A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]iwas not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.
- force potentiation
- excitation contraction coupling
- mechanical output
- skeletal muscle
in vivo discharge patterns of single motor units often start with a burst of two to three impulses at a high frequency. Such high-frequency bursts have been reported in humans (see e.g., Ref. 14) as well as in animals (see e.g., Ref. 10). The high-frequency burst is then followed by a much lower frequency of discharge. Studies with a high-frequency burst have shown sustained increases in force during isometric contractions (5) and in power output during concentric contractions (1).
It has been speculated that a high-frequency burst results in a maintained increase of the free myoplasmic [Ca2+] ([Ca2+]i) and, hence, increased activation of the contractile machinery (18, 24). A higher stimulation frequency undoubtedly results in increased [Ca2+]i and consequently increased force production (6, 22), but it is not clear whether the prolonged force increase after a high-frequency burst is due to a sustained increase of [Ca2+]i. To determine whether [Ca2+]i remains elevated after a high-frequency burst and whether this sustained elevation can explain the increased force, we performed simultaneous measurements of [Ca2+]i and force in single intact fibers isolated from mouse flexor digitorum brevis muscles. The fibers were stimulated with a triplet of 150-Hz impulses, given either at the start or in the middle of 20- to 60-Hz tetani. [Ca2+]i and force in these tetani were compared with measurements in control tetani without the triplet. The results showed a sustained force increase after the high-frequency triplet, whereas [Ca2+]i rapidly fell to the control level.
MATERIALS AND METHODS
Male mice (NMRI strain, weight ∼30 g) were killed by rapid neck disarticulation, and intact single fibers were dissected from the flexor digitorum brevis muscle of the hindlimb following a procedure described by Lännergren and Westerblad (12). The isolated fiber was mounted between an Akers 801 force transducer and an adjustable holder. Fiber length was adjusted to that giving maximum tetanic force. The fiber was stimulated by supramaximal current pulses (duration 0.5 ms) delivered via platinum plate electrodes lying parallel to the long axis of the fiber.
Experiments were performed at room temperature (24° C), i.e., somewhat less than the in situ temperature of the superficial muscle used (30.5°C) (4). The fiber was superfused by a Tyrode solution of the following composition (mM): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24.0 NaHCO3, 0.1 EDTA, and 5.5 glucose. Fetal calf serum (0.2%) was added to the solution, which was bubbled with 5% CO2-95% O2, giving a pH of 7.4.
[Ca2+]i was measured with the fluorescent Ca2+ indicator indo 1 as described previously (2). Briefly, indo 1 (Molecular Probes Europe, Leiden, The Netherlands) was microinjected into the fiber. The fluorescence of indo 1 was measured with a system consisting of a xenon lamp, a monochromator, and two photomultiplier tubes (PTI, Wedel, Germany). The excitation light was set to 360 ± 5 nm, and the light emitted at 405 ± 5 and 495 ± 5 nm was measured. The ratio of the light emitted at 405 nm to that at 495 nm (R) was converted to [Ca2+]i using the following equation (8) where K d is the apparent dissociation constant of indo 1, β is the ratio of the 495-nm signals at very low and saturating [Ca2+]i, Rmin is the ratio at very low [Ca2+]i, and Rmax is the ratio at saturating [Ca2+]i. Fluorescence and force signals were sampled online and stored on a desktop computer system for subsequent data analysis.
The kinetics of indo 1 are too slow to accurately follow rapid transients of [Ca2+]i (25). This problem can be reduced by kinetic correction of the indo 1 signal, and such a correction has a large impact on [Ca2+]i measurements performed at the onset of tetanic contractions. However, the effect of kinetic correction is limited during the tetanic plateau (23). In the present study, we compare [Ca2+]i measurements obtained during the tetanic plateau. This comparison will be little affected by the slow kinetics of indo 1, and, hence, no kinetic correction of the indo 1 signal has been performed.
To study the effects of a high-frequency burst on [Ca2+]i and force, the isolated fiber was stimulated with three impulses at 150 Hz at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus. The stimulation frequency during the rest of the tetanus was 20, 30, 40, or 60 Hz. Changes induced by the high-frequency triplet were assessed by measuring [Ca2+]i and force in tetani with triplet and in bracketing tetani produced at the same stimulation frequency but without the triplet. The order of the stimulation frequencies at which the effects of the triplet were studied was randomized. There was a resting period of 1 min between the individual contractions.
[Ca2+]i and force (in kPa) were measured as the mean between each stimulation pulse. Because this study focuses on sustained changes after the high-frequency triplet, force and [Ca2+]i were not compared in the time frame that included the triplet. Change in force and Ca2+ were calculated by subtracting data within the same time frame of a tetanic contraction with a triplet from the mean of bracketing contractions without a triplet. Change in force (Δforce) is expressed as a percentage of the maximal tetanic force during a contraction at 120 Hz. For each fiber, a force-[Ca2+]i curve was constructed from measurements of force and [Ca2+]i in tetani without a high-frequency triplet produced at 20 to 120 Hz as described previously (2).
To investigate the possible role of stretching of passive elements as a mechanism of the force potentiation after a high-frequency triplet, five fibers were stretched and the relative increase of force was compared at the different lengths. In these experiments, the basal stimulation frequency was 30 Hz while [Ca2+]iand force were measured in the first time frame following the triplet. At each fiber length, the force increase is expressed relative to the maximum active force without a triplet. The relative force increase at optimum length was set to 100% in each fiber. Fluorescence signals were measured with a fixed field of view. This means that the fiber volume within the field of view was reduced as the fiber was stretched, and, hence, the fluorescence signals became smaller, which resulted in more noise in the fluorescence ratio signals. Therefore, at long fiber lengths, fluorescence ratios were filtered at 10 Hz, which reduced the peak of [Ca2+]i transients and made them broader (see Fig. 6), whereas the mean [Ca2+]i was not affected. Thus a direct quantitative comparison between [Ca2+]itransients at normal and long length cannot be performed. However, our main objective was to determine whether there was a prolonged increase of [Ca2+]i after the high-frequency triplet, and this would not be affected in [Ca2+]i measurements at long length.
Data are presented as means ± SE. Paired t-tests were used to establish significant differences between [Ca2+]i and force in tetani with and without a high-frequency triplet. The significance level was set to 0.05.
High-frequency triplet at the start of a contraction.
Figure 1 shows typical [Ca2+]i and force records from one fiber stimulated at 20 Hz with and without the high-frequency triplet. The triplet resulted in a marked increase of both [Ca2+]i and force. However, while force remained elevated for about 200 ms after the triplet, [Ca2+]i rapidly declined to the level obtained without the triplet. Similar results were obtained at the other frequencies tested, and results from all fibers are summarized in Fig. 2. The change in Ca2+showed no consistent increase after the triplet. On the other hand, Δforce was significantly (P < 0.05) increased by 16.6 ± 2.8% at 20 Hz, 16.9 ± 1.1% at 30 Hz, 17.8 ± 2.0% at 40 Hz, and 19.9 ± 2.5% at 60 Hz (n = 7) in the first time frame after the triplet. Thereafter, Δforce decreased exponentially with an average rate constant of ∼20 s−1.
Figure 3 shows an example of a force-[Ca2+]i curve from one fiber obtained from tetani at 20–120 Hz. As can be seen, force increases in a well known sigmoidal manner with higher [Ca2+]i. Data points obtained after a high-frequency triplet given at the start of a 30 Hz tetanus are also shown. In the first time frame after the triplet, force is substantially increased, whereas [Ca2+]ishowed little change and, hence, the data point lies markedly to the left of the curve obtained under control conditions. The effect of the triplet then gradually fades, and, in the fourth and fifth time frames, data points lie on the control curve. A similar shift of the force-[Ca2+]i relationship was observed in all other fibers studied, and the mean shift in the first time frame after the triplet amounted to −0.18 ± 0.05 μM (n = 7), calculated by assuming a simple parallel shift of the force-[Ca2+]i curve.
High-frequency triplet in the middle of a tetanus.
Figure 4 shows representative [Ca2+]i and force records from one fiber stimulated for 700 ms at 20 Hz with and without a high-frequency triplet in the middle of the tetanus. The pattern is qualitatively similar to that observed when the triplet was given at the start of the contraction: the increase of [Ca2+]i induced by the triplet rapidly disappeared, whereas there was a sustained increase in force. Mean data from all fibers (n = 7) studied with a high-frequency triplet in the middle of the tetanus are shown in Fig. 5. Similar to the situation when a triplet was given at the start of tetani, there was no consistent increase in [Ca2+]i after the triplet. Δforce in the first time frame after the triplet was 13.2 ± 2.6% at 20 Hz, 18.2 ± 3.4% at 30 Hz, 9.4 ± 1.9% at 40 Hz, and 4.5 ± 1.5% at 60 Hz [i.e., there was a significant increase in force at all frequencies (P < 0.05)]. Fitting the decay of Δforce to a single exponential function in 20- and 30-Hz tetani (where the force increase was large enough to give a reliable measure) gave a rate constant of ∼20 s−1. Compared with the Δforce observed when the triplet was given at the start of a contraction, Δforce in the first time frame was markedly smaller when the triplet was given in the middle of tetani at 40 and 60 Hz. This can be explained by tetanic force before the triplet starting to approach maximum force, which sets the limit on how much force can increase. For instance, in control tetani produced to obtain force-[Ca2+]icurves (see Fig. 3), the force in 60-Hz tetani was 87.7 ± 1.9% of the force in 120-Hz tetani (i.e., maximum force). Therefore, the maximum force potentiation would be limited to about 12%.
Effects of high-frequency triplets at long fiber lengths.
The effect of a high-frequency triplet at the beginning of a 30-Hz tetanus was studied at optimal and increased length. Passive structures in the fibers were stretched at the increased length, resulting in a resting force of 50–200% of the maximum tetanic force at optimal length. As a result, the filament overlap within sarcomeres was reduced, resulting in a reduction of the active force in 30-Hz tetani to <20% of that at optimal length (Fig.6). There was a substantial force potentiation after the high-frequency triplet also in stretched fibers, although the relative force increase was somewhat smaller than at optimal length (77.1 ± 3.5%; n = 5). At long lengths, the high-frequency triplet resulted in an immediate increase of [Ca2+]i, but in the first time frame after the triplet, [Ca2+]i was not higher than in bracketing tetani without triplets. Thus a high-frequency triplet, while augmenting force production, did not result in a sustained increase of [Ca2+]i either at optimal or increased fiber length.
The main finding of the present study is that a high-frequency burst of impulses during a contraction at lower frequency results in a very brief increase in [Ca2+]i, which is followed by a prolonged force elevation. Thus a short period of high [Ca2+]i results in a prolonged increase in the activation of the contractile machinery, and possible mechanisms for this will be discussed in the following.
The present results show a leftward shift of the force-[Ca2+]i relationship after a high-frequency triplet (see Fig. 3). A similar shift in the force-[Ca2+]i relationship has been associated with increased myosin light chain phosphorylation (15,20), which is thought to underlie other types of force potentiation such as posttetanic potentiation (16, 19,21). Therefore, it may be speculated that a high-frequency triplet results in an increased level of myosin light chain phosphorylation. However, it is known that a relatively long tetanus is needed to induce a marked posttetanic potentiation (see e.g., Refs.13, 21). Moreover, a study by Moore and Stull (16) on rat gastrocnemius muscle showed that dephosphorylation of the myosin light chain takes ∼2 min, which is much longer than the duration of the effect of high-frequency triplets in the present experiments. Thus, considering the short duration of the present high-frequency triplets (∼13 ms) and the short-lasting force potentiation, it seems very unlikely that an increased level of myosin light chain phosphorylation can account for the present force elevation after the triplet.
Another possible explanation for the present force potentiation after a triplet could be an enhanced force transmission due to stretching of passive elements of the muscle fiber. Parmiggiani and Stein (17) showed that two stimuli separated by a short interval produced three times the force of a single twitch in cat muscles (nonlinear summation of force). Because the increase in force was larger than the increase in muscle stiffness, which was assumed to be a measure for actin myosin bonds, Parmiggianni and Stein concluded that improved transmission of internal force was the only means for producing a net facilitation of force production. To study whether such a mechanism could also account for the sustained force potentiation following the triplet, we performed experiments at increased fiber length. Passive elements would be stretched already at rest at long lengths, and the effect of a short period of increased active force production would be small. Therefore, the force potentiation after a high-frequency triplet should be severely reduced at long lengths if the potentiation was due to stretching of passive elements. The present results show a slightly smaller triplet-induced relative force increase (∼80%) in stretched fibers compared with fibers studied at optimal length. Thus improved force transmission through stretching of passive elements may account for ∼20% of the present triplet-induced force potentiation, and the greater part of the potentiation must be due to some other mechanism.
The force-[Ca2+]i relationship was shifted to the left after the high-frequency triplet (see Fig. 3), i.e., there was an apparent increase of myofibrillar Ca2+ sensitivity. The interaction between Ca2+ activation of the actin filament and cross-bridge force production is rather complex (for a recent review, see Ref. 7). It is possible that the high-frequency triplet affects this interaction. For instance, Ca2+ binding to the regulatory sites of troponin C may be facilitated by attached, force-producing cross bridges (11). Thus the transient increase of [Ca2+]i during the triplet will result in an increased number of active cross bridges. This might then increase the Ca2+ affinity of regulatory sites on troponin C, and the activation of the actin filament would remain high despite declining tetanic [Ca2+]i. However, while important in cardiac myocytes, this mechanism appears to be of little importance in skeletal muscle fibers (7). Moreover, the present results showed the same rapid decline of [Ca2+]i to the control level after the triplet both at optimal and long length, and, hence, there were no signs of different Ca2+ buffering in the two situations. A mechanism that encompasses the present results is that the high-frequency triplet affects the actin-myosin interaction via direct effects of attached cross bridges on actin filament activation. Recent models of cross-bridge activation in skeletal muscle fibers suggest that Ca2+ binding to troponin C makes it possible for myosin heads to move tropomyosin from the sites where cross bridges bind. Once a cross bridge has moved tropomyosin, the binding of additional cross bridges to neighboring sites will be facilitated (7). This mechanism implies that there will be a delay in the force decline when [Ca2+]i is reduced because new force-bearing cross bridges can be formed close to already attached cross bridges despite lowered [Ca2+]i. In line with this model, the decrease of force markedly lags behind the decline of [Ca2+]i during relaxation of contractions (22).
The elevated force after the high-frequency triplet declined with a rate constant of about 20 s−1, which would be similar to the rate of cross-bridge cycling under isometric conditions in fast-twitch mammalian muscle fibers (∼5 s−1 in rabbit psoas muscle fibers studied at 12°C) (9). Interestingly, the increased force after a burst of high-frequency stimulation is maintained for a markedly longer period in slow-twitch skeletal muscle (see e.g., Refs. 3 and 5), where the rate of cross-bridge cycling is lower than in fast-twitch skeletal muscle.
In conclusion, this study has shown that force potentiation after a high-frequency triplet is not the result of a sustained high [Ca2+]i. Rather, we suggest that the force potentiation is caused by an apparent increase in the myofibrillar Ca2+ sensitivity due to the facilitated formation of additional force-bearing cross bridges in the vicinity of already attached cross bridges.
This work was supported by grants from the Swedish Medical Research Council (Project 10842), the Swedish National Center for Sports Research, funds at the Karolinska Institutet, and the Netherlands Organization for Scientific Research.
Address for reprint requests and other correspondence: F. Abbate, Dept. of Physiology and Pharmacology, Karolinska Institutet, SE 171 77 Stockholm, Sweden (E-mail:).
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- Copyright © 2002 the American Physiological Society