcomere functionality under conditions of sarcomere length instability.


    GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present research was financed by Università degli Studi di Firenze.


    ACKNOWLEDGMENTS
 
We thank Dr. Stuart Taylor for valuable discussion of the results.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Bagni, Dipartimento di Scienze Fisiologiche, Università degli Studi di Firenze, Viale G. B. Morgagni, 63, I-50134 Firenze, Italy (E-mail: mangela.bagni{at}unifi.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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