Decreased motor unit discharge rate in the potentiated human tibialis anterior muscle.

AIM: The purpose of this study was to examine the influence of post-activation potentiation (PAP), the transient increase in low-frequency isometric force observed after muscle activity, on motor unit discharge rates measured during submaximal contractions. METHODS: A quadrifilar needle electrode was inserted into the tibialis anterior muscle to determine discharge rate of individual motor units while monopolar electrodes were used to monitor the root-mean-square (RMS) and mean power frequency (MPF) of the surface EMG signal. Control (unpotentiated) and experimental (potentiated) measures were obtained during a 5 s voluntary contraction at 50% of maximal. In between these measures, subjects performed a 10 s maximal voluntary contraction (MVC) to induce PAP. RESULTS: All subjects data are reported as means ± SEM (n = 10). Compared to baseline values measured prior to the MVC, isometric twitch force measured immediately after the MVC was increased by 260 ± 16% (day 3). Motor unit discharge rate in the potentiated tibialis anterior muscle decreased by approx. 10%, from 20.3 ± 0.8 (before) to 18.3 ± 0.99 pps (P = 0.01) (after). Moreover, the MPF was decreased by approx. 9% (from 58.1 ± 2.84 to 53.6 ± 2.85 Hz; P = 0.01) in the potentiated tibialis anterior. On the other hand, consistent with the absence of fatigue during the MVC, the RMS signal was not altered in the potentiated tibialis anterior (0.29 ± 0.03 vs. 0.33 ± 0.04 mV; P = 0.07). CONCLUSION: Motor unit discharge rates determined during a brief, submaximal contraction were decreased in the potentiated human tibialis anterior muscle.

Power fatigue of the rat diaphragm muscle.

We hypothesized that decrements in maximum power output (W(max)) of the rat diaphragm (Dia) muscle with repetitive activation are due to a disproportionate reduction in force (force fatigue) compared with a slowing of shortening velocity (velocity fatigue). Segments of midcostal Dia muscle were mounted in vitro (26 degrees C) and stimulated directly at 75 Hz in 400-ms-duration trains repeated each second (duty cycle = 0.4) for 120 s. A novel technique was used to monitor instantaneous reductions in maximum specific force (P(o)) and W(max) during fatigue. During each stimulus train, activation was isometric for the initial 360 ms during which P(o) was measured; the muscle was then allowed to shorten at a constant velocity (30% V(max)) for the final 40 ms, and W(max) was determined. Compared with initial values, after 120 s of repetitive activation, P(o) and W(max) decreased by 75 and 73%, respectively. Maximum shortening velocity was measured in two ways: by extrapolation of the force-velocity relationship (V(max)) and using the slack test [maximum unloaded shortening velocity (V(o))]. After 120 s of repetitive activation, V(max) slowed by 44%, whereas V(o) slowed by 22%. Thus the decrease in W(max) with repetitive activation was dominated by force fatigue, with velocity fatigue playing a secondary role. On the basis of a greater slowing of V(max) vs. V(o), we also conclude that force and power fatigue cannot be attributed simply to the total inactivation of the most fatigable fiber types.

Effects of rapid shortening on rate of force regeneration and myoplasmic [Ca2+] in intact frog skeletal muscle fibres.

1. The effect of rapid shortening on rate of force regeneration (dF/dtR) was examined in single, intact frog (Rana temporaria) skeletal muscle fibres (3.0 C). Step releases leading to unloaded shortening were applied after 500 ms of stimulation, during the plateau of an isometric tetanus. Initial mean sarcomere length ranged from 2.05 to 2.35 micrometer; force regeneration after shortening was at 2.00 micrometer. 2. Values for dF/dtR following a 25 nm half-sarcomere-1 release were 3.17 +/- 0.17 (mean +/- s.e.m., n = 8) times greater than the initial rate of rise of force before release (dF/dtI). As release size was increased from 25 to 175 nm half-sarcomere-1, the relationship between release size and dF/dtR decreased sharply before attaining a plateau value that was 1.34 +/- 0.09 times greater than dF/dtI. Despite wide variations in dF/dtR, the velocity of unloaded shortening remained constant (2.92 +/- 0.08 micrometer half-sarcomere-1 s-1; n = 8) for the different release amplitudes used in this study. 3. To investigate its role in the attenuation of dF/dtR with increased shortening, the effects of rapid ramp (constant velocity) shortening on intracellular free Ca2+ concentration ([Ca2+]i) were monitored using the Ca2+-sensitive fluorescent dye furaptra. Compared with an isometric contraction, rapid fibre shortening was associated with a transient increase in [Ca2+]i while force regeneration after shortening was associated with a transient reduction in [Ca2+]i. The greatest reductions in [Ca2+]i were associated with the largest amplitude ramps. 4. Cross-bridge-mediated modifications of the Ca2+ affinity of troponin C (TnC) may explain the fluctuations in [Ca2+]i observed during and after ramps. Associated fluctuations in TnC Ca2+ occupancy could play a role in the reduction of dF/dtR with increasing release size.

Potentiation of in vitro concentric work in mouse fast muscle.

Phosphorylation of myosin regulatory light chain (R-LC) is associated with potentiated work and power during twitch afterloaded contractions in mouse extensor digitorum longus muscle [R. W. Grange, C. R. Cory, R. Vandenboom, and M. E. Houston. Am. J. Physiol. 269 (Cell Physiol. 38): C713-C724, 1995]. We now describe the association between R-LC phosphorylation and potentiated concentric work when the extensor digitorum longus muscle is rhythmically shortened and lengthened to simulate contractions in vivo. Work output (at 25 degrees C) was characterized at sine frequencies of 3, 5, 7, 10, and 15 Hz at excursions of 0.6, 1.2, and 1.6 mm (approximately 5, 9, and 13% optimal muscle length) at a low level of R-LC phosphorylation. Muscles stimulated during the sine function with a single twitch at specific times before or after the longest muscle length yielded maximal concentric work near the longest muscle length at a sine frequency of 7 Hz (e.g., excursion approximately 9% optimal muscle length = 1.6 J/kg). Power increased linearly between sine frequencies of 3 and 15 Hz at all excursions (maximum approximately 29 W). After a 5-Hz 20-s conditioning stimulus and coincident with a 3.7-fold increase in R-LC phosphate content (e.g., from 0.19 to 0.70 mol phosphate/mol R-LC), work at the three excursions and a sine frequency of 7 Hz was potentiated a mean of 25, 44, and 50% (P < 0.05), respectively. The potentiated work during rhythmic contractions is consistent with enhanced interaction between actin and myosin in the force-generating states. On the basis of observations in skinned skeletal muscle fibers (H. L. Sweeney and J. T. Stull. Proc. Natl. Acad. Sci. USA 87:414-418, 1990), this enhancement could result from increased phosphate incorporation by the myosin R-LC. Under the assumption that the predominant effect of the conditioning stimulus was to increase R-LC phosphate content, our data suggest that a similar mechanism may be evident in intact muscle.

Increased force development rates of fatigued mouse skeletal muscle are graded to myosin light chain phosphate content.

Phosphorylation of myosin regulatory light chain (R-LC) increases the Ca2+ sensitivity of cross-bridge transitions, which determine rate of force development in skinned skeletal muscle fibers. The purpose of this study was to determine whether phosphorylation of R-LC is the molecular basis for the increased force development rates (+dF/dtmax) observed in fatigued mouse extensor digitorum longus muscle (EDL) (stimulated in vitro at 25 degrees C). Parameters of twitch and tetanic force were obtained after the application of different-frequency conditioning stimuli (CS), which were used to vary R-LC phosphorylation and reduce peak tetanic force (Po). Without CS, R-LC phosphorylation (in moles phosphate per mole R-LC) was not elevated above rest (0.11 +/- 0.02 vs. 0.13 +/- 0.02, respectively), and no aspect of the twitch (Pt) Po was altered. Stimulating muscles at 2.5-20 Hz increased R-LC phosphorylation in a frequency-dependent manner, from 0.23 +/- 0.04 to 0.82 +/- 0.03, respectively. Moreover, stimulation at 2.5-20 Hz potentiated Pt (range: 4 +/- 2-28 +/- 2%), increased the +dF/dtmax of potentiated twitches (range: 5 +/- 1-28 +/- 2%), and reduced Po (range: 6 +/- 1-21 +/- 1%). Higher-frequency stimulation (40 or 100 Hz) did not phosphorylate R-LC or potentiate Pt or twitch +dF/dtmax further. Stimulation at 40 and 100 Hz did, however, have different effects on Po compared with 20-Hz data (Po reduced 27 +/- 2 and 11 +/- 2%, respectively). The increased +dF/dtmax of potentiated twitches observed after different CS procedures were graded to R-LC phosphorylation (r = 0.97, P < 0.001). It is concluded that phosphorylation of R-LC increases extent of twitch force development in mouse EDL muscle fatigued by CS.

Phosphorylation of myosin and twitch potentiation in fatigued skeletal muscle.

Phosphorylation of myosin regulatory light chain (R-LC) increases the sensitivity of skinned skeletal muscle fibers to low Ca2+ activation. The purpose of this study was to determine whether phosphorylation of R-LC-mediated increases in Ca2+ sensitivity provides a molecular basis for potentiated twitch forces observed during fatigue of intact mammalian skeletal muscle. Tetanic stimulation for 120 s reduced peak tetanic force (Po) of mouse extensor digitorum longus (EDL) muscle by 74 +/- 2%. Despite high frequency fatigue (HFF), Pt was potentiated by 18 +/- 3% when R-LC phosphorylation (in moles phosphate per mole R-LC) was increased from 0.11 +/- 0.05 (rest) to 0.52 +/- 0.04 by 15 s of stimulation. Thereafter Pt declined below resting values despite high levels for R-LC phosphorylation (0.80 +/- 0.04 after 120 s of stimulation). In separate experiments, 10 min of stimulation, which reduced Po and Pt by 80 +/- 2 and 67 +/- 3%, respectively, was used to induce low frequency fatigue (LFF) in mouse EDL muscle. During LFF, long-lasting reductions in Pt were evident despite near-normal levels for Po (79 +/- 2 and 98 +/- 2% of controls, respectively). Application of conditioning stimuli (CS) increased R-LC phosphate content of fatigued muscles from 0.15 +/- 0.03 (rest) to 0.56 +/- 0.03 (stimulated) and potentiated Pt by 26 +/- 2% compared with LFF. Twitch potentiation during LFF was transient, lasting only as long as R-LC was phosphorylated above resting values for fatigued muscles. Overall, our data showing potentiated twitch forces concomitant with elevations in R-LC phosphate content during either HFF or LFF of mouse EDL muscle suggest that this molecular event counters reduced twitch forces during these forms of fatigue. Our results may be explained by R-LC phosphorylation induced increases in Ca2+ sensitivity for twitch force production in fatigued muscle.

Elevated catalase activity in red and white muscles of MyoD gene-inactivated mice.

MyoD is a myogenic transcription factor responsible for skeletal muscle differentiation during development. Muscle antioxidant enzyme status was determined in transgenic MyoD deactivated mice. While catalase activity was significantly (P < 0.05) elevated in soleus and extensor digitorum longus muscles from MyoD deactivated mice, superoxide dismutase and glutathione peroxidase activities were not. While this may imply a greater propensity for inherent oxidative stress, soleus glutathione status was similar between MyoD deactivated mouse and control soleus muscles. Catalase activity is localized primarily in peroxisomes. Therefore elevated catalase activity may also indicate the presence of factors associated with peroxisome proliferation in muscles from MyoD gene-inactivated mice.

Myosin phosphorylation augments force-displacement and force-velocity relationships of mouse fast muscle.

Two studies were conducted to examine the effect of myosin regulatory light chain (R-LC) phosphorylation on the rate and extent of shortening in submaximally activated mouse extensor digitorum longus muscles in vitro at 25 degrees C. For each study, R-LC phosphate content was increased fivefold by application of a 5-Hz, 20-s conditioning stimulus (CS) to 0.65-0.68 mol phosphate/mol R-LC; this level was sustained between 10 and 40 s after the CS. Maximum isometric twitch force and the maximum rate of force development (+dF/dtmax) were potentiated in the range 13-17% and 9-17% (P < 0.05), respectively, after the CS. In study 1, the maximal rate and extent of shortening were significantly enhanced by 10 and 21% (P < 0.001), respectively, when measured using a twitch zero-load clamp technique. In study 2, the force-velocity and force-displacement relationships were both augmented when determined with the twitch afterload technique. Displacement was enhanced between 20 and 82% for loads that ranged from 3 to 75% of active peak twitch force, whereas velocity was increased 6-8% over the same range (P < 0.05), including the predicted maximum velocity (Vmax; 5.08 vs. 4.69 muscle length/s). In both studies the increase in velocity likely represents a shift along the force-velocity relationship toward true Vmax that reflects a decrease in relative load due to force potentiation. Furthermore, with the decrease in relative load, displacement at a given load was also increased. Potentiated displacement and extent of R-LC phosphorylation also decreased in parallel when studied for 5 min after the CS. The increase in muscle shortening is a novel finding and suggests a function for R-LC phosphorylation with respect to movement because both peak work and power were also enhanced by up to 22%. These effects are consistent with an R-LC phosphorylation-induced increase in fapp, the apparent rate constant that describes the cross-bridge transition from the non-force-generating to the force-generating state.

Myosin phosphorylation enhances rate of force development in fast-twitch skeletal muscle.

Skeletal muscle force output is regulated through Ca(2+)-mediated alterations of the rate at which cross bridges make the transition from non-force-generating to force-generating states, defined by the rate constant fapp. In skinned-fiber models, phosphate incorporation by the regulatory light chain (R-LC) subunits of myosin increases fapp independent of Ca2+, thus increasing the Ca2+ sensitivity for the rate and extent of steady-state force development. The goal of this study was to determine whether phosphate incorporation by the R-LC subunits of skeletal muscle is related to the maximal rate of isometric force development (+dF/dtmax) in intact muscle. Changes in myosin phosphate content and contractile performance were analyzed at selected times after the application of a 5-Hz 20-s conditioning stimulus (CS) employed specifically to elevate R-LC phosphate content in mouse extensor digitorum longus at 25 degrees C. R-LC phosphate content (in mol phosphate/mol R-LC) increased from 0.13 +/- 0.04 at rest to 0.68 +/- 0.02 20 s after the CS and by 360 s after the CS R-LC phosphate content had decayed to 0.37 +/- 0.06. Values obtained for twitch and tetanic +dF/dtmax after the CS were strongly correlated to R-LC phosphate content (r = 0.97 and 0.96, respectively), suggesting that phosphate incorporation by skeletal myosin R-LC contributes to an enhanced rate of isometric force development in fast-twitch skeletal muscle.

Threshold for force potentiation associated with skeletal myosin phosphorylation.

Phosphate incorporation by the phosphorylatable light chains (P-LC) of myosin is associated with isometric twitch force potentiation in intact fast-twitch muscle. The purpose of this study was to examine the association between myosin P-LC phosphorylation and force potentiation at higher stimulation frequencies (1-150 Hz) using mouse extensor digitorum longus (EDL) muscles at 25 degrees C. Peak isometric force and the peak rate of isometric force development (+dF/dtmax) were measured at selected test frequencies before and after the application of a 5-Hz 20-s conditioning stimulation known to increase P-LC phosphate content. Associated with a ninefold elevation in myosin P-LC phosphate content (to 0.72 mol phosphate/mol P-LC), +dF/dtmax was increased at all test frequencies (mean 27%, range 20-37%). After the conditioning stimulus, peak isometric force was increased by approximately 15% for frequencies 1-15 Hz. However, at 20-150 Hz, the increase in +dF/dtmax was not associated with force potentiation, since peak force was diminished by 5-40%. These data reveal that the stimulation frequency limit for the potentiation of peak force production associated with myosin P-LC phosphorylation is < 20 Hz in mouse EDL at 25 degrees C. Furthermore, the data suggest that increases in the rate constant describing the rate of cross-bridge transition from a non-force-generating to a force-generating state mediated by myosin P-LC phosphorylation may be responsible for the general increase in +dF/dtmax and for the force potentiation at 1-15 Hz.

Physiological significance of myosin phosphorylation in skeletal muscle.

Each S-1 or head portion of the myosin molecule in skeletal muscle contains a subunit known as the regulatory or phosphorylatable light chain (P-LC). Phosphorylation of the P-LC is mediated by the second messenger Ca2+ and takes place when the muscle fibre is activated. In smooth muscle, phosphorylation of the P-LC is the principal mechanism that initiates contraction, but in skeletal muscle myosin P-LC phosphorylation is not required for contraction and a definitive role has not been established. It has been proposed that P-LC phosphorylation modulates the intrinsic nature of actin-myosin interactions, leading to force potentiation under suboptimal activation conditions. An example of this is posttetanic potentiation. This paper describes a P-LC phosphorylation induced mechanism for force enhancement during isometric contraction. In addition, it summarizes recent data revealing that P-LC phosphorylation is associated with enhanced work output of fast-twitch muscle during shortening and lengthening contractions.