INTRODUCTION

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MYOSIN REGULATORY LIGHT CHAIN (R-LC) phosphorylation is a molecular event strongly associated with isometric posttetanic twitch potentiation and staircase in fast and mixed fast mammalian skeletal muscles (8). Incorporation of phosphate by the R-LC occurs when Ca²⁺ released by the sarcoplasmic reticulum forms a Ca²₄⁺-calmodulin complex to activate a specific enzyme, myosin light chain kinase (2). The fractional activation of myosin light chain kinase is dependent on the frequency and duration of muscle activation (16). In turn, the extent of R-LC phosphorylation is dependent on the relative activities of this kinase and myosin light chain phosphatase (protein phosphatase 1-myofibrillar), which dephosphorylates the R-LC (22). During low-frequency muscle activation the resultant elevation of R-LC phosphate content is congruent with augmented isometric twitch tension (e.g., staircase). Potentiation of both isometric twitch tension (26) in intact muscle and steady-state force in skinned fibers activated at submaximal Ca²⁺ concentrations (18) is thought to result from an R-LC phosphorylation-induced increase in f_app, the rate constant that describes the transition of cross bridges from the weak to the strong binding states, while the rate constant for the reverse transition, g_app, remains unchanged (24). However, a decrease in g_app during steady-state contractions of skinned fibers at submaximal Ca²⁺ activation has also been reported (10), suggesting that if cross bridges left the strong binding states more slowly, force would be enhanced. Thus, at a given submaximal Ca²⁺ activation, a greater fraction of the cycling cross bridges would be in the strong binding states, resulting in an increased extent and rate of isometric force development (26, 27).

Recently, we reported that a 5-Hz 20-s conditioning stimulus that yielded potentiated isometric twitch force coincident with a fivefold increase in R-LC phosphate content also enhanced the work and power of afterloaded contractions by ~22% in mouse extensor digitorum longus muscles (EDL) in vitro. When a potentiated force moves a given submaximal load, upward shifts along the force-displacement and force-velocity relations yield increased work and power (6). However, determination of work and power from force-displacement and force-velocity relations may not reflect muscle performance in vivo (9, 25), because muscles in vivo do not normally move a constant load, nor do they shorten at a constant velocity. For example, muscle displacement in vivo for cats walking on a treadmill demonstrates that muscle length changes (e.g., gastrocnemius) above compared with those below a reference length are not uniform in magnitude, and the periodicity is very much like a sine function (19). Thus a more realistic alternative to assess potentiation of dynamic contractions is the work cycle technique, which can mimic muscle action in vivo in an isolated muscle model. This method has been employed to measure work and power from muscles that generate cyclic movements (e.g., locomotion) in a variety of insects, fish, and mammals (9). If R-LC phosphorylation is to serve a functional purpose in vivo, then work and power should be potentiated during cyclic contractions. To mimic cyclic muscle actions in vitro, but to keep our work cycle model simple, we employed equal-length changes above and below optimal length (L_o) and employed twitch stimulations based on our previous report of potentiated work by mouse EDL during afterloaded contractions (6). The purpose of this study was to determine the work and power responses of mouse EDL in vitro to single-twitch stimulations during rhythmic lengthening-shortening sine cycles at 25°C, with the myosin R-LC in a nonphosphorylated and a phosphorylated state.

	METHODS

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Muscle Preparation

Both EDL were obtained from anesthetized mice (pentobarbital sodium, 80 mg/kg body wt ip) and prepared for contractile measures as described previously (26). Briefly, individual EDL were suspended vertically by securing one tendon in a clamp at the base of a jacketed organ bath containing an oxygenated (95% O₂-5% CO₂) Krebs solution (pH 7.4, 25°C) and tying the other tendon with 6-cm of 4-0 suture to the lever arm of a dual-mode servomotor (model 300H, Cambridge Technology, Waterton, MA). Each muscle was equilibrated in the bath for 10 min at a load of 1 g, and then muscle length was adjusted to determine peak active twitch force (L_o). Muscles were field stimulated via platinum electrodes with square pulses of 200-µs duration at a voltage eliciting maximal isometric twitch force (model S48, Grass Instruments, Quincy, MA).

Study 1: Work and Power Across Sine Frequency and Excursion

Muscles were rhythmically cycled above and below L_o by a computer-generated sine function driving the arm of the Cambridge servomotor. Data were collected on-line (sample frequency 2,000 Hz) while custom software controlled the frequency and excursion of the sine function and the timing (phase) of the twitch stimulation (Fig. 1) (25). Excursion describes the muscle length change in response to the peak and nadir of the servo arm displacement. Phase describes the relative delay as a percentage of the sine period (e.g., 5, 10, and 15%) between the time of maximum muscle length and the time at which the maximum rate of force development (T_dF/dtmax) would occur after a twitch stimulation. At time 0 each sine cycle began by lengthening from L_o. Equation 1 was used to determine the time for the twitch stimulation (T_stim) relative to time 0 for a given phase; the example calculates T_stim for a phase of 10% at a sine frequency of 5 Hz. With the assumption of a latent period at 25°C of 2-3 ms (6) and a delay from the onset of force development to achieve maximal rate of isometric stress development (dF/dt_max) of 2-3 ms (26), a value of 6 ms was employed for T_dF/dtmax. T_cycle is the sine period (e.g., 200 ms), and T_Lmax is the time relative to time 0 at which the muscle achieves its maximum length and then begins to shorten [i.e., 0.25 of T_cycle; modified equation of Syme and Stevens (25)]

(1)

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Stimulation during sine function () for phases of 5 to 30% and changes in muscle length over excursion (Exc) of sine function about a reference muscle length Lo. Equation 1 was employed to determine time of stimulation.

Each work determination at a given phase required a pair of consecutive sine cycles: the first with no stimulation to account for the passive force response of the muscle to movement of the servo arm and the second with a single-twitch stimulation at a specific phase (Fig. 2). Between each pair of cycles the muscle rested quietly at L_o. Eight different sine cycle pairs were evoked, each for a different phase. Phases were ordered randomly and varied from 5% ( just before the longest length of the muscle) to 30% (approximately L_o) in increments of 5% that yielded predominantly concentric contractions (Fig. 1). A complete work set for a given excursion and sine frequency consisted of eight sine cycle pairs for the eight concentric contractions, for a total collection duration of 8 s (Fig. 2A).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: work cycle protocol. There were 8 randomly ordered concentric phases. Data collection for each phase consisted of a pair of sine cycles: one with no stimulation (ns) and another with a stimulation to evoke a twitch response (st). Only one phase stimulation was used per pair of sine cycles. Muscle was quiescent until next pair of sine cycles, as indicated by timing scheme. B: raw data force (top trace) and displacement (bottom trace) responses from Cambridge servomotor during paired sine cycles for phase of 0%. , Stimulation.

Study 2: Work and Power Potentiation

Data Analysis

2

2

1

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A: determination of net force response. Nonstimulated muscle force (bottom solid trace, shown inverted) in response to Cambridge servomotor is added to stimulated response (top solid trace) to yield net force response (dashed trace). B: work loops produced at different phases when net force is plotted against muscle length change (muscle work is area inside loop). Arrows, direction of given loop; , change.

Statistics

	RESULTS

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Study 1: Effect of Phase, Excursion, and Sine Frequency on Work and Power

Phase. Active work outputs for mouse EDL stimulated by single twitches at sine frequencies that varied from 3 to 15 Hz and mean excursions of ~5, 9, and 13% L_o are illustrated in Fig. 4. At 3 and 5 Hz, work rose to a plateau by a phase of 5% and remained relatively constant before decreasing (Fig. 4A). At frequencies of 7, 10, and 15 Hz, over the range of phases employed, work at a phase of 5% was maximal and was enhanced with increasing sine frequency (7 Hz < 10 Hz < 15 Hz; Fig. 4B). After a phase of 0%, work at 7 Hz decreased slightly and then plateaued; work at 10 and 15 Hz for most phases decreased by as much as 80% (e.g., 1.6-mm excursion at 15 Hz decreased from ~2.0 to 0.4 J/kg) and then increased slightly only at a phase of 30% (Fig. 4B).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Work and power output for mouse extensor digitorum longus muscles at 25°C in response to twitch stimulations during rhythmic shortening-lengthening cycles. A: work output at 3 different excursions and 3 different frequencies at 8 different phases. B: work output as described in A for 7 Hz and 2 additional frequencies; symbols as in A. C: work response at 3 phases across 5 frequencies; symbols as in A. D: power vs. cycle frequency for 3 phases (5, 0, and 5%) in C; symbols and lines as in A and C, respectively.

Excursion. Active work output increased with increased excursion at a phase of 5% at all sine frequencies. For frequencies of 7, 10, and 15 Hz, at a phase of 0%, the work output was very similar independent of excursion. However, at phases of 5-20%, active work output decreased with increasing excursion at a given frequency (Fig. 4B). In general, at sine frequencies of 7 Hz the magnitude of work output from greatest to least with respect to the excursion magnitude was ordered 5% > 9% > 13% L_o at phases of 5-25% (Fig. 4B).

Sine frequency. Work output at 3 and 5 Hz tended to plateau at phases of 5-20% (Fig. 4A); at 7, 10, and 15 Hz, work was greatest at phases of 5, 0, and 5% (Fig. 4B). Work output at these phases for a given excursion begins to plateau at a cycle frequency of 7 Hz and remains relatively constant, with only a slight rise (5%) or slight decline (0 and 5%) at 10 or 15 Hz (Fig. 4C). In general, work was maximal for most phases at 7 Hz at each excursion (Fig. 4B); therefore, this frequency was selected to compare work output at all phases with the myosin R-LC in a nonphosphorylated and a phosphorylated state. Power increased linearly with increased sine frequency up to 15 Hz (Fig. 4D).

Study 2: Work With and Without Elevated Myosin R-LC Phosphate Content

R-LC phosphate content. We showed previously that R-LC phosphate content 10-30 s after a 5-Hz 20-s CS is 0.60-0.70 mol phosphate/mol R-LC (6, 15, 26, 27). In the present study, R-LC phosphate content determined in muscles freeze clamped at times equivalent to 17 and 60 s after a CS in the no-CS condition were at a basal level of 0.16 ± 0.06 (n = 4) and 0.19 ± 0.03 (SE) mol phosphate/mol R-LC (n = 4), respectively, similar to basal values reported previously (0.08 ± 0.02 to 0.14 ± 0.04 mol phosphate/mol R-LC) (6, 26, 27). These basal levels suggest that R-LC phosphate content after a CS would be increased ~3.7-fold compared with the no-CS condition (e.g., 0.70/0.19 = 3.68).

Isometric twitches. Isometric twitch contractile properties were unchanged by the work cycles (Table 1; see data for control groups for no-CS and CS conditions). Mean stress for isometric twitches obtained 44 s after compared with 1 min before the control or the experimental work cycle series of the no-CS condition and the control work cycle series of the CS condition was not potentiated (Table 1). Consistent with the hypothesis of the present studies, this was interpreted as a low degree of R-LC phosphorylation. In contrast, mean stress and dF/dt_max for isometric twitches obtained 44 s after the completion of the experimental work cycle series in the CS condition were potentiated a mean of 14 and 12%, respectively (Table 1). These potentiated responses were interpreted as evidence of elevated myosin R-LC phosphate content. Half relaxation time was significantly less after compared with before the work cycle series for the CS condition (P < 0.05).

Work and power. The work response pattern at 7 Hz in study 2 was similar to that observed in study 1 with respect to phase and excursion (data not shown). Figure 5 illustrates representative concentric work loops at a phase of 15% for the same muscle in a phosphorylated and a nonphosphorylated state from the CS condition. Work potentiation at each phase is presented in Fig. 6A for the ~9% L_o excursion. Significant relative potentiation of work output for the CS condition was evident at all phases, whereas only minimal enhancement or depression in work output was observed for the no-CS condition (Fig. 6B). At the ~9% L_o excursion, relative work was potentiated a mean of 44% for phases of 0-25% (Fig. 6B), at the ~5% L_o excursion a mean of 25% for phases of 5-30% (representative data, Table 2), and for the ~13% L_o excursion a mean of 50% for phases of 0-25% (representative data, Table 2). Because work was increased in the CS vs. the no-CS condition at the same sine frequency (7 Hz), power was also increased. For example, at an excursion of ~13% L_o and a phase of 15%, mean power was increased 51% (8.9 vs. 5.9 W/kg; Table 2).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Overlay of concentric work loop produced from same muscle in a nonphosphorylated (dashed trace) and phosphorylated (solid trace) condition. Excursion = 1.2 mm (~9% Lo, phase = 15%, sine frequency = 7 Hz). Arrows, direction of loops.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   A: work output at an excursion of 1.2 mm (mean displacement ~9% Lo) and a sine frequency of 7 Hz at 8 different concentric phases. First pair of bars in each group of 4 represents work response obtained 5 min before (pre; open bars) and 17-25 s after (post; filled bars) no conditioning stimulus (no-CS) condition (n = 4). Second pair of bars represents pre (cross-hatched bars) and post (hatched bars) work outputs of 5-Hz 20-s CS condition (n = 7). * Significant increase in work output for post vs. pre work values in CS condition (P < 0.05). B: relative work for no-CS (open bars) and CS (filled bars) conditions. In each case, post work value (W*) is divided by pre work value (W) and 1 is subtracted to yield fraction increase/decrease in work. * Significant increase in work output for post vs. pre work values in CS condition (P < 0.05).

	DISCUSSION

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The purpose of this report was to examine the work and power output of submaximally activated isolated mouse EDL at 25°C during rhythmic lengthening-shortening sine cycles at a low and a high level of R-LC phosphate content. At a basal level of R-LC phosphate content, specific characteristics described the work and power responses of the EDL with respect to sine frequency, phase, and excursion when the EDL was stimulated by single twitches (Fig. 4). Power increased linearly with increased cycle frequency up to 15 Hz. Because concentric work output was maximal for most phases and for each excursion at a sine frequency of 7 Hz, this frequency was selected to determine work and power across all phases at an elevated level of R-LC phosphorylation. Coincident with a 3.7-fold increase in R-LC phosphate content, muscle concentric work and mean power were potentiated from 25 to 50% across mean excursions of ~5-13% L_o. Peak isometric twitch force and maximal rate of force development were potentiated 14 and 12%, respectively. These data clearly indicate that the utility of a force-potentiating mechanism to muscle function is best revealed under dynamic conditions (e.g., work cycle), instead of from a single point on the isometric force profile (i.e., the peak). As expected, when no conditioning stimulus (no-CS) was employed and there was no increase in R-LC phosphate content, neither concentric work, isometric force, nor maximal rate of force development was augmented.

Work output coincident with an elevated R-LC phosphate content was determined at a constant cycle frequency of 7 Hz; therefore, the potentiated net work at each of the three excursions after the 5-Hz 20-s CS was due to augmented force. Although possible, it is unlikely that fatigue mechanisms significantly depressed force output. We previously reported minimal metabolic displacement of stimulated EDL from that of rested muscles after this CS, with only a modest depression in ATP and phosphocreatine (22-27%) and a modest increase in lactate (2-fold) (26). Thus, if elements associated with fatigue such as increased P_i were attenuating force output, their effects were easily masked by mechanisms of force potentiation. Two possible mechanisms to account for the potentiated force after the CS are alterations in Ca²⁺ dynamics, such as increased residual cytosolic Ca²⁺ concentration, or changes in the rates that describe the cross-bridge transitions between the non-force-generating and force-generating states.

Half relaxation time was significantly decreased for isometric twitches obtained after the experimental work series in the CS condition (Table 1); therefore, it is unlikely that increased sarcoplasmic reticulum Ca²⁺ release [e.g., due to increased P_i (28)] or elevated residual cytosolic Ca²⁺ concentration in the period after the CS can account for potentiated force, work, and power (6, 26, 27). Rather, the diminished half relaxation time suggests less activating Ca²⁺ per twitch stimulation. Therefore, although the potential modulatory effects of Ca²⁺ in the period after the CS cannot be completely discounted because Ca²⁺ was not directly measured, the significant decrease in half relaxation time for the potentiated isometric twitches suggests that force potentiation was not due to Ca²⁺ but likely another mechanism or mechanisms. One such possibility is the increased incorporation of phosphate by the R-LC. This possibility is supported by data obtained from mouse EDL that demonstrate the extent of potentiated displacement (and therefore work) during twitch afterloaded contractions (6) and potentiated work and power during cyclic contractions (unpublished observations) are closely correlated to the extent of R-LC phosphate content over a period of 5 min after the CS.

A current hypothesis to account for steady-state force potentiation in skinned fibers and twitch force potentiation in intact muscle suggests that myosin heads covalently bound with negatively charged phosphate become disordered from the negatively charged thick filament backbone and come in closer proximity to actin, thereby facilitating transition of the cross bridges from the weak to the strong binding states (1, 12, 14, 17). In the cross-bridge model of Brenner (3), this transition is described by the apparent rate constant, f_app; a second rate constant, g_app, describes the reverse transition (3). Although in this model Ca²⁺ binding to troponin C is considered the primary regulator of force output by altering the kinetics of actin-myosin interaction through modulation of f_app and not g_app (3), increased R-LC phosphate content appears to have similar modulatory effects independent of Ca²⁺. Coincident with an elevated R-LC phosphate content in skinned fibers of rabbit psoas during submaximal Ca²⁺ activations (e.g., pCa = 5.4) at 15°C, f_app was increased, g_app was unchanged, and the rate of force development and the extent of steady-state force (24) were potentiated. The Ca²⁺ regulation of g_app, however, may be temperature dependent, because at 21°C a decreased g_app was reported in skinned fibers of rabbit during submaximal steady-state force contractions (10). Thus, if cross bridges left the strong binding states more slowly rather than, or in addition to, entering these states more quickly, force would be enhanced. To clarify this possibility, the combined influence of temperature and R-LC phosphorylation on rate constants and on steady-state force potentiation should be further characterized. Within the context of the above hypothesis, increased incorporation of phosphate by R-LC could reasonably account for augmented isometric twitch force production and dF/dt_max (26, 27), as well as augmented peak work and power from force-velocity and force-displacement relations (6), if f_app (and possibly g_app) is modulated in intact muscle as it is in skinned fibers during steady-state force contractions. Furthermore, if elevated R-LC phosphate content has a similar modulatory effect during cyclic submaximal contractions of intact muscle, then work output should be increased. Thus, based on the assumption that increased phosphate incorporation by the R-LC is the predominant response of the muscle to the 5-Hz 20-s CS, we propose, on the basis of available literature, that this biochemical event represents a reasonable mechanism by which work and power during cyclic contractions can be potentiated.

Modulation of muscle force by a potentiating mechanism during dynamic contractions could enhance in vivo skeletal muscle function (6). Movement of the body (e.g., locomotion) is the outcome of a carefully sequenced activation of motor units within appropriate skeletal muscles to provide the force and displacement requirements to articulate the limbs. Muscle work and power to effect movement result from contractions that are characterized by force production and length change over a discrete time interval. The twitch contraction, which represents the fundamental response of a motor unit to a single above-threshold stimulus (5), can clearly be potentiated in humans and in small mammals, yielding increases in isometric force (8) and in work and power (6). Force potentiation in isolated mouse EDL and in human quadriceps appears to be limited to a stimulation frequency threshold of <20 Hz where the force profiles are characterized by unfused twitches (7, 26); thus work potentiation is likely also limited to a similar stimulation frequency threshold. This frequency range has been observed with activation of motor units in human muscles. For example, 60% of the peak force generated by a pool of motor units from human toe extensors was achieved at a stimulation frequency of <20 Hz (4). It should be noted that fusion occurred at a lower stimulus frequency [between 5 and 8 Hz (4)] than in the quadriceps (7), suggesting that the potentiation threshold may vary among muscles.

In humans, muscles are composed of a mixed-fiber population (20), in which slow and fast myosin R-LC can be phosphorylated (21), suggesting that force potentiation may not be limited to only fast or mixed fast motor unit populations, as it is for small mammals (8). With the assumption of a stimulation frequency below the potentiation threshold, if a majority of activated motor units during a movement were potentiated, it would be reasonable to expect a collective potentiated work/power output from the muscle. From a muscle function perspective, a pool of potentiated motor units could yield a substantial gain in work/power output for the same level of neural input. Conversely, to maintain a desired submaximal work/power output, motor unit recruitment and/or firing frequency could decrease, possibly mediated by a mechanical or biochemical feedback pathway.

In summary, we have assumed that increased R-LC phosphorylation is the predominant outcome of a 5-Hz 20-s CS. Potentiation of isometric twitch force in intact muscles (11, 13, 15) or steady-state force in single fibers at suboptimal Ca²⁺ concentration (14, 23, 24) is strongly associated with an elevated phosphate incorporation by the R-LC. Recently, we reported an association between R-LC phosphorylation and potentiated displacement, work, and power on the basis of the force-velocity relation (6). In the present study, coincident with an elevated R-LC phosphate content, work and power were also potentiated during twitch activations of a rhythmically cycled muscle. Our findings suggest that R-LC phosphorylation could act to modulate isometric and dynamic muscle contractions during submaximal activations.