Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature.

KEY POINTS: Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi ] in the myoplasm. High [Pi ] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi ] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi ] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low- or no-force state and show that fibre length changes perturb the Pi -sensitive force generation of the crossbridge cycle. ABSTRACT: Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi ] in the myoplasm. Experiments in skinned fibres at low temperature (10-12°C) have shown that [Pi ] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi ] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi ] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.

The selective and non-selective cyclooxygenase inhibitors valdecoxib and piroxicam induce the same postoperative analgesia and control of trismus and swelling after lower third molar removal.

We compared the clinical efficacy of orally administered valdecoxib and piroxicam for the prevention of pain, trismus and swelling after removal of horizontally and totally intrabony impacted lower third molars. Twenty-five patients were scheduled to undergo removal of symmetrically positioned lower third molars in two separate appointments. Valdecoxib (40 mg) or piroxicam (20 mg) was administered in a double-blind, randomized and crossed manner for 4 days after the surgical procedures. Objective and subjective parameters were recorded for comparison of postoperative courses. Both agents were effective for postoperative pain relief (N = 19). There was a similar mouth opening at suture removal compared with the preoperative values (86.14 +/- 4.36 and 93.12 +/- 3.70% of the initial measure for valdecoxib and piroxicam, respectively; ANOVA). There was no significant difference regarding the total amount of rescue medication taken by the patients treated with valdecoxib or piroxicam (173.08 +/- 91.21 and 461.54 +/- 199.85 mg, respectively; Wilcoxon test). There were no significant differences concerning the swelling observed on the second postoperative day compared to baseline measures (6.15 +/- 1.84 and 8.46 +/- 2.04 mm for valdecoxib and piroxicam, respectively; ANOVA) or on the seventh postoperative day (1.69 +/- 1.61 and 2.23 +/- 2.09 mm for valdecoxib and piroxicam, respectively; ANOVA). The cyclooxygenase-2 selective inhibitor valdecoxib is as effective as the non-selective cyclooxygenase inhibitor piroxicam for pain, trismus and swelling control after removal of horizontally and totally intrabony impacted lower third molars.

The selective and non-selective cyclooxygenase inhibitors valdecoxib and piroxicam induce the same postoperative analgesia and control of trismus and swelling after lower third molar removal

We compared the clinical efficacy of orally administered valdecoxib and piroxicam for the prevention of pain, trismus and swelling after removal of horizontally and totally intrabony impacted lower third molars. Twenty-five patients were scheduled to undergo removal of symmetrically positioned lower third molars in two separate appointments. Valdecoxib (40 mg) or piroxicam (20 mg) was administered in a double-blind, randomized and crossed manner for 4 days after the surgical procedures. Objective and subjective parameters were recorded for comparison of postoperative courses. Both agents were effective for postoperative pain relief (N = 19). There was a similar mouth opening at suture removal compared with the preoperative values (86.14 ± 4.36 and 93.12 ± 3.70 percent of the initial measure for valdecoxib and piroxicam, respectively; ANOVA). There was no significant difference regarding the total amount of rescue medication taken by the patients treated with valdecoxib or piroxicam (173.08 ± 91.21 and 461.54 ± 199.85 mg, respectively; Wilcoxon test). There were no significant differences concerning the swelling observed on the second postoperative day compared to baseline measures (6.15 ± 1.84 and 8.46 ± 2.04 mm for valdecoxib and piroxicam, respectively; ANOVA) or on the seventh postoperative day (1.69 ± 1.61 and 2.23 ± 2.09 mm for valdecoxib and piroxicam, respectively; ANOVA). The cyclooxygenase-2 selective inhibitor valdecoxib is as effective as the non-selective cyclooxygenase inhibitor piroxicam for pain, trismus and swelling control after removal of horizontally and totally intrabony impacted lower third molars.

Analgesic and anti-inflammatory dose-response relationship of 7.5 and 15 mg meloxicam after lower third molar removal: a double-blind, randomized, crossover study.

Fifty patients were scheduled to undergo removal of symmetrically positioned lower third molars in two separate appointments. Meloxicam 7.5 or 15 mg was once daily administered in a double-blind, randomized and crossover manner after the surgery for 4 days. Objective and subjective parameters were recorded for comparison of postoperative courses. Patients treated with 7.5mg meloxicam who underwent osteotomy reported higher pain scores at 1.5, 3, 4, 10, 12 and 16 h (P<0.05) and ingested a greater amount of rescue analgesic medication (P<0.05) than those who did not require osteotomy. A higher percentage of patients who underwent osteotomy medicated with 7.5mg meloxicam needed rescue medication as compared to those who did not require osteotomy (P<0.05). There was a similar mouth opening at suture removal compared with preoperative values for both doses (P>0.05). There were no significant differences concerning swelling observed on the 2nd or 7th postoperative days in comparison with baseline (P>0.05) between the two doses. Pain, trismus and swelling after lower third molar removal not requiring osteotomy can be successfully controlled by a dose regimen of 7.5mg meloxicam once daily. For more aggressive extractions 15 mg meloxicam is advisable.

Effects of the number of actin-bound S1 and axial force on X-ray patterns of intact skeletal muscle.

Effects of the number of actin-bound S1 and of axial tension on x-ray patterns from tetanized, intact skeletal muscle fibers were investigated. The muscle relaxant, BDM, reduced tetanic M3 meridional x-ray reflection intensity (I(M3)), M3 spacing (d(M3)), and the equatorial I(11)/I(10) ratio in a manner consistent with a reduction in the fraction of S1 bound to actin rather than by generation of low-force S1-actin isomers. At complete force suppression, I(M3) was 78% of its relaxed value. BDM distorted dynamic I(M3) responses to sinusoidal length oscillations in a manner consistent with an increased cross-bridge contribution to total sarcomere compliance, rather than a changed S1 lever orientation in BDM. When the number of actin-bound S1 was varied by altering myofilament overlap, tetanic I(M3) at low overlap was similar to that in high [BDM] (79% of relaxed I(M3)). Tetanic d(M3) dependence on active tension in overlap experiments differed from that observed with BDM. At high BDM, tetanic d(M3) approached its relaxed value (14.34 nm), whereas tetanic d(M3) at low overlap was 14.50 nm, close to its value at full overlap (14.56 nm). This difference in tetanic d(M3) behavior was explicable by a nonlinear thick filament compliance which is extended by both active and passive tension.

Myosin lever disposition during length oscillations when power stroke tilting is reduced.

M3 reflection intensity (I(M3)) from tetanized, intact skeletal muscle fiber bundles was measured during sinusoidal length oscillations at 2.8 kHz, a frequency at which the myosin motor's power stroke is greatly reduced. I(M3) signals were approximately sinusoidal, but showed a "double peak" distortion previously observed only at lower oscillation frequencies. A tilting lever arm model simulated this distortion, where I(M3) was calculated from the molecular structure of myosin subfragment 1 (S1). Simulations showed an isometric lever arm disposition close to normal to the filament axis at isometric tension, similar to that found using lower oscillation frequencies, where the power stroke contributes more toward total S1 movement. Inclusion of a second detached S1 in each actin-bound myosin dimer increased simulated I(M3) signal amplitude and improved agreement with the experimental data. The best agreement was obtained when detached heads have a fixed orientation, insensitive to length changes, and similar to that of attached heads at tetanus plateau. This configuration also accounts for the variations in relative intensity of the two main peaks of the M3 reflection substructure after a length change. This evidence of an I(M3) signal distortion when power stroke tilting is suppressed, provided that a large enough amplitude of length oscillation is used, is consistent with the tilting lever arm model of the power stroke.

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

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 Ca(2+) concentration. In the experiments reported here, we investigated the possible role of Ca(2+) in static stiffness by comparing static stiffness measurements in the presence of Ca(2+) release inhibitors (D600, Dantrolene, (2)H(2)O) and cross-bridge formation inhibitors [2,3-butanedione monoxime (BDM), hypertonicity]. Both series of agents inhibited tension; however, only D600, Dantrolene, and (2)H(2)O decreased at the same time static stiffness, whereas BDM and hypertonicity left static stiffness unaltered. These results indicate that Ca(2+), 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.

Frequency-dependent distortion of meridional intensity changes during sinusoidal length oscillations of activated skeletal muscle.

Bundles of intact, tetanized skeletal muscle fibers from Rana temporaria were subjected to sinusoidal length oscillations in the frequency domain 100 Hz to 3 kHz while measuring force and sarcomere length. Simultaneously, intensity of the third-order x-ray reflection of the axial myosin unit cell (I(M3)) was measured using synchrotron radiation. At oscillation frequencies <1 kHz, I(M3) was distorted during the shortening phase of the sinusoid (i.e., where bundle length was less than rest length). Otherwise, during the stretch phase of oscillations at all frequencies, during the shortening phase of oscillations above 1 kHz, and for bundles in the rigor state, I(M3) was approximately sinusoidal in form. Mean I(M3) during oscillations was reduced by 20% compared to the isometric value, suggesting a possible change in S1 disposition during oscillations. However, the amplitude of length change required to produce distortion (estimated from the phase angle at which distortion was first evident) corresponded to that of a step release sufficient to reach the maximum I(M3), indicating a mean S1 disposition during oscillations close to that during an isometric tetanus. The mechanical properties of the bundle during oscillations were also consistent with an unaltered S1 disposition during oscillations.

Crossbridge kinetics in single frog muscle fibres in presence of ethylene glycol.

Single fibres isolated from frog muscle were tetanically stimulated at 14 degrees C to produce isometric tetani at a sarcomere length of about 2.16 microm, using a striation follower device to measure the sarcomere length of a selected segment of fibre. Force-velocity data were obtained by applying ramp releases at pre-set velocity at the tetanus plateau. Sarcomere stiffness was measured at isometric plateau and during isotonic shortening by using sinusoidal length changes at 2 kHz frequency and about 1 nm per half sarcomere (hs) peak to peak amplitude. A correction method was used to compensate for the force truncation due to the quick recovery. After data collection, the bathing solution was substituted with Ringer plus ethylene glycol (EG) at 2 M (11.2% v/v). When the fibre was fully equilibrated with the new solution, the measurements were repeated. Ethylene glycol reduced the speed of the tetanus rise and tetanus relaxation without altering the isometric tension, and reduced the maximum shortening velocity by about 20%. During isotonic contraction tension and stiffness at each given shortening velocity were reduced by about the same amount, so that the stiffness/tension ratio remained almost unaltered. Force-velocity and stiffness data in both standard and EG Ringer were analysed in terms of a two state model (Huxley, 1957). The analysis showed that our results can be accounted for by assuming that EG at 2 M concentration reduces all the rate constants for crossbridges interaction by about the same amount.

Sarcomere tension-stiffness relation during the tetanus rise in single frog muscle fibres.

The sarcomere stiffness was measured in single muscle fibres during the development of tetanic tension using a method insensitive to fibre inertia and viscosity. The stiffness was calculated by measuring the ratio between tension and sarcomere length during a period of fast sarcomere elongation at constant velocity. Tension changes were corrected for force truncation by the quick recovery mechanism. The results show that the relation between force and stiffness deviates from the direct proportionality less than previously reported. If the deviation is due to the presence of a linear myofilament compliance in series with the cross-bridges, our data suggest that myofilament compliance accounts for about 30% of the sarcomere compliance. This value is significantly smaller than 50-70% determined by X-ray diffraction measurements. These two different findings, however, may be reconciled by assuming that the myofilament compliance is non-linear increasing appropriately at low tension.

Mechanical properties of frog muscle fibres at rest and during twitch contraction.

Data reported in the literature suggest that crossbridges in rapid equilibrium between attached and detached states (weakly binding bridges), demonstrated in relaxed skinned fibres at low ionic strength, could be present also in intact fibres under physiological conditions. In addition, it was suggested that the well known leading of stiffness over force during the tension development in stimulated muscle fibres could be due to an increased number of weakly binding bridges induced by the stimulation. The experiments reviewed in this paper were made to investigate these possibilities. Fast ramp length changes were applied to single frog muscle fibres at rest and during the early phases of activation. The corresponding force changes were analysed, searching for the components expected from the presence of weakly binding bridges. The results showed no mechanical indication for the presence of weakly binding bridges in both skinned and intact fibres, either at rest or during activation. It was also found that a portion of the fibre stiffness increase induced by stimulation leads the formation of crossbridges.

Force responses to fast ramp stretches in stimulated frog skeletal muscle fibres.

Force responses to fast ramp stretches at various velocities were recorded from single muscle fibres isolated from either lumbricalis digiti IV or tibialis anterior muscle of the frog (Rana esculenta) at sarcomere length between 2.15 and 3.25 microns at 15 degrees C. Stretches were applied at rest, at tetanus plateau and during the tetanus rise. Stretches with the same velocity but different accelerations were imposed to the fibre to evaluate the effect of fibre inertia on the force responses. Length changes were measured at sarcomere level with either a laser diffractometer or a striation follower apparatus. The force response to a fast ramp stretch could be divided into two phases. The initial fast one (phase 1) lasts for the acceleration period during which the stretching velocity rises up to the steady state. The second slower phase (phase 2) lasts for the remainder of the stretch and corresponds to the well-known elastic response of the fibre. Most of this paper is concerned with phase 1. The amplitude of the initial fast phase was proportional to the stretching velocity as expected from a viscous response. This viscosity was associated with a very short (about 10 microseconds) relaxation time. The amplitude of the fast phase increased progressively with tension during the tetanus rise and scaled down with sarcomere length approximately in the same way as tetanic tension and fibre stiffness. These data suggest that activated fibres have a significant internal viscosity which may arise from crossbridge interaction.

Myofilament compliance and sarcomere tension-stiffness relation during the tetanus rise in frog muscle fibres.

The sarcomere stiffness was measured in single muscle fibres during the development of tetanic tension using a method insensitive to fibre inertia and viscosity. The stiffness was calculated as the ratio between tension changes and sarcomere length changes during a period of fast sarcomere elongation at constant velocity. The results show that, unlike previous measurements with step or sinusoidal length changes, the relation between relative force and relative stiffness on the tetanus rise is linear. Consequently, the development of stiffness upon stimulation is synchronous with the development of force. Since a substantial fraction of sarcomere compliance is localized in the myofilaments, this result can be accounted for by assuming that either myofilament compliance is highly non-linear or that crossbridges stiffness during the tetanus rise is not proportional to crossbridge tension.

Studies on the 14.5 nm meridional X-ray diffraction reflection during length changes of intact frog muscle fibres.

The intensity of the 14.5 nm meridional reflection (M3) from activated skeletal muscle fibres was studied in both single fibres and fibre bundles during the imposition of length changes. During shortening at small load, the intensity of the reflection decreased within 2 ms to less than 20% of isometric intensity, then recovered partially during the remainder of the shortening. When shortening was terminated, recovery of intensity was delayed. Small shortening steps (0.5% fibre length) produced a fall in M3 intensity (IM3) delayed by ca. 250 microseconds compared to the fall in tension. For larger step releases (1% fibre length), the fall in IM3 was not delayed. The fall in IM3 could be almost completely reversed by a subsequent restretch applied within 1.5 ms. Beyond 10 ms after the initial release, the restretch caused a further fall in intensity. A rapid step stretch (0.5% fibre length) also caused a fall in IM3 without delay, which was partially reversed by a release applied within 10 ms. A second small release applied 3 ms (or less) after the first caused a second fall in M3 intensity, but without delay and with faster time course. Small amplitude sinusoidal length oscillations (0.15-0.2% sarcomere; 1 kHz) caused a sinusoidal change in M3 intensity, which was 180 degrees out of phase with the force oscillations, and lacked distortion during its release phase.

Crossbridge viscosity in activated frog muscle fibres.

Force responses to fast ramp stretches at various velocities were recorded in single muscle fibres isolated from tibialis anterior muscle of the frog (Rana esculenta) at a sarcomere length between 2.15 and 3.25 microns at 15 degrees C. Stretches were applied at the tetanus plateau and during tetanus rise. Length changes were recorded at the sarcomere level using either a laser diffractometer or a striation follower apparatus. The immediate force response to the stretch is not simply elastic, as is usually assumed, but is composed of the sum of at least two components: (i) elastic (force proportional to the amount of stretch); and (ii) viscous (force proportional to the rate of stretch). The viscous response is associated with a short (about 10 microseconds) relaxation time. The amplitude of the viscous component increases progressively with tension during the tetanus rise and scales down with sarcomere length approximately in the same way as the tetanic tension. These results suggest that the viscosity of activated fibres may arise from crossbridge kinetics.

Expression, purification, and characterization of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase.

A genetic construct consisting of the synthetic gene coding for human muscle acylphosphatase linked to the gene for glutathione S-transferase has been prepared. This gene was transformed into and expressed by the Escherichia coli strains DB1035 and TB1, respectively. The fusion protein was purified by affinity chromatography and subsequently cleaved to the fully active acylphosphatase, which was further purified by gel filtration chromatography. Such a purification procedure is very rapid and suitable for obtaining considerable amounts of enzyme at a very high yield. The purified human muscle acylphosphatase was fully active and showed structural features, as well as kinetic and stability parameters, identical to those of the native enzyme.